AMER. ZOOL., 37:269-310 (1997)
The Rise of Fish Embryology in the Nineteenth Century1
JOHN P. WOURMS
Department of Biological Sciences,
Clemson University, Clemson, South Carolina 29634-1903
SYNOPSIS. The nineteenth century was critical for the empirical and conceptual
growth of developmental biology. Fishes played a central role in this process. The
study of fish development, mainly that of teleosts but also chondrichthyans, can be
traced back to classical times. In the nineteenth century, it merged with modern
descriptive embryology, continued with the rise of comparative embryology associated with evolutionary studies, and moved into the experimental and physiological analysis of development. Any consideration of fish development must take into
account that fishes phylogenetically are the most diverse group of the vertebrates
and also the most speciose. These features are reflected in the diversity of their
development. The descriptive embryology of fishes is reviewed from Aristotle to
the beginning of the nineteenth century. The study of chondrichthyans, especially
viviparous species, was characteristic of this period. During the nineteenth century,
there was a progressive development of knowledge of the descriptive embryology
of teleosts and chondrichthyans. Teleosts came to the fore because artificial fertilization ensured a ready supply of material and their transparent eggs were well
suited for microscopy. The subsequent development of embryological microtechnique made possible the examination of sectioned material and moved research to
a more cellular level. By the end of the century, an in-depth description of development was in place. Interest in the comparative embryology of fishes was stimulated by Haeckel's melding of embryology and evolution and led to a description
of development of agnaths, chimaeras, lungfish, and primitive actinopterygian fishes. Experimental and analytical methods of inquiry began to be used at mid-century. The experiments of Ransom on the contractility of egg cytoplasm, Lereboullet's experimental teratology, chemical studies of embryonic nutrition in viviparous
fishes, in vitro observation of blastomeres, His's concrescence theory of embryo
formation and Kastschenko's and Morgan's testing of it are considered.
INTRODUCTION
and assorted invertebrates. For obvious reaIt is particularly appropriate to write sons > t h e s t u d y o f fish development continabout the history of fish embryology at this u e d to be pursued by fisheries biologists,
time. History is often said to move in cy- With the popularization of the zebrafish Dacles, and this seems to be true of the study nio rerio as a "model of vertebrate develof fish development. After a period of opment" and a revival of interest in the reprominence during the nineteenth and early lationship between development and evotwentieth centuries, the study of fish devel- lution, the cycle has come full turn and the
opment, except in the hands of a stalwart study of fish development again has a place
few such as Oppenheimer and Trinkaus in the sun.
working with Fundulus and Ballard and
My objective is to present an outline of
Devillers working with the trout and other the historical development of the science of
fishes, was overshadowed by developmen- fish embryology in the nineteenth century,
tal studies of other organisms, such as am- broadly defined as 1789-1914. My plan is
phibians, the domestic fowl, sea urchins, to assess the state of knowledge at the outset of the period, to document the advance1
From the symposium Forces in Developmental Biology Research: Then and NoW presented at the Annual Meeting of the Society for Comparative and Integrative Biology, 26-30 December 1995, at Washing-
a n d e lasmobranchs,
ment of descriptive embryology of teleosts
to Consider the rise of
.
Comparative e m b r y o l o g y of fishes aSSOCl-
ton, D.c.
ated with evolutionary studies, and to
269
270
JOHN P. WOURMS
Ostelchthyes
Actlnopterygll
Sarcopterygll
Agnatha
Chondrichthyes
ir
Aclpenserlformes
Dipnoi
I
ir
2
UJ
FIG. 1.
i
s
o
•o
11
!
3 S
Neopterygll
II
I I
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1 I
o
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Phylogeny of living craniates adapted from Bemis et al. (1977).
search out the beginning of experimental,
physiological, and biochemical analyses of
fish development.
When discussing the development of
fishes, it is prudent to recall that fishes are
the most diverse group of vertebrates (Fig.
1). They should not be treated as a distinct,
relatively homogeneous taxonomic unit.
Rather, the term "fishes" applies to a grade
of ectothermic, aquatic craniates and vertebrates and not to a distinct taxonomic
group (Atz, 1985; Bruton, 1990; Nelson,
1994). Extant fishes are divided into five
classes, namely Myxini (hagfish), Cephalaspidomorphi (lampreys), Chondrichthyes
(cartilaginous fishes), Sarcopterygii, and
Actinopterygii (Nelson, 1994). Chondrichthyes contains two taxa, namely the subclass Elasmobranchii (living sharks, rays,
and skates) and the Holocephaiii (ratfishes
or chimaeras) that diverged at a relatively
early time, i.e., the Devonian-Carboniferous
boundary (Compagno, 1990). The class
Sarcopterygii (flesh-finned fishes) contains
the Actinistia (the living coelacanth), the
Dipnoi (lungfishes) and the tetrapod vertebrates. The class Actinopterygii (ray-finned
fishes) comprises five groups, namely Cladistia (bichir and reedfish), Chondrostei
(sturgeons and paddlefish), Ginglymodi
(gars), Halecomorphi {Amia calva, the bowfin), and the Teleostei (teleost fishes) (Lauder and Leim, 1983; Atz, 1985; Nelson,
1994). Thus, the five classes of fishes contain eleven major taxonomic groups with an
estimated total of 25,000 species. Each
class is as distinct from the others as it is
from the four other classes of vertebrates.
The theme of eleven major taxonomic
groups of fishes that is used in the ensuing
presentation to emphasize the broad phylogenetic relationships of fishes and other
vertebrates, tends, however, to obscure the
extraordinary phylogenetic diversity within
the teleosts. Teleostei, a monophyletic
group, contains 24,000 species in 38 orders
RISE OF FISH EMBRYOLOGY
and 126 families compared to the 1,000
species, 19 orders, and 56 families in the
ten other groups of fishes and the 25,000
species of tetrapods (Nelson, 1994).
Not only are fishes the most diverse
group of craniates, but they also display the
greatest diversity of life history styles (Balon, 1975, 1984; Bruton, 1990; Compagno,
1990). Most fishes are oviparous, but viviparity is estimated to have independently
evolved 42 times in five of the eleven major
groups of fishes, all within the Chondrichthyes and Osteichythes (Wourms, 1981;
Wourms et al. 1988; Wourms, 1994). The
phylogenetic diversity of fishes is reflected
in the diversity and variability of their developmental patterns (Collazo et al, 1994;
Wourms, 1994). Craniate development
based on cleavage and subsequent patterns
of gastrulation and embryogenesis is holoblastic, meroblastic, or transitional between
the two. In holoblastic eggs, cleavage of the
ooplasm is complete by the blastula stage.
The morphogenetic movements of gastrulation occur in the three-dimensional space
of the entire egg and involve most of the
embryonic mass. They often lead to the occlusion of the blastocoele. Neurulation is
synchronous in time and space. Holoblastic
development is considered the less specialized and more primitive craniate developmental pattern because it is characteristic of
most echinoderms and non-craniate chordates as well as the more primitive representatives of various craniate lineages,
namely lampreys, sturgeons, bichirs, lungfish, and most amphibians.
In the meroblastic egg, most of the yolk
mass remains uncleaved at the blastula
stage. Morphogenetic movements are confined to a portion of the egg volume, i.e.,
the blastoderm, and involve only a portion
of the embryonic mass. Gastrulation and
neurulation tend to be spatially and temporally asynchronous. Evolution of the
meroblastic pattern appears to have involved qualitative and quantitative changes
in the ooplasm and yolk components of the
egg, namely an increase in total yolk content, an increase in the amount of yolk relative to ooplasm, a change in the type of
yolk, and a change in the relative disposition of ooplasm and yolk (Soin, 1981; Elin-
271
son, 1989). Not only did the changes in
yolk-cytoplasm relationships of the egg affect cleavage patterns, but they also imposed a set of physical or physiological
constraints on the morphogenetic movements of embryo formation, the so-called
holoblastic-meroblastic barrier (Elinson,
1989). Consequently, new or highly modified patterns of gastrulation and embryogenesis co-evolved with the evolution of
the meroblastic egg. The meroblastic pattern of development appears to have independently evolved in six craniate lineages,
namely hagfish, sharks and chimaeras, teleost fishes, the living coelacanth (?), one
group of amphibians (the caecilians) and
amniotes. In each instance, different morphological patterns of gastrulation and embryogenesis have evolved. Thus, although
meroblastic cleavage is superficially similar
in hagfish, elasmobranchs, and teleosts, the
morphological events of embryo formation
differ considerably. These points and others
are summarized in part by Collazo et al.
(1994) who present a phylogenetic perspective and an evolutionary scenario for teleost
gastrulation. Paradoxically, whether development proceeds according to a holoblastic
or meroblastic pattern, the end product is a
stereotypic craniate embryo. Recent advances in the study of the molecular basis
of embryogenesis should help resolve this
paradox (De Robertis et al., 1994).
DESCRIPTIVE EMBRYOLOGY
Beginning with Aristotle, various authors
have summarized the existing knowledge of
fish development. In most instances, they
did so in conjunction with larger works that
dealt with the natural history or systematics
of fishes, e.g., Rondelet (1554), Willughby
and Ray (1686), Bloch (1782-1795) and
Agassiz (1857), or else as part of a monograph that treated a selected aspect of development, e.g., Tilesius von Tilenau
(1802), von Baer (1835), Duvernoy (1844),
Van Bambeke (1876), Kingsley and Conn
(1883), Henneguy (1888), and Scammon
(1911). Comprehensive treatment of fish
development was provided in several embryological textbooks, e.g., Balfour (1880),
Ziegler (1902), and Kerr (1919). Access to
older, classical literature was provided by
272
JOHN P. WOURMS
TABLE 1. Chronology of some significant contributions to the study of the descriptive embryology of fishes from the late seventeenth to the late nineteenth centuries, arranged according to author.
1. Steno (1673)—Rediscovery of shark yolk sac placenta.
2. Lorenzini (1678)—Blastoderm-neurula—Torpedo
3. Jacobi (1763)—Artificial fertilization
4. Bloch (1785)—Stages in early embryonic development; viviparity
5. Monro (1785)—Skate embryos
6. Cavolini (1787)—Teleost blastoderm
7. Forchhammer (1810)—Embryonic shield; heart
development
8. Rathke (1825-1827, 1833)—Sharks, teleosts; urogenital system; gill slits and arches
9. von Baer (1835, 1837)—Epiboly; germ ring
10. Rusconi (1836)—Cleavage stages; epiboly
11. Vogt and Agassiz (1842)—Cellular nature of embryo
12. Muller (1842)—Shark placenta
13. Lereboullet (1862; 1863)—Periblast; teratology
14. von Kupffer (1866; 1878)—Kupffer's vesicle;
photographs of herring embryos
15. Goette (1873)—Involution during teleost gastrulation
16. Balfour (1878)—Elasmobranch development
17. Agassiz and Whitman (1884)—Yolk syncytial
layer
18. Ryder (1884a); Henneguy (1888); Wilson
(1891)—Descriptive accounts based on sectioned
material
19. Ruckert (1892)—Lampbrush chromosomes
Dean's Bibliography of Fishes (Dean,
1916-1923). It was not until the late Jane
Oppenheimer (1936) published her "Historical Introduction to the Study of Teleostean Development" that the topic was subject to historical analysis. Only recently has
the history of the study of elasmobranch development been summarized (Wourms and
Demski, 1993). Little historical documentation has been done for the other groups
of fishes (cf. Table 1 for an outline of descriptive embryology).
When considering early studies of fish
development, one soon realizes that many
of these studies were carried out on viviparous species of elasmobranchs and teleosts
rather than oviparous ones, and that both
egglaying and livebearing elasmobranchs
received a disproportionate amount of attention, considering their relatively low diversity compared to teleosts. First, elasmobranch eggs are considerably larger than
those of most teleosts and their develop-
ment can be easily observed with either the
unaided eye or with a hand lens. Secondly,
it has always been relatively easy to obtain
gravid specimens of viviparous fishes as
part of routine fishing efforts. Moreover, as
museum or natural history collections of
fishes were made during the course of explorations, specimens of curious, new species of gravid viviparous fishes became
available for study, e.g., the four-eyed fish
Anableps. In contrast, the eggs of oviparous
teleosts are small and not easily collected
in the wild. Artificial fertilization of teleost
eggs did not begin until the late eighteenth
century (Jacobi, 1763; cited in Oppenheimer, 1936) and the regulated spawning of
captive fish species and the rearing of their
eggs under "controlled" conditions for scientific purposes was first used only toward
the mid-nineteenth century (Vogt, 1842). In
addition, the technology for maintaining
fishes in aquaria and the possibility of
spawning them under aquarium conditions
did not emerge until the mid-late nineteenth
century. In passing, one should recall that
the Chinese have been rearing captive goldfish, including mutant strains, for over a
thousand years. Other factors also influenced decisions to study certain viviparous
instead of oviparous fishes. Viviparity in
fishes, especially when there is a placental
relationship, is an intrinsically interesting
process. Finally, as a subject for the general
study of development, oviparous teleosts
have been secondary to the chicken egg
ever since the Renaissance because of the
greater size of the chicken's egg and its
ready availability as an article of food (Oppenheimer, 1936).
Early history: Aristotle to Bloch
Because the research of early naturalists
provided the foundation for the development of fish embryology in the nineteenth
century, a brief outline of their work is presented here, beginning with Aristotle. Aristotle, the founder of the science of embryology, was the first to recognize and record
many important features of fish reproduction and development (Aristotle, 1942,
1965, 1970). He distinguished oviparous
(egg-laying) animals from viviparous (livebearing animals. Aristotle described vivi-
RISE OF FISH EMBRYOLOGY
parity in the dogfish shark (Mustelus), the
thresher shark (Alopias), and the electric
ray (Torpedo). He did not know of viviparity in teleost fishes because there are no viviparous teleosts in the coastal waters of the
Mediterranean or the fresh waters of Europe and Asia Minor. His attribution of viviparity to the needlefish, a teleost, is actually a case of misinterpretation. The needlefish of Aristotle is a pipefish or sygnathid. Male pipefishes brood embryos in a
pouch of abdominal skin. This form of parental care is analogous to viviparity and in
its most highly evolved forms, the parentalembryonic relationships can be as complex
as in viviparous fishes. The body of Aristotle's observations concern chondrichthyan
fishes and include: (1) the distinction between oviparous and viviparous modes of
reproduction in sharks, rays, and skates; (2)
description of the female reproductive system; (3) description of shark and skate egg
cases; (4) description of egg structure and
some observations on embryonic development; (5) first description of the oviducal or
nidamental (=shell) gland but without any
knowledge of its function in egg case formation or sperm storage; (6) hatching from
the egg case in utero in viviparous species;
(7) description of the male reproductive
system; (8) notes on breeding seasons and
inshore migration of gravid females for
"pupping"; and (9) description of the yolk
sac placenta in sharks. Writing of the
smooth dogfish Mustelus canis in the 5th
century B.C., Aristotle states, "the young
are produced with the umbilical cord attached to the uterus so that as the substance
of the egg gets used up, the embryo's condition appears to be similar to what is found
in quadrupeds. The umbilical cord, which
is long, is attached to the lower parts of the
uterus; each one is, as it were, fastened to
a cotyledon and is attached to the embryo
by the middle where the liver is situated.
Each embryo has a chorion and membranes
of its own round it, just as in quadrupeds.
If the embryos are cut open, a situation is
disclosed exactly similar to quadrupeds;
whatever internal organs they have, such as
the liver, are large and supplied with blood"
(Aristotle, 1970, p. 565b).
Aristotle's observations on teleosts are in
273
large part concerned with reproduction,
e.g., his exquisite account of brood protection by male catfish. He realized that in
contrast to the chondrichthyans, the overwhelming majority of teleosts are oviparous. His observations on the development
of teleosts are more limited, due primarily
to the small size of most teleostean eggs.
Aristotle observed developing teleost eggs,
recording their small size and rapid development. He states, "The embryo appears at
the upper end of the egg and is enveloped
in a membrane; the eyes, large and spherical, are the first organs visible. The embryo
and the egg (yolk) are enveloped by a common membrane, and just under this is another membrane that envelops the egg
(yolk) by itself; between the two membranes is a liquid" (Aristotle, 1970, p.
561b). He described the organization of the
teleost egg, distinguishing the perivitelline
fluid from the yolk mass. [As an aside, Oppenheimer (1936) pointed out that some
celebrated nineteenth century embryologists, viz. von Baer and Rathke, erred by
considering the perivitelline fluid to be an
albumen such as occurs in avian eggs.]
Lastly, in comparing the development of
teleostean and avian eggs, Aristotle recognized that teleosts lack the allantois.
Relatively little was added to the pioneering efforts of Aristotle by subsequent
Greek and Roman scholars. With the decline of classical civilization, the study of
science in general as well as the study of
fish development languished for almost
1,000 years. Slowly the tide turned. According to Needham (1959), the first account of fish development in medieval Europe was given by Albertus Magnus, a thirteenth century scholastic. During the Renaissance, there was a revival of interest in
natural history. The early zoological encyclopedists, especially Belon (1517—1564),
Rondelet (1507-1566), and Aldrovandus
(1522-1605), investigated fishes and described aspects of their reproduction and
development. Again, there was a decided
bias toward viviparous chondrichthyans.
Rondelet (1554) illustrated an egg case and
dissection of the female reproductive system of an oviparous catshark. He also illustrated part of the ovary and an egg case of
274
JOHN P. WOURMS
a skate. He depicted a female shark, prob- with that of other vertebrates (Glass, 1959).
ably Mustelus canis, connected to a well- In essence, he used sharks as a "model"
developed pup by an elongated yolk stalk system for mammals.
that passes from the pup through the cloaca
In 1678, Stephen Lorenzini, a pupil of
of the female. Although the placenta is not Steno and Redi, published a set of obserillustrated, Aristotle's research on the shark vations on the viviparous electric ray Toryolk sac placenta is cited in the text. Pro- pedo (Lorenzini, 1678). This appears to be
gressing to the seventeenth century, Fabri- the first monographic treatment of a single
cius (1600), who is best known for his stud- fish species. Lorenzini's work is of considies of the chick, described and illustrated erable interest because almost one third of
the female reproductive system and uterine it is devoted to an account of urogenital
embryos with large yolk sacs in a non-pla- anatomy, reproductive biology, and develcental species of dogfish shark.
opment of Torpedo, as well as several other
By 1673, Niclaus Steno had rediscovered rays. Amongst his observations, those of
the yolk sac placenta in the smooth dogfish, primary interest concern oogenesis and
M. canis, investigated its anatomy, and pub- ovulation, the blastoderm, and embryonic
lished an illustrated account of it. He also nutrition. Describing the ovary, he notes
distinguished between placental viviparity differences in size and structure between
in the smooth dogfish shark, and aplacental young and mature fishes. Maturing oocytes
viviparity in the spiny dogfish shark, Squa- and ova vary in size, composition, and collus. In the latter, he discovered that the yolk or. Oocytes are connected to the ovarian
stalk terminates in an intestine character- matrix by a stalk through which several
ized by a spiral valve (Steno, 1673). His blood vessels pass, an arrangement similar
rediscovery appears to have gone unappre- to that described by Fabricius in the hen's
ciated. During this period, inability to dis- egg. He states that these vessels provide nutinguish among species of dogfish sharks trients to the eggs. The maturing egg is inthat had either aplacental or placental vested by a "membrane" except for the
modes of viviparity caused the very exis- pole opposite the site of attachment to the
tence of a yolk sac placenta in sharks to be stalk. This pole constitutes the pore or stigdoubted. This controversy was not fully re- ma through which the mature egg is resolved until the time of Johannes Miiller leased from the follicle. During oocyte mat(Miiller, 1842; Singer, 1921). Steno's stud- uration, changes in membrane adhesion
ies on shark development had important were documented. Lorenzini speculates that
ramifications for the study of mammalian mature eggs are released from the follicle
reproduction. On the basis of his observa- by contraction of fibers in the ovarian strotion of large eggs in the uterine oviduct and ma. The ovulated egg is "received" by the
large eggs in the ovary of the spiny dogfish, oviduct and passes into the uterine region
he concluded that the embryo does not form where embryonic development is completde novo in the oviduct, but comes form an ed. Following Fabricius (1537-1619), De
egg released by one of the ovaries. From Graaf (1641-1673), and Harvey (1578this, he postulated that mammalian devel- 1657), Lorenzini refers to the residual poropment proceeded in a similar fashion, ex- tion of the follicle as the "calyx." Calyces,
cept that the mammalian egg is much small- which apparently are the corpora lutea, ocer. In his examination of the mammalian cur in various sizes and shapes throughout
ovary, unfortunately, he mistook the entire gestation, and are lost or regress some time
follicle for the mammalian egg. These stud- after parturition. Lorenzini found a correies were undertaken at a time when a con- lation between the number of young in a
troversy had arisen between animalculists brood and the number of calyces in the ovaand ovists with respect to the primacy of ry. Ovulation in the Torpedo is considered
the sperm in reproduction and the absence to conform to De Graaf's account in other
of any knowledge of the mammalian egg. species. Among the more interesting of
Steno's observations, although flawed, Lorenzini's observations are those made on
brought mammalian development in line the early stages of embryonic development.
RISE OF FISH EMBRYOLOGY
He describes and illustrates two neurula
stages. The earlier stage depicts an early
neural groove on the surface of the blastoderm. He describes the characteristic chondrichthyan blastoderm, "a leaden gray
disc" that is clearly distinct from and surmounts the "yellow yolk mass." In another
egg, he describes and depicts a slightly
more advanced neurula stage (equivalent to
Ballard et al., 1993, Stage 15). Here Lorenzini correctly shows the splayed, outspread cephalic margins of the medullary
plate, a characteristic feature of chondrichthyan embryos. Lorenzini also observed
the blastoderm in three other species of
rays. Lorenzini seems to be the first to have
depicted the chondrichthyan blastoderm.
His observations appeared only six years
after Malphighi's initial account of the avian (chick) blastoderm (Needham, 1959).
Lorenzini made one of the first major contributions to the knowledge of embryonic
nutrition in non-placental viviparous fishes.
Late stage embryos with yolk sacs lie free
in the uterine region of the oviduct where
they are bathed by uterine fluid. A yolk
stalk passes from the external yolk through
the abdominal wall and empties into a large
internal yolk sac that in turn empties into
the intestine. The intestine is filled with yellow material apparently derived from the
yolk and brown globules. The same brown
globules occur in the uterine fluid and in
the mouth, esophagus, and stomach of the
embryos. Lorenzini concluded that advanced embryos are nourished both by yolk
passed to the intestine and by uterine fluid
which they imbibe. He believed that the nutrients in uterine fluid are derived from the
blood and conveyed to the uterine fluid by
vascularized glandular structures which he
compared to chorionic villi. These structures are now known as trophonemata
(Wourms, et al., 1988). Villi increase in size
during gestation; their size is correlated
with the amount of uterine fluid. They regress and are absent in non-gravid fish. In
addition, Lorenzini also made significant
observations on the male reproductive system, reproductive cycles, and the duration
of gestation. In the closing years of the seventeenth century, Willughby and Ray
(1686) summarized much of what was
275
known of fish reproduction and development in a work primarily devoted to systematic s.
Considering its promising beginning, it is
surprising that the study of chondrichthyan
development made relatively little progress
in the eighteenth century. There are many
possible reasons. Those scholars interested
in fish biology may have been attracted to
systematic studies because of the revolutionary efforts of Willughby and Ray, Artedi (1705-1735), and Linnaeus (17071778) as well as the influx of new species
of fishes from the tropics. Changing fashions in science also enter the picture, especially the rise of microscopy and microscopic anatomy. Perhaps in terms of the
material, questions, and technology, research on chondrichthyans had reached an
impasse. There are some exceptions. Bohadsch (1776) investigated the structural organization of eggcases. Monro (1785) published detailed descriptions accompanied by
elegant illustrations of the urogenital anatomy of the oviparous skates. He also described and illustrated for the first time the
spermocysts, spherical aggregations of Sertoli cells and differentiating sperm cells that
are a characteristic feature of chondrichthyan spermatogenesis. Descriptions and
illustrations of microdissections of the internal anatomy of mid- and late-stage embryos are given. The extensive vitelline circulation and the connection of the yolk sac
and the small intestine are documented. External gill filaments, another feature of
chondrichthyan development, are described
for the first time. The eighteenth century
concluded with Bloch's magnum opus
(1782-95) which added little to the study
of chondrichthyan development other than
information on the embryos of the viviparous sawfish Pristis.
The study of teleostean development, as
previously noted, lagged well behind that of
both oviparous and viviparous chondrichthyan fishes, and little was done prior to
the mid-late eighteenth century. To be more
accurate, the little that was done was done
with viviparous teleosts. Schoenveld (1624)
first described viviparity in the embryos of
the eelpout, Zoarces viviparus, a fish common to the marine waters of northern Eu-
276
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JOHN P. WOURMS
rope. Late stage embryos are illustrated.
Impressed by the substantial growth of the
embryos during gestation, Schoenveld postulated that they obtain nutrients by ingesting ovarian fluid. (In viviparous teleosts, an
oviduct or uterus is absent and gestation
takes place in the ovary. Cf. Wourms, et ai,
1988). Somewhat later, Willughby and Ray
(1686) apparently observed the development of live Zoarces embryos. Interest in
teleost development during the seventeenth
and eighteenth centuries seems to have
been generated by the viviparity of exotic
species discovered during the explorations
of the Old and New World Tropics. Anableps, the four-eyed fish, conspicuous because of its size, unusual anatomy, and behavior in its Central and South American
habitats, was described as viviparous as early as 1738 (Artedi, 1738). Gronovius
(1754-1756) was the first to illustrate advanced embryos of Anableps with their
characteristic rugose "yolk sac" (actually
the abdominal trophoderm). Gronovius may
also have been the first to describe viviparity in the clinid, Clinus superciliosus. Bloch
(1782-1795) described and illustrated the
external and internal reproductive anatomy
of Anableps, its embryos in situ, and the
embryos themselves. He also depicted the
adults and embryos of Zoarces and Clinus.
One of the first sets of field observations
was made by de Alzate y Ramyrez in 1769
(cited in Gill, 1882) who discovered viviparity in a Mexican poeciliid and made microscopic observations of blood circulation
in living, near-term embryos.
During this period, the foundations were
laid for the study of early development, i.e.,
fertilization through embryogenesis. In
1763, Jacobi (cited in Oppenheimer, 1936)
published an extensive letter in which he
reported that he had artificially fertilized the
eggs of trout and salmon by removing the
ripe eggs and adding sperm to them. Although this is the first known written account of artificial fertilization, it is probable
that the technique had been in use for some
time, for fish culture had been practiced in
Europe since the late fifteenth century. Jacobi may have observed the entry of sperm
into the egg. He also recorded the formation
of double embryos in artificially fertilized
eggs, a condition that he attributed to the
entry of two spermatozoa. In 1787, Cavolini published a monograph on the "generation" of fishes and crabs. The bulk of
this work was devoted to the description of
spawning habits, hermaphroditism, fertilization, and contemplations on the nature of
development. However, he did make one
observation of considerable significance. In
the eggs of the pipefish, he described a
structure on the egg which he referred to as
the "cicatrix" or in modern terminology,
the blastoderm. He identified the blastoderm with the same structure in the chick
and assumed that the embryo originated
from the blastoderm. The study of teleost
development in the eighteenth century was
brought to a close with the appearance of
Bloch's (1782-1795) massive, twelve-volume monograph on the natural history of
fishes. This work is one of the linchpins of
systematic ichthyology and summarized
what was known about fishes up to that
time. It contains much information on fish
reproduction and development. As previously noted, aspects of the development of
viviparous chondrichthyans and teleosts are
described and illustrated. Of greater interest
are his pioneering studies of the early stages
of development of an oviparous teleost. For
the first time, he describes and illustrates
consecutive stages in development, from
post fertilization through hatching to a late
larval stage, and provides a time base for
the description of developmental stages. He
appreciated the eccentric position of the
yolk mass relative to the blastoderm. He
made observations on the development of
the heart, and illustrates the contractile,
simple tubular stage of heart development.
There were, of course, errors of interpretation, one of which was the identification of
the perivitelline fluid of the teleost egg with
the albumen layer of the avian egg.
Development of teleostean fishes
As the nineteenth century dawned, the
foundations had been laid for the study of
fish development, and the stage had been
set for the extraordinary progress that
would soon be made. The rise of fish embryology was vitally linked to the origins
of embryology as a formal science and its
RISE OF FISH EMBRYOLOGY
AU805B97 277
subsequent growth, diversification, and in- first to describe the embryonic shield, the
teraction with other branches of biology region in which the embryo forms. His ob(Needham, 1959; Horder et al, 1985; Chur- servations on the contractility of the excised
chill, 1991). Scientific embryology is gen- embryonic heart constitute one of the earerally considered to begin with the publi- best attempts to study isolated embryonic
cation of the first volume of von Baer's organs. He also observed that yolk ulticomparative studies of animal development mately is absorbed into the liver. Rathke
(von Baer, 1828). As Oppenheimer (1936) (1833) also published a monograph on the
has pointed out, even though von Baer's development of the viviparous blenny, as
work codified embryology as a science and part of a much larger serial work on the
profoundly influenced its future directions, development of animals and humans. Connonetheless, it was the culmination of a ceptually, Rathke's studies parallel those of
course that had been pioneered by Dollin- Dollinger and von Baer (Oppenheimer,
ger (1770-1841) and Rathke (1793-1860). 1936). Although he described and analyzed
Oppenheimer (1936) states that Dollinger early stages of development, he was pri"was the originator of the appreciation of marily concerned with older stages. His obcomparative development by working out servations on the developmental anatomy
carefully the succession of steps in the de- of the brain, heart, gut, and other organs
velopmental processes in various vertebrate established a new standard of excellence in
forms." Dollinger not only influenced von the field.
Baer (1792-1876) but also guided Pander's
The first volume of von Baer's three-vol(1793-1860) revolutionary studies of chick ume treatise on embryology appeared in
development and inspired in Louis Agassiz 1828 but fishes were not dealt with until the
(1807-1873) a life-long commitment to a second volume (von Baer, 1837). (Cf, Blayprogram of comparative embryological re- ker, 1982, for an English summary of von
search. Dollinger's approach to embryology Baer's great classic and his monograph on
had its origins in the influence of German fish development.) In the interim, he pubNaturphilosophie on the study of compar- lished a separate communication devoted to
ative anatomy. Thus influenced, anatomists the development of fishes based on his studattempted to systematize, describe, and ies of species of Cyprinus (von Baer, 1835).
classify all organisms or parts of organisms He made several fundamental observations
as variations of a single type. Dollinger, such as the sharp distinction between the
who was a proponent of Naturphilosophie, pattern of development in large-yolked and
thus accepted and extended suggestions of small-yolked teleostean eggs and the great
the anatomist Meckel (1781-1833) that a differences in the duration of development,
unity of plan manifests itself in develop- viz- three months in trout, a few weeks in
ment as well as in structure (Oppenheimer, Zoarces and a few days in Cyprinus. His
1936).
choice of Cyprinus, an egg-laying species,
For reasons of convenience and also be- made it possible to obtain newly fertilized
cause their study progressed at different eggs, thus giving him access to the early,
rates and in different ways, I have chosen i.e., cleavage-epiboly, stages of developto treat teleosts and chondrichthyans sepa- ment and providing a chronological base
rately. In retrospect, it is amazing to con- for the consecutive stages of development,
template the almost explosive increase in von Baer (1835) was the first to describe
knowledge of teleostean development that clearly and illustrate one of the most chartook place in the period 1819-1842. The acteristic features of teleostean developfirst major work from this period was ment, viz. epiboly or overgrowth of the yolk
Forchhammer's (1819) dissertation on the by the blastoderm. Not only did he describe
development of the viviparous blenny it, but also he provided a timetable for the
Zoarces. He described no stages younger process. He was also the first to describe
than pre-motile embryos. The main body of the germring, i.e., the thickened edge of the
the work comprises descriptions of older blastoderm, von Baer failed to observe
embryos and organ formation. He was the cleavage and incorrectly homologized tele-
278
JOHN P. WOURMS
ost development with that of other vertebrates by mis-identifying the early embryonic shield as primitive streak and the late
shield as a broad neural plate (Oppenheimer, 1936). von Baer concluded his monograph on fish development with an appendix in which he discussed the embryological origin and function of the swimbladder
and compared it to the lungs of other vertebrates. Studies very much similar to those
of von Baer were being carried out by Rusconi (1836) on two species of Cyprinus.
Rosconi gives a detailed account of his
method of artificial fertilization. Developing eggs, thus obtained, were of known age
and were subsequently examined microscopically. His major contribution is that he
appears to have been the first to describe
cleavage in the teleostean egg. His detailed
description is illustrated by a plate which
"rivals all other in the literature for accuracy and beauty in presenting the early
stages of teleostean development" (Oppenheimer, 1936, p. 138). He also made extremely accurate observations on the embryonic shield and its role in the formation
of the embryo. To aid observation, he used
an acid to stop development and render the
embryo opaque without affecting the transparency of the yolk. His figures depict the
concentration of cells in the midline of the
embryonic shield that are the precursor of
notochord and spinal cord. Elsewhere, he
states for the first time that the teleostean
nerve cord originates from a single structure rather than by the folding of a neural
plate.
Embryological research on teleosts
moved to France during the middle years of
the century (Oppenheimer, 1936). The first
important work was Vogt's (1842) contribution to the embryology of the whitefish
(Coregonus), which was part of Agassiz's
elaborate series of monographs on the
freshwater fishes of central Europe. The
embryology portion consists of a text volume and a folio of colored lithograph
plates. Although considered part of the
French School by Oppenheimer (1936), it
could just as well be considered a product
of the Dollinger-Wurzburg School. Agassiz
was in close contact with Dollinger at Munich between 1827 and 1830 and was
studying teleost development (Oppenheimer, 1936). The role of Agassiz, if any, in
Vogt's study is not clear, for the dispute
over authorship was the first in a series of
controversies that would occur between Agassiz and his collaborators (Oppenheimer,
1986). In addition to its scientific methods
{vide infra), this work had an extraordinary
impact because of the clarity and beauty of
its illustrations. The figures were drawn by
professional artists and reproduced, many
in color, by lithography, a printing process
capable of rendering fine detail. Not only
were new observations effectively presented, but also the known details of development took on a new lucidity. The Vogt-Agassiz study is another study in which developing eggs were obtained by artificial
fertilization, cultured within a set temperature range (5-10°C), and examined microscopically. For the first time, the technique
of dechorionation is mentioned as an aid for
observation. Vogt also used the technique
of acid fixation to enhance the visibility of
the embryo. A series of well-illustrated,
closely spaced stages in the normal development of the whitefish was presented. This
work appears to be the first study to apply
a cellular approach to the interpretation of
developmental events, especially oogenesis,
cleavage, epiboly, and embryogenesis. Information is provided on oogenesis, the
structure of the mature egg, and fertilization. According to Oppenheimer (1936),
Vogt was the first to inquire into the mechanics of embryo formation. He also speculated on the role of cell movement and cell
migration in development. A considerable
portion of the work was devoted to the microanatomy of the later stages of embryonic
and larval development. In this respect,
Vogt gives an account of the formation of
a number of organ systems, tracing their development from the earliest cellular condensations or anlagen to completion. Illustrations of melanocytes and the cellular
components of the notochord and connective tissue are remarkable for their detail
which included nuclei.
The next figure of consequence in the
French school was Lereboullet, a pupil of
Duvernoy, who in turn was a protege of
Cuvier. Lereboullet made substantial con-
RISE OF FISH EMBRYOLOGY
tributions in the field of descriptive embryology and subsequently in experimental
studies which were the first great attempt to
analyze the mechanism of embryo formation (Oppenheimer, 1936). His monograph
on the development of the pike and perch
established new standards in terms of the
accuracy with which his observations were
depicted (Lereboullet, 1862). He provided
the first detailed description of the microscopic organization of the egg and a more
accurate description and illustration of the
embryonic shield. He stated that it is the
precursor of the entire embryo and not just
the central nervous system. His most important single observation was the description of the yolk syncytial layer (periblast)
which he termed "feuillet muqueux," literally mucous lamina. He mistakenly ascribed to it the fate of forming elements of
the digestive system. He regarded the germ
ring, i.e., the thickened margin of the blastoderm, as a dynamic force during the formation of the embryo. This viewpoint, reinforced by his studies on experimental teratology {vide infra), led him to interpret the
process of embryo formation in terms of
concrescence, i.e., the right and left portions
of the germ ring assemble into the two
halves of the body. Lereboullet arrived at
this conclusion a decade before His (1874)
formally promulgated the concrescence theory. Finally, in his monograph on the comparative embryology of the trout, a lizard
and a snail (Lereboullet, 1863a), he refuted
the concept of a unity in the plan of development propounded by Meckel and Serres
(Oppenheimer, 1936).
Three important structures, characteristic
of teleostean development, still need to be
dealt with, namely the yolk syncytial layer
or periblast, the enveloping layer, and Kupffer's vesicle. Although Lereboullet (1862)
first described the yolk syncytial layer, it
was von Kupffer (1868) who suggested that
the cells of the blastoderm not only give
rise to the tissue cells of the embryo but
also are progenitors of the yolk syncytial
layer. Agassiz and Whitman (1884, 1915)
proved beyond any doubt that yolk syncytial nuclei are derived from the marginal
cells of the blastoderm. The enveloping cell
layer is a tightly sealed epithelial monolayer
279
that forms the outermost boundary of the
blastoderm. Priority for its discovery is unclear, for descriptions of it under various
names appeared more or less simultaneously in a number of independent studies.
Kupffer's vesicle is a transient structure that
is only found in teleost embryos. It was discovered by von Kupffer (1866), who
thought that it might be an allantoic rudiment. It is a small, fluid-filled epithelial sac
that appears during the somite stages. Kupffer's vesicle develops mid-ventrally in a region below the tail bud and at the interface
between the embryo and yolk mass. Even
now, its function, if any, still remains unknown (Brummet and Dumont, 1978).
At this point, a digression to consider the
development of viviparous teleosts is in order. Following the pioneering studies of
Forchhammer (1819) and Rathke (1833) on
Zoarces, knowledge of viviparity in teleosts
progressed steadily through the rest of the
century. In 1844, Duvernoy described the
development of a freshwater poeciliid fish
from Surinam. His work was mainly concerned with the later stages of development,
but did provide some important information
on early development. The ovary is the site
of gestation. Fertilization takes place in the
follicle where the embryo is invested by an
egg envelope. Development to term takes
place in the follicle. Duvernoy illustrated
for the first time an early stage follicular
embryo in its egg envelope. In his Memorias, published 1851-55, the Cuban naturalist Poey was able to extend the number of
viviparous species to include all ten species
of Cuban poeciliids. Of greater significance
were his observations for the first time of
sperm storage in these fishes. Unfortunately, he interpreted his observations in terms
of parthenogenesis rather than sperm storage and superfetation (Jacobson, 1977).
The problem was not satisfactorily resolved
until 1908, when Phillipi discovered that in
poeciliids, sperm is packaged into spermatozeugmata that are transferred to the male
reproductive tract. He also presented evidence for specific structural pathways,
which he called "delle," that allow sperm
to move from the lumen into close proximity with the egg and fertilize it within the
follicle. In 1856, Wyman produced a defin-
280
JOHN P. WOURMS
itive study on the embryology of the foureyed fish Anableps. It, too, is a fish with
follicular gestation. He depicts the elaborate
follicular epithelium and the highly vascularized abdominal trophoderm of the embryo. Because of the massive increase in
size of the embryo during gestation, he postulated that the developing embryos are
nourished by secreted maternal proteins. He
extended his speculations to include embryonic nutrition in the surfperch and the electric ray Torpedo.
In 1853, Agassiz first described viviparity in the surfperches or embiotocids from
the Pacific coast of North America. In a
more extensive publication, Girard (1858)
described and illustrated 17 species. He
provided the first detailed information about
their reproduction and development. Gestation is ovarian: embryos develop in the
ovarian lumen and are enfolded by sheaths
of ovarian tissue. Initial egg size is minute,
but term embryos are large (5 cm). A series
of developmental stages is described and illustrated. Figures on brood size which varies (10-80 young) according to species are
given. The duration of gestation is estimated at 5-6 months. The initial descriptions
of Agassiz and Girard culminated in a definitive study of surfperch development by
Eigenmann (1894) which is tour de force of
descriptive embryology. He noted that the
early cleavage stages of the small, yolkpoor eggs verge on being holoblastic. He
described the presence of sperm in the ovarian fluid and the imbibition of ovarian fluid
by the embryos. Male embryos undergo
precocious sexual development. In an earlier separate publication, he (Eigenmann,
1891) described the precocious segregation
of germ cell determinants at an early cleavage stage, an overlooked, early contribution
to concepts of germ cell development.
Throughout the remaining years of the century the occurrence of viviparity was described in various groups of teleost, e.g., the
rockfish, Sebastes (Kr0yer, 1845), halfbeaks, Hemirhamphus (Peters, 1865), and
the Lake Baikal oilfish, Comephorus (Dybowski, 1873). One should recall that such
studies are still continuing, with viviparity
in the living coelacanth Latimeria not discovered until 1975 (Smith et al., 1975). By
1886, the field had progressed to the extent
that Ryder (1886a) could review it and add
to it some of his own original observations.
He concluded that the developing embryos
of surf perches derive nutrients by absorbing imbibed ovarian fluid through elongated
intestinal villi found in the hypertrophied
hindgut. He also concluded that the other
characteristic feature of surfperch embryos,
the unique vascular apparatus of the enlarged vertical fins, serves a respiratory
rather than a nutritive function. Much of the
research on viviparity, both in teleosts and
chondrichthyans during the latter part of the
century was concerned with the elaboration
of highly diverse maternal and embryonic
structural adaptations for viviparity and attempts to correlate these structures with
physiological function, e.g., nutrition, gas
exchange. (For details on viviparity, cf.
Wourms, 1981, 1994, and Wourms et al,
1988.)
Advances in technology had profound effects on embryological research during the
latter half of the century. Because of the
transparency of most teleostean eggs (Kimmel et al., 1995) observations had usually
been made on intact eggs or microdissections of embryos. Beginning in 1860, a series of discoveries and innovations in histological microtechnique made it possible
to fix, embed, section, stain, and subsequently examine serial sections of embryonic material with microscopes equipped
with high resolution, apochromatic, oil-immersion objectives (cf. Ryder, 1884b and
Whitman, 1885 for the availability of techniques in embryology, and Horder et al.,
1985, for a chronology of technological and
conceptual innovations). In this respect, the
application of photography to the study of
fish development occurred surprisingly early in the history of photography. Von Kupffer is credited with the first extensive use
of photomicrographs to illustrate an embryological monograph in his 1878 study of
herring development (cited in Dean, 1900).
By 1898, His had published micrographs of
sectioned trout blastomeres that illustrated
aspects of chromosome behavior during cell
division.
The consequences of these advances in
embryological technology was to trigger a
RISE OF FISH EMBRYOLOGY
281
veritable explosion of embryological stud- most teleostean eggs and embryos was well
ies of teleosts based on the microscopic suited for the microscopic examination of
study of sectioned material, e.g., Van Bam- external and internal features of developbeke, 1876; Hoffman, 1881; Henneguy, ment. Accordingly, nineteenth century stud1888; Ryder, 1884a; Kowalewsky, 1886; ies of chondrichthyan development can be
and Wilson, 1891. By the end of the nine- divided into two phases. Phase One
teenth century, an excellent descriptive spanned the period 1800-1860. During this
knowledge of teleost development had been phase, research progressed along previously
achieved. But there was a trade-off. The established lines with an emphasis on reemphasis on the study of sectioned material productive biology, the study of viviparous
detracted from the observation of living species and adaptations for viviparity, and
embryos, especially cell behavior during the later stages of embryonic development.
early development, a field that was just be- Phase Two spanned the period 1860-1911.
ginning to be explored by His (vide infra). Research in this phase was shaped by the
This trend led to subsequent difficulties in introduction of new embryological and histhe interpretation of morphogenetic move- tological microtechniques to the study of
ments and embryo formation (Trinkaus, chondrichthyans (see previous section on
1984a; Solnica-Krezel et al, 1995). Even teleosts for detail).
as this trend was established, its advantages
Research of the eighteenth century
and limitations were becoming obvious. passed imperceptibly into the nineteenth
Harrison (1901) published his histological century with little or no change of agenda.
study of the differentiation and axonal In addition to his magnum opus, Bloch pubgrowth of spinal neurons in trout embryos lished a set of separate papers in obscure
in which he focused on the patterns of journals, some of which dealt with chongrowth of individual nerve cells and their drichthyan reproduction and development.
role in establishing neural connections. The Tilesius von Tilenau (1802) published a
constraints of the histological approach led monograph on the egg cases of sharks and
him to develop tissue culture and use it six skates. It is noteworthy because it repreyears later to investigate neuronal out- sents the first attempts to determine the
growth (Harrison, 1907).
chemical composition of egg cases, now
known to be a type of "tanned" protein. In
addition, he also reported on the urogenital
Development of chondrichthyan fishes
anatomy
and the reproduction and develAt the onset of the nineteenth century,
opment
of
sharks and skates. The major sigthe study of chondrichthyan development
was already farther advanced than that of nificance of Mitchell's (1803) report on the
teleosts. However, the very features that anatomy of early (7-8 cm) pre-implantation
had initially favored the study of chondri- shark embryos is that it is one of the earliest
chthyan fishes, e.g., large eggs and embry- embryological papers and probably the first
os, viviparity, and internal fertilization, sub- paper on fish embryology published in the
sequently worked against their further United States.
Home's (1810) paper had two major asstudy. It was difficult to obtain access to
early stages of development in viviparous pects: one embryological and the other respecies and there was no really effective productive. He provided anatomical deway to maintain egg-laying species in cap- scriptions of the male and female reproductivity so as to obtain newly laid eggs. The tive system and described the structural
rate of development was slow. The large, changes that occur as mature individuals
meroblastic eggs were opaque, so that only pass from a reproductively inactive to an
external features of development and those active state. His study of the male urogendetails revealed by microdissections of em- ital system and claspers (modified caudal
bryos could be observed. In contrast, the portion of each pelvic fin) represents the
introduction of artificial fertilization made first step toward understanding the process
the early stages of teleostean development of chondrichthyan insemination and fertilreadily available and the transparency of ization. He gives a fine illustration of the
282
JOHN P. WOURMS
claspers with their terminal processes expanded as they would be in copulation. He
also describes the urogenital papilla, the terminal end of the male reproductive duct,
but mistakenly refers to it as a "penis" and
states that it is the intromittent organ.
Claspers were first reported by Aristotle,
and their gross anatomy described by Lorenzini (1678) and Bloch (1782-1795). A
controversy developed over the centuries
concerning their function either as an intromittent organ or an organ for holding the
female fast during copulation Further contributions were made by Davy (1839). He
described the internal anatomy of the clasper that makes possible its function in sperm
transfer. On the basis of his own work and
a review of the literature, he concluded that
the clasper functions both as a holdfast and
intromittent organ. He also described the
accessory clasper gland of skates and rays,
a structure now known to produce prostaglandins (Lacy, personal communication).
Davy (1839) appears to be the first to
have observed chondrichthyan sperm. In
the electric ray (Torpedo) and a skate, he
described sperm with an elongated head
and flagellum. In vitro microscopic examination revealed that their motion was "serpentine and vibratory." Details on the process of spermatogenesis (Moore, 1895) and
the structure of the mature sperm (Retzius,
1902) came later. In 1871, Agassiz stated
that when the claspers (of a skate) are rotated forward and upwards, an opening in
them was brought opposite the spermatic
duct ( = urogenital papilla). The clasper
could then be introduced into the oviduct
and spermatic fluid could pass up the clasper and into the oviduct. Bolau (1881) appears to be the first to actually observe and
describe the mating activity of sharks. The
current view of insemination is that of Gilbert and Heath (1972, p. 97): "In mating,
one clasper is flexed medially, inserted, and
is anchored in the oviduct by a complex of
cartilages at the clasper tip. Sperm pass
from the urogenital papilla into the clasper
groove and are washed into the oviduct by
seawater and secretions expressed from a
siphon sac." The actual cytological events
of fertilization in chondrichthyans did not
become known until Riickert (1892b) demonstrated physiological polyspermy.
Embryology was the second aspect of
Home's (1810) paper. He compared stages
in development of the oviparous catshark,
Scyliorhinus with the non-placental viviparous spiny dogfish shark, Squalus. He illustrated an advanced yolk sac embryo of the
catshark in its egg case. He illustrated and
described the slits that open in the egg case
to permit the flow of seawater through the
egg case during mid-late phases of development. He gave an excellent rendering of
an oviduct of gravid Squalus with its ostium tubae, nidamental (shell) gland, and
posterior uterus lined with longitudinal vascular ridges. Within the uterus, the "candle" is illustrated for the first time. It is an
attenuated egg case that contains three developing eggs and a quantity of transparent
jelly. Neonates of both the catshark and
spiny dogfish are illustrated. To determine
the physical and chemical properties of the
oviducal jelly of the spiny dogfish, Home
(1810) enlisted the aid of the chemist W.
Brande. The jelly was extremely hydrophilic, being able to absorb many times its
volume of water. Chemically the jelly was
found to be neither gelatin nor albumen. It
is interesting to note that Home, under the
influence of Humphrey Davy, speculated on
the requirements of developing fish eggs for
aeration and how this process might be accomplished with different patterns of embryonic circulation.
Rathke was von Baer's successor at K6nigsberg. His study (Rathke, 1825) of the
"sexual apparatus" in fishes and other vertebrates was the first major step in understanding the development of the urogenital
system, the differentiation of the gonads
and accessory ducts, and the clarification of
relationships in the vertebrate urogenital
system (Churchill, 1991). His monograph
(Rathke, 1827) on the development of
sharks and rays was primarily concerned
with organogenesis and the microanatomy
of the developing embryo. His comparative
study of the development of the gill slits
and arches forms the basis for homologizing the pharyngula phase of vertebrate development and was used extensively by the
RISE OF FISH EMBRYOLOGY
proponents of the biogenetic law later in the
century.
John Davy, the brother of Humphrey
Davy, published an account (Davy, 1834)
of the embryonic development of the electric ray Torpedo and a series of experiments
on its electric organs. In his description of
the female reproductive system, he noted
that the absence of a nidamental (shell)
gland accounts for the absence of an egg
envelope in this fish. He presents an illustrated series of developmental stages which
includes information on the electric organ.
His earliest embryo was 18 mm long. It had
short, external gill filaments but lacked an
electric organ. The electric organ had begun
its development in 28 mm embryos. Pectoral fin development began in 39—35 mm
embryos, and by 62 mm the embryo looked
like a miniature adult. Living full-term embryos were surgically removed from the female and maintained in sea water for up to
six months. These neonates had functional
electric organs. Activity was measured with
a galvanometer. At six months, the young
rays still had vestiges of an internal yolk
sac and showed no interest in food. Davy
suggests that they depend on yolk reserves
during this period. Davy's (1834) contributions to the study of embryonic nutrition
will be discussed in another section. Almost
simultaneously, Leuckart (1836), a protege
of Rathke, published a comparative study
of the external gill filaments of several species of sharks and the electric ray. This paper has added interest because Leuckart's
incorporation of an extensive series of measurements of embryonic structures represents an early attempt to quantify embryological research. Based on his own work
and that of others, Leuckart concluded that
external gill filaments are transitory structures that are common to the embryos of all
chondrichthyan fishes. He suggested that
passage of the embryos through an external
gill filament phase is a type of metamorphosis. In addition, for the first time he describes the yolk stalk appendiculae of the
pre-implantation embryos of the bonnethead shark, Sphyrna tiburo. They are vascularized villiform projections that festoon
the umbilical cord of this placental species.
Johannes Miiller was one of the pivotal
283
figures in mid-nineteenth century biology
as a pioneer marine biologist, a founder of
comparative physiology, an accomplished
embryologist, and a mentor of illustrious
students such as Henle, Kolliker, Haeckel,
His, Virchow, and Du Bois-Reymond. He
helped to lay the foundations for the experimental approach to embryology. Miiller's
classical paper of 1842 is considered a
benchmark of chondrichthyan research. In
it, he employed a comparative approach to
the study of oviparous and viviparous species. He described and compared the egg
cases of sharks, skates, and chimaeras.
More importantly, he provided a detailed
description of the yolk sac placenta in the
smooth dogfish shark, Mustelus canis and
the blue shark, Prionace glauca. He gave
an in-depth account of the maternal and fetal portions of the placenta and their vascularization. Prior to Miiller's definitive account, knowledge of the shark yolk sac placenta had languished since Steno's (1673)
rediscovery of it, over 170 years previously
(Singer, 1921). Shortly thereafter, Leydig's
(1852) monograph on the histology and embryology of rays and sharks appeared. In it
he described five stages in the early development of the spiny dogfish, Squalus acanthias. A detailed description and accurate
illustration of the microanatomy of the earliest stage (equivalent to Stage 23 of Ballard et al., 1993) is given. His research on
the yolk sac placenta extended that of Miiller. The embryonic portion of the placenta
of M. laevis at its attachment site is illustrated and a schematic illustration of the
histology of the maternal embryonic placental interface is provided. In another advance, Leydig (1852) described the differentiation of six tissues, viz- neurons, notochord, connective tissue, lens fibers, heart
muscle, and striated muscle. In the latter tissue, cell fusion was depicted.
Although the eggs and embryos of viviparous species continued to be used for the
study of development (Ziegler and Ziegler,
1892; Scammon, 1911), the study of viviparity per se entered a period of stasis that
lasted well into the twentieth century
(Wourms, 1977; Wourms and Demski,
1993). However, some progress was made.
Bruch (1860) published the first accurate
284
JOHN P. WOURMS
descriptions and illustrations of the uterine
villi of several viviparous rays. The extensive vascularization and glandular organization of these structures were noted. These
observations were confirmed and extended
by Trois (1876). Wood-Mason and Alcock
(1891) discovered that the uterine villi of
the butterfly ray (Gymnura) are grossly hypertrophied in one region of the uterus and
enter the spiracles of the embryo. This juxtaposition of maternal and embryonic tissues was subsequently termed "a branchial
placenta" (Wourms et al., 1988). WoodMason and Alcock coined the term "trophonemata," literally growth threads, to describe the hypertrophied uterine villi. They
ascribe a function of nutrient production to
the trophonemata. Developing embryos
would imbibe the nutrients. Although studies of the yolk sac placenta also lagged, Ercolani's (1879) observations on shark placentae were incorporated into his general
classification of the types of vertebrate placentae. Parker (1889) described the fetal
membranes of Mustelus antarcticus and
provided an analysis of the periembryonic
fluid. Alcock (1890) described the histology
of the spatulate extensions that occur on the
umbilical cord of the yolk sac placenta in
the hammerhead shark Sphyrna blochi. He
uses, apparently for the first time, the term
"appendiculae" to categorize these processes. Their function was enigmatic. Alcock speculates on a possible role as a lymphatic gland or involvement in the "purification" of embryonic blood. (For details
on maternal and embryonic adaptations for
viviparity, see Wourms et al., 1988;
Wourms and Lombardi, 1992.)
Advances in embryological microtechnique and microscopy that so profoundly
altered the study of teleostean embryology
had an even more pronounced effect on the
study of chondrichthyans because of their
large, opaque, meroblastic eggs. Research
shifted to the histological and cellular levels. The consequences are especially apparent in research on early development, i.e.,
fertilization through early development, as
well as organogenesis and cell differentiation.
Although the blastoderm and neurula
stages had been recognized by Lorenzini
(1678), little progress had been made in the
intervening years in defining the blastula
and gastrula stages. Coste (1850) appears to
be the first to describe but not illustrate
cleavage. His report was extended by Gerbe
(1872) who illustrated surface views of
stages from the first cleavage through a late
blastula and compared them to the cleavage
stages of birds and reptiles. These observations were confirmed by Balfour (1878)
who also included illustrations of sectioned
blastulae. The definitive study of early development from fertilization through the
early gastrula was conducted by Riickert
(1899). Surface views of the blastoderm
were accompanied by sections through it.
In 1878, Balfour published his monograph, On the Development of Elasmobranch Fishes. This work raised the study
of chondrichthyan development to heretofore unattained heights by establishing new
standards of excellence for accuracy and
comprehensive description. It has been categorized as "a famous landmark in the history of vertebrate development" (Ballard et
al., 1993). Balfour explored all aspects of
elasmobranch embryology from early
cleavage through mid-late development and
established a staged series of embryos,
mostly sharks, that was employed in research for many years. Illustrations of sectioned material were used extensively. Balfour described the organization of the mature egg and cleavage of the zygote, commenting on yolk platelets, the germinal
vesicle and its fate, the process of cleavage,
nuclear division, the origin of yolk nuclei,
and asymmetry of the blastoderm. An in-depth account of gastrulation, germ layer
formation, and the general details of embryonic development is presented. Detailed
descriptions of the development of the major organ systems included: (1) brain, spinal
and sympathetic nerves; (2) head and associated structures; (3) digestive tube and
associated organs; (4) heart and vascular
system; and (5) gonads, kidney, Wolffian
and Miillerian ducts and their derivatives.
In passing, it should be noted that Balfour
conducted much of his research at the Naples Zoological Station. His research was
facilitated by the director, Anton Dohrn,
who was pursuing a major research pro-
RISE OF FISH EMBRYOLOGY
gram in evolutionary physiological anatomy (Ghiselin, 1996). This program included studies of shark embryology by Dohrn
and his associates in order to resolve
Dohrn's ideas about vertebrate origins.
Observations by Ziegler and Ziegler
(1892) on the development of the electric
ray Torpedo continued Balfour's pioneering
approach. Their work is characterized by
the use of serial sections and photographs
of surface views of model reconstructions
of selected embryonic stages. They investigated the equivalent of Balfour stages B
to K, and considerably advanced descriptive knowledge of gastrulation, formation
of the medullary plate, closure of the medullary plate and early embryo formation,
and the organization of gill slit-tail bud
stage embryos. Balfour had made major advances in the study of chondrichthyan development and the introduction of a series
of normal stages was useful. However, there
were defects in his normal series. The series
was intended to illustrate development in
Scyliorhinus, but it was necessary to use
embryos of the shark Galeus and the ray
Torpedo to fill in gaps. Moreover, there was
no time base to the series (Ballard et al.,
1993). As part of Keibel's series of monographs on the normal stages of development
of vertebrates, Scammon (1911) published
his monograph on the spiny dogfish shark,
Squalus acanthias. It is a reasonably complete survey of the development of a single
elasmobranch species. Ballard et al. (1993,
p. 334) refer to Scammon's monographs as
"hitherto unrivaled in its completeness,
[and it] records in handsome drawings, wax
reconstructions and serial sections the anatomy of dozens of randomly collected but
closely spaced specimens." Scammon
(1911) also included a bibliography arranged by subject of the important literature
on chondrichthyan development. The appearance of Scammon's monograph represents the apex of nineteenth century studies
of chondrichthyans.
A major conceptual difficulty encountered in the study of chondrichthyan development is the lack of correspondence of the
processes of gastrulation and embryo formation with those of other vertebrates with
meroblastic eggs, in particular, teleosts and
285
reptiles and birds. (For details, cf. Balfour,
1878; Emmert, 1900; His, 1877; Hoffman,
1896; Kastschenko, 1888; Kopsch, 1898;
Ruckert, 1885, 1887, 1889, 1899; Samassa,
1895; and Ziegler and Ziegler, 1892). Suffice to say that the posterior rim of the
chondrichthyan blastoderm thickens to
form a medial embryonic shield with two
lateral crescent-like arms. The arms eventually close to form the embryonic axis.
The embryo-forming region overhangs the
yolk mass. Initially, the anterior end of the
embryonic axis is a fixed point and lengthening of the axis is accomplished by posterior growth brought about by the joining
together of the right and left arms of the
shield (Ballard et al., 1993) Although considerable effort has been invested in the descriptive and experimental study of the
problem, according to Ballard et al. (1993,
p. 328), "No consensus has been reached
as to how the morphogenetic cell movements are taking place in this or any other
elasmobranch fish."
Another area of inquiry in which the advances in embryological microtechnique
provided spectacular insights was the study
of oogenesis. Although lampbrush chromosomes were first seen by Flemming in
1878, it was Ruckert (1892a) who first gave
evidence that these structures are chromosomes (Callan, 1986). He investigated the
fate of the chromosomal material during the
growth of the oocyte nucleus in the ovaries
of three elasmobranchs, Scyllium ^
iorhinus), Torpedo, and Pristiurus ^
eus). Ruckert made three major contributions. He was the first to devise a method
of isolating intact germinal vesicles and
staining lampbrush chromosomes in situ.
Secondly, he described the characteristic
structure of the lampbrush chromosomes,
especially the lateral loops, and was the first
to use the term "lampbrush." Finally, by
examining stages in oogenesis, he described
the genesis of lampbrush chromosomes during early stages of oocyte growth (18 u,m
to 2 mm), their unique structural configuration at full development (2-3 mm), and
the retraction of the lateral loops and contraction of the chromosome axis as these
chromosomes give rise to normal condensed meiotic bivalents during later stages
286
JOHN P. WOURMS
TABLE 2. Major contributions, both historical and modern, to the study of the comparative embryology of
fishes, arranged according to taxonomic group.
1. Hagfish
Dean (1898, 1899); Doflein (1898); Gorbman (1983); Gudger and Smith (1931); Northcutt and Bemis (1993)
2. Lampreys
Balfour (1880); Goette (1890); Hatta (1907); Piavis (1971); Schultze (1856); Selys-Longchamps (1910)
3. Chimaeras
Dean (1906); Schauinsland (1903)
4. Sharks, Skates, and Rays
Balfour (1878); Ballard et al. (1993); His (1877); Hoffman (1896); Kopsch (1898, 1950); Leuckart (1836);
Leydig (1852); Miiller (1842); Ranzi (1932); Ruckert (1899); Scammon (1911); Ziegler (1902); Ziegler and
Ziegler (1892)
5. Lungfish
Budgett (1901); Kemp (1982); Kerr (1900, 1901); Semon (1893, 1901a, b); Wourms and Kemp (1986)
6. Coelacanth
Fricke and Frahm (1992); Northcutt and Bemis (1993); Smith et al. (1975); Wourms et al. (1991)
7. Bichir (Polypterus)
Arnoult (1964); Bartsch and Britz (1996); Budgett (1901); Kerr (1907)
8. Sturgeons and Paddlefish
Ballard and Ginsburg (1980); Ballard and Needham (1964); Bemis and Grande (1992); Bolker (1993a, b)\
Dean (1895); Detlaff et al. (1993); Ginsburg and Detlaff (1991); Kowalewsky et al. (1870); Ryder (1890);
Salensky (1878, 1881)
9. Garpike (Lepisosleus)
A. Agassiz (1878); Balfour and Parker (1882); Dean (1895); Eycleshymer (1899); Long and Wourms (1991)
10. Bowfin (Amia)
Ballard (1986a, b); Dean (1896a, b); Eycleshymer and Wilson (1906); Lanzi (1909); Whitman and Eycleshymer (1897)
11. Teleosts
(A) Larvae—Marine Fishes
A. Agassiz (1882); A. Agassiz and Whitman (1884); Ehrenbaum (1909); Mclntosh and Prince (1890);
Moser (1984); Ryder (1884a, 1887)
(B) Organ Systems
Eigenmann (1903); Ryder (1886c)
(C) Viviparity
Duvernoy (1844); Dybowski (1873); Eigenmann (1894); Phillipi (1908); Ryder (1886a); Stuhlmann
(1887); Wourms et al. (1988); Wyman (1856)
(3—13 mm) of oogenesis. Riickert's findings
were fully confirmed by Mare'chal (1907)
in an elegantly illustrated study of two
sharks and two teleosts.
Within the context of the reproductive
system, some other notable contributions
should be mentioned. Woods (1902) carried
out a quantitative study in which he discovered that the germ cells of the spiny dogfish
shark Squalus acanthias originate in endoderm associated with the yolk sac and migrate apparently by amoeboid movement to
the germinal ridge of the developing gonad.
Borcea's (1905) classical study of the urogenital system contains the definitive statement on the structure of the nidamental
(=shell) gland of the oviduct and its role in
the secretion and morphogenesis of the egg
case.
By the end of the nineteenth century, the
descriptive aspects of chondrichthyan development were well known (Ziegler, 1902;
Kerr, 1919). The state of knowledge cornpared favorably with what was available for
the more intensively studied domestic fowl
and selected amphibians. Building on this
foundation, future investigators would be
able to formulate a series of questions based
upon a functional and analytical approach
to the study of development,
COMPARATIVE EMBRYOLOGY
Comparative studies of fish development
constitute one of the glories of nineteenth
century embryology (Table 2). The comparative method as a formal concept in
morphology has its roots in German Naturphilosophic and was extended to embryology by Dollinger and von Baer (Oppenheimer, 1936). Subsequently, Johannes Miiller
RISE OF FISH EMBRYOLOGY
and others incorporated it into physiology.
Already well established, comparative embryology was thrust into a position of
prominence with the publication of Darwin's On the Origin of Species. Darwin
stated that much could be learned about
evolution through embryological studies.
Fritz Miiller (1864) and Haeckel (1866)
considered that embryology, especially
comparative embryology, was paramount in
understanding evolution, i.e., "ontogeny recapitulates phylogeny" (Gould, 1977). The
new evolutionary impetus combined with
the pre-existing research agenda drove
comparative studies of fish development
ahead at a rapid clip. Fishes provided a intellectual feast for evolutionary comparative embryologists. The four major groups
of fishes occupy key positions in vertebrate
evolution (Fig. 1). Agnaths (hagfish, lampreys) are the most primitive representatives of the craniate-vertebrate lineage.
Chondrichthyans (sharks, rays, chimaeras)
are among the oldest extant jawed vertebrates and incorporate the definitive vertebrate Bauplan and developmental program.
Sarcopterygians comprise lungfishes, coelacanths, and tetrapods. The Actinopterygian or ray-finned fishes (Polypterus, sturgeons, gars, Amia, and teleosts) represent a
group of bony fishes that evolved in a lineage that culminated in the teleosts, one of
the most successful groups of vertebrates.
Not only was there evolution at the phyletic
level, but the process of development itself
underwent evolutionary change (c/. Introduction and Collazo et ah, 1994).
Once embarked upon the comparative
venture, the availability of study material
became a problem. Many fishes of interest
were found only in the fresh waters of the
tropics, and some, such as the Australian
lungfish Neoceratodus, were only discovered in the last quarter of the nineteenth
century. Coincident with the rise of interest
in comparative embryology, geographical
regions that harbored fish species of interest
became more accessible, the fishes themselves became better known, and the technology of travel improved. Even so, studying the embryology of fishes under primitive field conditions was a major task. In-
287
dividuals such as Budgett, Kerr, and Semon
are part of the golden age of exploration.
The hagfishes and lampreys are the most
primitive representatives of the craniatevertebrate lineage. Both are jawless. Hagfishes are common, marine, bottom-dwelling fish in cold, inshore waters and in deeper waters off continental shelves. Recently,
hagfishes have been considered to be the
sister group to all remaining vertebrates
(Nelson, 1994). Virtually everything that is
known about their embryonic development
is derived from collections of Eptatretus
(=Bdellostomd) stoutii eggs obtained by
Bashford Dean and Franz Doflein in Monterey Bay, California, on the Pacific coast
of North America in the 1890's. For all intents and purposes, there have been no
modern embryological studies of hagfish
except for Gorbman (1983), Gorbman and
Tamarin (1986), and Wicht and Northcutt
(1992), who used Dean's material. For this
discussion, research on the Atlantic hagfish
Myxine will not be considered because
there is so little of it. Doflein (1898) and
Dean (1898, 1899) described embryonic
development. Dean's 1899 extraordinary
monograph gives a definitive account,
sumptuously illustrated with color lithographs, of hagfish development from early
cleavage through hatching. Unfortunately,
because the monograph is not readily accessible, this research has been overlooked.
Most of Dean's account is based on the observation of whole mounts. Sections of
blastula and gastrula stages are illustrated.
The eggs are large ellipsoids about 22 mm
in length by 8 mm in width. They are heavily yolked, and there is a germinal disc at
the animal pole. Cleavage is meroblastic.
The process of embryo formation coincides
with the epibolic movement of the blastoderm over the yolk mass. The equivalent of
a germ ring is present and the embryo is
formed along an anterior-posterior axis as
the blastoderm moves over the yolk mass.
The only section of a gastrula (Dean, 1899,
fig. 24) reveals the absence of bottle cells,
although this could be a function of the
plane of section. Both a cellular "periblast"
and an underlying yolk syncytium are depicted. The neural tube is formed by the
closure of the medullary folds. Much infor-
288
JOHN P. WOURMS
mation is given on organogenesis and the
later phases of embryonic development.
Additional descriptions of early cleavage
stages are given by Gudger and Smith
(1931). A description of development of the
head, brain and associated organs based on
the analysis of sectioned material was published by von Kupffer (1899).
Lampreys are widely distributed throughout the waters of the northern and southern
temperate zones. They were known to zoologists such as Home, Rathke, and Miiller
who worked on aspects of their reproductive anatomy. It is surprising, then, that embryological research was only undertaken
in the mid-nineteenth century and that comprehensive studies only appeared in the latter part of the nineteenth and in the early
twentieth centuries. The first substantive
description of lamprey development was
that of Schultze (1856), who amongst other
observations demonstrated the somewhat
unequal holoblastic cleavage of the egg.
Aspects of gastrulation were described by
Balfour (1880), Goette (1890), and Hatta
(1907). Both Goette and Hatta demonstrated the presence of bottle cells and involution of surface cells through the blastopore.
Other contributions of note are those of
Scott (1887), von Kupffer (1890), and Selys-Longchamps (1910). Older literature on
lamprey development is listed in Ziegler
(1902). Piavis (1971) provides a modern
account of development and a list of the
more recent literature.
Two main lines of evolution are recognized within the class Chondrichthyes: the
holocephalans and elasmobranchs (Nelson,
1994; Didier, 1995). When chondrichthyan
development was discussed previously,
only the elasmobranchs (sharks, rays, and
skates) were considered. Now the holocephalans (chimaeras or ratfishes) will be
considered. Chimaeras are oviparous, bottom-dwelling marine fishes usually found in
the deep waters off the continental shelf,
except in those regions where the inshore
waters are cold, e.g., the northwest coast of
North America and New Zealand. Because
of their deep water habitat, it has been difficult to procure their eggs for study. The
major sources of information on chimaeroid
development are Schauinsland's (1903) and
Dean's (1906) monographs. Schauinsland
studied Callorhinchus ntilii, whereas
Dean's study was mostly based on the eggs
and embryos of Hydrolagus (=Chimaera)
colliei collected in Monterey Bay, California. Dean's monograph is remarkably complete. Dean begins with an account of the
breeding habitats of Hydrolagus and egg
case formation and then describes in detail
development from fertilization through
hatching. Chimaeras are oviparous. Their
eggs, moderately large, oblate spheroids (25
X 20 mm), are enclosed in an elaborate egg
case composed of structural proteins that
differ considerably from those of elasmobranchs (Wourms, 1977). Cleavage and
subsequent development is meroblastic and
closely resembles that of sharks. A closely
spaced series of stages from early cleavage
through the mid-phases of development is
illustrated with surface views of the blastoderm and drawings of the embryos. This
series is supplemented with many illustrations of sectioned material, especially from
the blastula and gastrula stages. One developmental characteristic that sets chimaeras
apart from elasmobranchs is the pronounced elongation of the post-anal region
of the body, which begins at the tailbud
stage and soon gives the young embryo an
almost eel-like appearance. The studies of
Schauinsland and Dean remained unchallenged for many years. Only in the late
twentieth century has interest in chimaeroid
development revived (Didier, 1995).
The osteichthyans or bony fishes include
the sarcopterygians and actinopterygians.
The sarcopterygians (fleshy-finned fishes)
are now considered to include the tetrapods,
two extinct groups of fishes, and two extant
groups of fishes: coelacanths and dipnoans
(lungfishes) (Nelson, 1994). These fishes
are of considerable interest because the
"lungfishes, coelacanths, or both together
are the closest living relatives of tetrapods"
(Nelson, 1994, p. 69).
The living coelacanth Latimeria chalumnae is the sole surviving representative
of a once common group. The only known
population occurs in the Comoros Islands
off the east coast of Africa. No embryological research was carried out on Latimeria
during the nineteenth century because it
RISE OF FISH EMBRYOLOGY
was not discovered until 1938. However, in
1975 Smith et al. discovered that Latimeria
is viviparous when they found five nearterm pups in the oviduct. Up to this time
only two gravid females have been collected. Wourms et al. (1991) described aspects
of viviparity and the maternal-embryonic
relationship using the embryos and maternal tissues from the Smith et al. (1975)
specimen. This material was also used by
Bemis and Northcutt (1991) in their study
of the innervation of cranial muscles and
Northcutt and Bemis (1993) in their study
of cranial nerves. Fricke and Frahm (1992)
described full-term pups obtained from the
second gravid female. Coelacanth eggs are
enormous, up to 9 cm in diameter and with
a dry weight of 184 g. They are heavily
yolked and contain yolk platelets (reviewed
in Wourms et al., 1991). Although no information is available on early embryonic
development, it is reasonable to assume that
development is meroblastic. Because the
coelacanth is on the verge of extinction, it
is highly unlikely that any substantial body
of embryological knowledge will be forthcoming.
The dipnoans or lungfishes comprise the
other extant group of sarcopterygian fishes.
There are three genera of living lungfishes:
Protopterus in Africa, Lepidosiren in South
America, and Neoceratodus in Australia.
Lepidosiren and Protopterus were discovered nearly simultaneously in the late
1830's and Neoceratodus in 1870. Lungfishes as "intermediate forms" readily fitted into the emerging evolutionary paradigm of the nineteenth century (Bemis et
al., 1987). Thus, the study of lungfish development became one of the most sought
after goals of comparative embryology. It
entailed expeditions to three continents that
involved considerable risk and in some instances loss of life, viz. Budgett (Semon,
1899; Shipley, 1901, Kerr, 1950).
The major accounts of lungfish development appeared during a brief sixteen-year
span at the end of the nineteenth and in the
early twentieth centuries. The embryology
of the lungfish Neoceratodus was first described by Semon (1893) in a series of 47
stages that extend from fertilization through
post-hatching juveniles. This report, which
289
was based on the description of external
features, was complemented by a study of
gastrulation based on the analysis of sectioned material (Semon, 1901a). Much of
the previous research was summarized and
new material was incorporated into a publication that was part of Keibel's series of
normal tables of development (Semon,
\90\b). Recently, Kemp (1982) and others
have re-investigated the development of
Neoceratodus. In much the same fashion,
Kerr (1900) published the first account of
the development of the South American
lungfish, Lepidosiren. An account of external development of closely spaced states
from fertilization through post-hatching juveniles was given. Unlike the Australian
lungfish, embryos of Lepidosiren develop
external gill filaments before hatching and
retain them for at least 40 days. A second
part of Kerr's account (Kerr, 1901) included
a comparison with the development of the
African lungfish Protopterus. The first account of its development was given by
Budgett (1901). This preliminary report
contained information on the reproduction
and development of other African fishes,
most notably Polypterus. Budgett died in
1904 from malaria contracted in Africa.
Kerr, Assheton, and others acted as Budgett's scientific executors, ensuring the publication of his research. The definitive report on the development of Protopterus was
made by Kerr in the same volume of the
Keibel series that contained the normal table of Lepidosiren development (Kerr,
1909).
Thus far, little has been said about lungfish development. Eggs are approximately
3 mm in diameter with a moderate amount
of yolk. Cleavage is holoblastic and unequal. A blastopore develops in a subequatorial position. Bottle cells are present. Surface cells involute through the blastopore.
The neurula is characterized by a flat neural
plate that folds to form the neural tube. In
general, lungfish development, especially
that of the Australian lungfish, bears an uncanny resemblance to that of urodele amphibians (Wourms and Kemp, 1986). Caution, however, is advised because of the
need for a fine-grained analysis of the early
(blastula-neurula) phases of development
290
JOHN P. WOURMS
using contemporary methods (Wourms and
Kemp, unpublished).
The actinopterygian or ray-finned fishes
are of particular interest to comparative embryologists. They constitute a well-defined
evolutionary lineage that culminated in the
teleosts, one of the most successful, diverse,
and species-rich groups of vertebrates (Nelson, 1994). Based on their evolutionary relationships, they provide a unique opportunity for investigating the transition from
the primitive, holoblastic pattern of fish development to the specialized meroblastic
pattern characteristic of teleosts (Collazo et
al., 1994). The actinopterygian fishes comprise three subclasses: Cladistia, Chondrostei, and Neopterygii (Atz, 1985; Nelson,
1994).
The Cladistia are the most basal group of
the (Lauder and Liem, 1983; Atz, 1985).
The surviving cladistians are represented by
two genera, Polypterus and Calamoichthys,
which are in the order Polypteriformes. In
some classifications, e.g., Nelson (1994),
the Polypteriformes are placed in the subclass Chondrostei together with the sturgeons. Both Polypterus, the sturgeons and
paddlefish are considered to be part of the
lineage that led to the teleosts (Nelson,
1994). Polypterus, the bichir, and Calamoichthys are from Africa. Little or nothing is
known about the development of Calamoichthys. The situation is somewhat better
with respect to Polypterus. The first, very
fragmentary report of its development was
made by Budgett (1901). Following Budgett's death in 1904, Kerr worked up Budgett's collections and notes and published the
only extensive account of the development
of Polypterus (Kerr, 1907). Almost all aspects of development from fertilization
through hatching and also post-hatching
larvae are described in 36 stages. Eggs are
small, slightly flattened spheres (0.9 X 1.3
mm along the major and minor axes), moderately yolked, and contain yolk platelets.
Cleavage is holoblastic and unequal. Unfortunately, information on the critical
events of gastrulation is scanty. At the onset
of gastrulation, what appears to be a very
wide blastopore occupies an equatorial position. Subsequently, the lateral margins of
the blastopore extend and meet to form a
circle nearly as large as the circumference
of the egg. This marks the advancing
boundary of the surface cells of the animal
pole during epiboly. Epiboly is pronounced
and initially a very large yolk plug is
formed. Subsequently, it is reduced in size.
The marginal quality of the single section
of a gastrula stage illustrated by Kerr
(1907) does not allow comment on the
presence or absence of bottle cells. Better
sections through early neural plate stages
confirm the presence of a blastopore that is
continuous with a large archenteron. During
neurulation, a flat neural plate folds to form
a neural tube. The embryo passes through
typical tail bud stages and eventually hatches as a larva with external gill filaments.
According to Arnoult (1964), the rate of development is quite rapid, i.e., 48 hours from
fertilization to hatching. Although there are
differences, the development of Polypterus
resembles that of the sturgeon. Obviously,
Polypterus is a prime candidate for further
study. The prospects are excellent for it is
in the aquarium trade and can be spawned
in captivity (Arnoult, 1964). In point of
fact, Bartsch and Britz (1996) have just
published a popular account of spawning
and development that includes scanning
electron micrographs of the neural plate
stages. Some of their material was used in
Piotrowski and Northcutt's (1996) study of
cranial nerves.
The subclass Chondrostei (Atz, 1985;
Grande and Bemis, 1996) has one extant
order, the Acipenseriformes, that contains
the sturgeons and paddlefishes. They are
considered an offshoot of the lineage that
led to the teleosts. Sturgeons occur in North
America and Eruasia. Paddlefish are restricted to the United States and China. The
development of sturgeons was first outlined
by Kowalewsky et al. (1870) for the European sterlet (Acipenser ruthenus). A more
detailed account of early development and
embryogenesis was given by Salensky
(1878, 1881). Somewhat later, similar studies were carried out on the eggs of the
North American Atlantic sturgeon A. oxyrhinchus (Ryder, 1890; Dean, 1895). Recently, Ginsburg and Dettlaff (1991) and
Dettlaff et al. (1993) have presented a detailed contemporary account of the devel-
RISE OF FISH EMBRYOLOGY
opment of the Russian sturgeon A. gueldenstaedtii from fertilization through hatching.
Eggs are 3.0—3.5 mm in diameter. Cleavage
is holoblastic. A dorsal lip develops in an
equatorial position as the apices of bottle
cells contract. Involution of surface cells
takes place through the blastopore. The
neurula stage is characterized by a flat neural plate that subsequently folds to form the
neural tube. According to Bolker (1993a),
gastrulation is similar to that of the amphibian Xenopus. Morphogenetic movements have been analyzed by Ballard and
Ginsburg (1980). The mechanisms of gastrulation and mesoderm morphogenesis
have been investigated by Bolker (1993a,
1993fc). The embryology of the paddlefish
(Polydon) did not become known until the
1960s (Ballard and Needham, 1964). The
early- and mid-phases of paddlefish development closely resemble that of sturgeons.
During the later phases of development,
embryos develop their characteristic elongated rostrum. A scanning electron microscopy study by Bemis and Grande (1992)
has substantially extended the description
of Polyodon development.
The subclass Neopterygii contains three
divisions, viz- Ginglymodi (gars), Halecomorphi {Amid), and Teleostei (Nelson,
1994). Gars {Lepisosteus) occur in eastern
North America and Central America. The
first account of the development of gars was
by Alexander Agassiz (1878), a report that
dealt exclusively with late embryonic and
post-hatching stages. Using material provided by Agassiz, Balfour and Parker
(1882) gave an account, albeit fragmentary,
of some of the earlier phases of development. Most of their report, however, contains the first detailed account of embryogenesis. The egg is about 3 mm in diameter
and heavily yolked. Cleavage is meroblastic. Sections of embryos reveal that the spinal cord originally is a solid rod that secondarily becomes hollow. Dean (1895) provided critical information on the early development of Lepisosteus from fertilization
through gastrulation and compared its development with that of the sturgeon. Dean
was the first to provide a timed series of
developmental stages for both fishes. In addition to providing surface views of the ex-
291
ternal features of early development, Dean
also based his descriptions on sections of a
developmental series. Eycleshymer (1899)
provided additional information on the
cleavage process. Recently, Long and
Wourms (1991) examined early development with scanning electron microscopy.
From these studies, the following view of
early development of Lepisosteus emerges.
Meroblastic cleavage produces a blastoderm atop a large, yolk sphere in which
abortive cleavage furrows are present. Further studies are needed to ascertain whether
the yolk mass undergoes any division.
Epiboly carries the blastoderm margin toward the vegetal pole preceded by microvilli-bearing zone of the yolk surface similar to the yolk syncytial layer of teleosts.
As epiboly of the blastoderm reaches the
equator, a blastopore begins to form at one
point along the rim of the blastoderm. Bottle cells are associated with the dorsal lip
of the blastopore. By this time, cells of
blastoderm are organized into an embryonic
shield and a wide germ ring. As epiboly
proceeds to completion, the germ ring narrows, the embryonic shield elongates, and
a shallow, neural groove forms similar to
that of teleosts.
Amia calva, the bowfin, is the sole extant
representative of the order Amiiformes
(Nelson, 1994). It is confined to the freshwaters of eastern North America. Its development was made known in a flurry of papers that appeared during 1896-1906 {cf.,
Ballard, l9S6a,b, for references). Dean
(1896a) reported on early development, i.e.,
fertilization through gastrulation. External
views of successive stages were supplemented with sections of the corresponding
stages. The later stages of embryonic development as well as the development of
the post-hatching larvae were reported on
separately (Dean, 18966). Almost simultaneously, Whitman and Eycleshymer (1897)
reported on reproduction and the cleavage
stages of development. Again, external
views of the whole egg were supplemented
with sections of the corresponding stages.
It is interesting to note that Whitman raised
the issues of both priority and unfair rivalry
with Dean because Dean obtained eggs of
Amia from the same small lake in Wiscon-
292
JOHN P. WOURMS
sin in which Whitman had previously discovered spawning colonies (Whitman and
Eycleshymer, 1897). Ballard (l9S6a,b) has
published a series of stages and rates of
normal development and has investigated
morphogenetic movements. He has also
produced a provisional fate of development
in Amia. Superficially, the development of
Amia resembles that of Lepisosteus but with
several important differences. The egg is an
elongated sphere (2.2 X 2.8 mm along the
major and minor axes). A germinal disc occupies about one-third of the egg volume.
Cleavage is holoblastic but quite unusual.
The yolk mass divides slowly and with apparent difficulty whereas the germinal disc
is rapidly divided into many small cells. In
late cleavage, a blastoderm surrounds a
yolk mass divided into giant blastomeres.
Cells of the blastoderm can be distinguished as enveloping layer cells, epiblast,
or hypoblast. As epiboly commences, morphogenetic movements within the blastoderm lead to the formation of the germ ring
and embryonic shield. The early phases of
embryo formation take place during epiboly
of the blastoderm. One of the most significant differences between the development
of Amia and Lepisosteus is that Amia lacks
both a blastopore and bottle cells (Ballard,
1986). It is disconcerting that Lanzi (1909)
illustrated sections of a groove at the leading edge of the blastoderm that contains
cells that appear to be bottle cells. For obvious reasons, ultrastructural studies are in
order. Collazo et al. (1994) discuss in detail
the phylogenetic implications of these comparative embryological studies of primitive
actinopterygian fishes with respect to teleost gastrulation.
Teleosts constitute the remaining major
group of actinopterygian fishes. The history
of teleostean embryology has been discussed. The main emphasis of early studies
was placed on defining the meroblastic pattern of teleostean development and piscine
features of embryogenesis and organ and
tissue formation. Some attention was given
to the diverse embryonic adaptations for viviparity in different taxa. By the end of the
nineteenth century, it was apparent that the
early development of most teleosts was
very similar but that mid- and late-phases
diverged, yielding diverse embryonic morphology and larval forms adapted to specific life styles and habitats. Thus, the later
phases of embryonic development, larval
development, and life histories became the
subjects of comparative studies of teleostean development during the late nineteenth
century and the twentieth century.
Several factors favored the growth of a
comparative approach, especially the advent and rise of marine biology, biological
oceanography, and fisheries biology. Fishes
are the dominant group of marine vertebrates and are commercially important.
Thus, the study of life histories and the embryonic and larval stages of inshore, pelagic, and deep-sea fishes became a research
agenda for the marine sciences. The results
were often quite spectacular, e.g., the leptocephalus larvae of eels and the bizarre
larvae of the stomiatoid fish Idiacanthus
with its stalked eyes and trailing gut (see
papers in Moser, 1984). Studies of this type
were pioneered by Alexander Agassiz
(1882) and Ryder (1884a) in the United
States, Mclntosh and Prince (1890) in Great
Britain, and Ehrenbaum (1909) in Germany, amongst others. Similar activities were
undertaken at limnological stations and in
freshwater fisheries biology. Popularization
of the aquarium hobby and the successful
reproduction in captivity of freshwater fishes imported from Africa, South America,
and Asia such as the zebrafish, Danio rerio,
established another source of material for
comparative studies. Given their numbers
and diversity, a wealth of information,
mostly of a descriptive nature, was produced on the development of a great many
species of teleosts (reviewed in Breder and
Rosen, 1966).
Increased activity in the study of teleostean development was accompanied by the
elaboration of several concepts and theoretical approaches which both aided and impeded further progress. In 1877, Haeckel
set forth his Gastrea theory. Based on his
observations of teleostean development, he
applied the concept to both teleosts and
chondrichthyans. Much of the literature on
fish development from his time to the present has been concerned with the morphogenetic movements and embryo-forming
RISE OF FISH EMBRYOLOGY
mechanisms that are associated with gastrulation, a literature too voluminous to review. One major problem that arose from
overzealous and inept efforts to use embryological studies to support evolutionary theory was the attempt to homologize the
events and processes of gastrulation in teleosts and chondrichthyans with amphibians
and amniotes. In this respect, it was Goette
(1873) who first proposed that involution,
i.e., flowing of a sheet of cells over an inpocketing such as a blastopore, has a prominent role in teleost gastrulation. (For discussion and examples cf. von Kupffer,
1884; Wilson, 1891; Sumner, 1900; Pasteels, 1936; Ballard, 1981; Shardo, 1995;
and Collazo et al, 1994.)
At the risk of oversimplification, many of
these studies were comparative in name
only. There was little or no conceptual basis
for interpreting the mass of information. Attempts, such as that of Schultz (1910), to
establish a well-defined conceptual basis for
comparative embryological research were
overlooked. (In passing, Schultz's 1910
monograph appears to be a direct precursor
of de Beer's 1930 study of embryology and
evolution.) Although the body of evolutionary theory was available, the influence of
Haeckel's biogenetic law interfered with the
productive application of it to many developmental issues. A growing anti-evolutionary bias tended to discourage this type of
inquiry. Studies of individual species were
often exceedingly well done, and the development of a single organ system was often compared in several different taxa and
interpreted in the context of the evolutionary paradigm. Examples of the latter include Ryder's (1886c) study of heterocercy
and the evolution of the fins and fin rays of
fishes and Eigenmann's (1903) study of the
abortive development of the eye in blind
cave fishes. Eigenmann compared the development and degeneration of the eye in
the blind cave fish Amblyopsis with that of
the surfperch Cymatograster, a fish with a
well-developed eye. In Amblyopsis, development of the eye begins normally, but then
the lens degenerates and the entire eye regresses. Recently, Jeffery (1996), using the
embryos of another blind cave fish Astyanax, has shown that programmed cell death
293
of the lens eliminates lens induction from
the cascade of inductive interactions required for normal eye development.
In general, however, the comparative
method was not used to explore the evolution of development and the factors that
brought about evolutionary change. It was
not until the maturation of evolutionary
thought in the twentieth century that a new
set of concepts and approaches were produced and used to exploit the potential of
comparative studies. Paramount among
these are: (1) revived interest in the evolution of development and the role of development in evolution (de Beer, 1958; Gould,
1977; Moser, 1984; Hall, 1992; Raff and
Raff, 1987; Raff, 1996); (2) application of
evolutionary theory to life histories (Clutton-Brock, 1991; Stearns, 1992); and (3) attempts to order the bewildering diversity of
fish reproduction in terms of systematics or
reproductive guilds (Breder and Rosen,
1966; Balon, 1975).
ANALYSIS OF DEVELOPMENT
The term "analysis of development"
comprises a set of conceptual and methodological approaches that are used to study
development, for example, functional morphology, biomechanics, physiology, biochemistry, and cellular biology. These approaches possess a strong experimental and
causal bias. It is difficult to say when such
analytical approaches to the study of development were introduced. Although its
origins are obscure, the experimental-analytical approach certainly was recognizable
in the eighteenth century studies of Spallanzani (1729-1799) on insemination,
Trembley (1710-1784) on hydra regeneration, and Broussonet (1789) on regeneration
in fishes (cf. Gasking, 1970). By the nineteenth century, experimental physiology
under the aegis of Claude Bernard and Johannes Miiller had become a formalized
science. Miiller was a pivotal figure for he
was both a physiologist and an embryologist. It is probably through him and his
many students that an analytical-experimental approach was transferred from physiology to development and became established in the mid-nineteenth century. For
example, Haeckel (1869), a student of
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JOHN P. WOURMS
TABLE 3. A chronological outline of some pioneer
nineteenth century studies of the analysis of fish development, arranged according to subject.
1. Fertilization—Micropyle—Doyere, 1849; Ransom, 1854, 1867
2. Egg Contractility—Myosin—Ransom, 1866; 1867
3. Viviparity—Embryonic Nutrition—Davy, 1834;
Stuhlmann, 1887; Kolster, 1905; Ranzi, 1932
4. Experimental Teratology—Lereboullet, 1864
5. Concrescence Theory—His, 1874; Kastchenko,
1888; Morgan, 1895; Kopsch, 1896
6. Precocious Segregation of Germ Cells—Eigenmann, 1891
7. Lampbrush Chromosomes—Riickert, 1892
8. Nuclear—Ooplasmic Transfer—Van Bambeke,
1893
9. Effects of Ions—Loeb, 1893
10. Movements of Blastomeres in Vitro—His, 1899
11. Neuronal Growth—Harrison, 1901
12. Experimental Cyclopia—Stockard, 1907
13. Radiation Effects (Radium)—Tur, 1896; Oppermann, 1913
Miiller, divided the larvae of the siphonophore Crystallodes in two and obtained
smaller, but perfect larvae. It was only later
in the nineteenth century that a more narrowly defined, conceptual approach
emerged in the Entwicklungsmechanik of
Roux and the mechanistic views of Loeb.
Here I will discuss a few examples that
demonstrate the analytical approach to fish
development (Table 3).
The introduction of new embryological
techniques opened up new areas of inquiry,
especially at the cellular level, and led to a
series of important observations, a few of
which will be discussed here. The organization of the teleostean egg, which differs
considerably from that of most other vertebrates, offers unique opportunities for the
study of fertilization. The mature ovum is
invested with an extracellular matrix, the
egg envelope. At the animal pole of the
egg, there is a funnel-shaped channel
through the egg envelope termed the micropyle. Fertilization is accomplished by the
passage of a single spermatozoan through
the micropyle and its subsequent fusion
with the ooplasm of the germinal disc. The
micropyle was first described by von Baer
(1835) and reinvestigated by Doyere in
1849 (cited in Ransom, 1867), who appreciated its significance in fertilization but did
not have any experimental evidence for its
role. Ransom (1867), in an extraordinary
paper on the eggs of teleostean fishes, not
only experimentally demonstrated fertilization, but also conducted experimental and
analytical studies of egg organization and
post-fertilization ooplasmic contractility (cf.
Ransom, 1854, for a preliminary report). In
a series of experiments, in which he was
able to restrict or facilitate access of spermatozoa to the micropyle, Ransom (1867)
observed the passage of a single spermatozoan through the micropyle and the subsequent events of the fertilization reaction,
namely, changes in structure of the micropyle, disappearance (discharge) of the cortical "granules," the appearance of the perivitelline space, and the movement of cytoplasm into the germinal disc. He did not
document the cytological details of spermegg fusion. This was done later by Hoffmann (1881) who depicted aster formation,
pronuclear fusion, and mitosis.
Ransom (1866, 1867) also carried out experimental studies to ascertain the physical
and chemical properties and structural organization of maturing oocytes, mature
ovum, and fertilized egg. He consciously
approached the egg from a cellular point of
view. The eggs of some fishes, especially
the stickleback Gasterosteus and the pike
Esox, exhibit strong rhythmic contractions
of the yolk mass shortly after fertilization
and throughout the early cleavage stages.
Contractile waves, originating at the animal
pole, bring about transitory deformations of
the yolk mass. Ransom (1867) found that
the contractions are independent of fertilization and appear to reside in what is now
known as the yolk cytoplasmic layer. He
conducted elaborate experiments to ascertain the "conditions" that regulate both
contraction of the yolk mass and cytokinesis. The effects of a number of factors, viz.
metabolic poisons, temperature, electricity,
carbon dioxide, and oxygen were determined. He devised a system whereby he
could subject eggs to electrical stimulation
while simultaneously observing them
through a microscope. Electrical stimulation excited peristaltic waves that were distinguishable from normal ones by their site
of origin, rapidity of formation and progress, and greater deformation. Cleavage was
RISE OF FISH EMBRYOLOGY
not affected. Cold temperatures delayed the
fertilization reaction and slowed cleavage
whilst moderately warm temperatures accelerated cleavage. High temperatures irreversibly arrested contractions and cytokinesis. Cyanide had no effect on yolk contraction, but delayed cytokinesis. Morphine
and chloroform inhibited contraction. Carbon dioxide was found to be a potent inhibitor of both contraction and cytokinesis.
On the basis of a series of experiments on
oxygen requirements, he concluded: (1) oxygen at low levels is necessary for contraction and cytokinesis; (2) cytokinesis requires more oxygen than do yolk contractions; (3) embryonic stages involving cell
multiplication and differentiation require
much more oxygen than do early cleavage
stages; and (4) the embryonic heart has a
higher tolerance to oxygen deficiency than
does striated trunk muscle. In a separate
publication that also dealt with rhythmic
yolk contractions, Ransom (1866) reported
on the isolation of a protein "allied to myosin" in the yolk of all fishes, amphibia,
and birds that he had examined. He did not,
however, state that myosin is involved in
yolk contraction. This apparent omission
may be an instance of understatement.
In addition to his fertilization and contractility research, Ransom also described
and isolated the germinal vesicle, the enlarged nucleus of early oocytes, and illustrated the multiple nucleoli within it (Figures 16-19 in Ransom, 1867). His pioneering efforts were not fully realized until the
twentieth century. Most of the research during the latter half of the century involved
histological studies of oocytes and eggs. In
this respect, there were some interesting developments in the study of nuclear-cytoplasmic relationships and chromosomes. In
a histological study of oogenesis, Van Bambeke (1893) depicts lampbrush chromosomes without recognizing them and reports on the "elimination of nuclear elements" from the nucleus into the ooplasm
of early oocytes. It appears that he was describing ribosome production and storage.
Lampbrush chromosomes were first described in the eggs of elasmobranchs (vide
infra) and subsequently discovered in teleosts (Marechal, 1907). The discovery of the
295
behavior of chromosomes during cell division was soon extended to include the
cleavage stages of teleosts (Kingsley and
Conn, 1883; Wilson, 1891).
The ability to maintain and manipulate
cells in vitro is an important tool of modern
biology. Ross Harrison (1907) is usually
credited with the "discovery" of cell-tissue
culture. However, he had several forerunners, one of whom was Wilhelm His. His
(1899) isolated single blastomeres and
clumps of blastomeres from salmonid blastulae and maintained them in the transparent fluid portion of the egg yolk. Under
these conditions, he was able to make longterm observations on the in vitro behavior
of blastomeres. He compared the behavior
of blastomeres from 0-4 day post-fertilization eggs (cleavage stages) with blastomeres from 5—12 day post-fertilization eggs
(gastrula stages). He observed "amoeboid"
movement and the extension and retraction
of finger-like processes which contained
clear cytoplasm (=hyaloplasm) in the 5-12
day blastomeres, but not in the 0—4 day
blastomeres. In the later stage blastomeres,
he also observed a transition in organization from single cells with finger-like processes, loosely arrayed in clusters, to round
cells that are joined together to form epithelial sheets. He compared amoeboid
movement and contractility of blastomeres
to muscle contraction. The cytoplasmic organization of living and fixed blastomeres
was shown to be similar. The leading edge
(lobopodia) of the blastomere consists of
hyaline cytoplasm which may contain elements of a sparse fibrous reticulum (ER, cytoskeleton?) whereas the perinuclear cytoplasm is dense and contains both granular
elements and a fibrous reticulum. Dynamic
changes in the regional organization of the
cytoplasm in living cells was noted. These
observations were illustrated with both
drawings and photomicrographs of sectioned cells. The in vitro observations of
His are remarkably similar to those carried
out by Trinkaus on the blastomeres of Fundulus many years later (Trinkaus, 1984a,
1990).
The first attempt to analyze the mechanism of embryo formation in teleosts was
undertaken by Lereboullet in a set of de<|AN9f R LIBRARY
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JMLLBRSVILLE, PA 17551
296
JOHN P. WOURMS
scriptive and experimental studies of teratology (Lereboullet, 1863fc, 1864). He used
different temperature regimes and chemicals to induce abnormal development, and
also tested the effect of pressure on development. Although he used over 200,000
eggs in his experiments, the results were inconclusive because the incidence of induced abnormalities in treated eggs was no
greater than that in untreated eggs. Oppenheimer (1936) speculated that Lereboullet
worked with too many eggs and should
have monitored each egg. Careful individual analysis of the many "spoiled eggs" in
the experiments would have produced more
conclusive results. Lereboullet's descriptive
studies were more fruitful. By tracing the
progress of developmental anomalies, especially duplicated or partially duplicated
embryos from their first visible origin, Lereboullet obtained insight into both their development and the normal process of embryogenesis. These studies caused him to
focus on the embryo-forming dynamics of
the germ ring. Eventually, he concluded
that embryo formation in teleosts took
place by the process that His (1874) later
called concrescence.
The pioneering efforts of Ransom and
Lereboullet were capitalized on by Loeb,
Stockard, and others. They used a variety
of physical and chemical parameters either
to determine the physiological conditions
required for normal development or to investigate the mechanisms of developmental
regulation by perturbing those conditions.
Loeb began his work on fish embryos in
the 1890's primarily using the cyprinodontid Fundulus, found in abundance at Woods
Hole. In a series of early experiments
(Loeb, 1893), he found that embryos, the
hearts of which had been immobilized by
potassium chloride, developed structurally
normal circulatory systems but ones lacking
blood circulation. Chromatophores that normally migrated onto the surface of the vitelline vessels of the yolk sac failed to do
so. Loeb suggested that migration would
only occur if there was blood circulating in
the vessels. Later, (Loeb, 1900) used his research on the effects of ions on embryos of
Fundulus as the basis for his theory of
physiologically balanced salt solutions.
Stockard (1906, 1907) studied the effects of
magnesium and lithium ions on the development of Fundulus in order to compare
them with the development-perturbing effects already well known in sea urchins and
frogs (Atz, 1986). At high ionic concentrations, cyclopean monsters with a single median eye were produced. By varying ion
concentrations, stages intermediate between
normal and cyclopean could be produced.
The ionic perturbation interfered with the
separation of the early, single eye-forming
field into the right and left eye anlagen.
Although radioactivity was only discovered in the latter years of the nineteenth
century, radioisotopes were soon employed
in embryological research. For example,
Tur (1896) studied the deleterious effects of
radium radiation on catshark Scyliorhinus
development, and Oppermann (1913) described a variety of developmental abnormalities that resulted from the fertilization
of trout eggs with radium-irradiated spermatozoa.
Fundulus has been a favorite experimental animal at Woods Hole and other marine
laboratories on the east coast of the United
States for over 100 years (Atz, 1986). Given its popularity, it is natural to inquire into
its history. The first accounts of the development of Fundulus were given by Alexander Agassiz (1882) and Ryder (1886fo).
Boyer (1892) was the first to specifically
state that he had obtained eggs by stripping
ripe females and artificially fertilizing them,
and he also gave a more extensive account
of the early stages of development. Who,
then, introduced the eggs and embryos of
Fundulus as experimental material and developed the methods for obtaining them? I
speculate that John Ryder was the driving
force behind Fundulus. Ryder was an embryologist's embryologist. In his brief life,
he published numerous (215) papers, many
of which dealt with fishes. From 1880 to
1886, he was in charge of embryological
research at the U.S. Fish Commission Laboratory at Woods Hole. Much of his research on fish embryology required artificial fertilization of eggs. The Fish Commission Laboratory was far better equipped
for research than was the fledgling Marine
Biological Laboratory. Consequently, many
RISE OF FISH EMBRYOLOGY
embryologists such as Boyer, Morgan, and
others started their work there and continued as long as the cultural atmosphere was
conducive to basic scientific research (Linton, 1915). Ryder left the Fish Commission
Laboratory for a professorship at the University of Pennsylvania which he held until
his death in 1895 (Allen, 1896). Both Morgan and Loeb were on the faculty of Bryn
Mawr and in close proximity to Ryder. Did
their paths cross?
In terms of what has come to be regarded
as classical experimental embryology, the
first experiments of consequence on embryonic fishes are based on the concrescence
theory. A different approach to the study of
fish embryology had been taken by His,
who was more concerned with the events
and mechanisms of development than their
possible evolutionary significance (His,
1879; Maienschein, 1985). In his quest for
a mechanistic theory, His (1874) proposed
his "Konkreszenz Theorie" to explain the
observed relationship between embryo formation, and the cellular material in the lateral arms of the germ ring. As originally
conceived by His, concrescence was a process by which the right and left lateral margins of the germ ring became apposed to
one another in the forming embryo, so that
as the two lateral margins progressively
fused in a medial plane, the embryo grew
backwards. In this theory, the embryo was
formed by the union of the two halves of
the germ ring so that successive regions
along the anterior-posterior axis of the embryo were originally located in successively
distal regions along the circumference of
the germ ring (Sumner, 1900). His applied
his theory both to teleostean (His, 1876)
and chondrichthyan development (His,
1877). Although not implicitly stated, His's
theory is a form of mosaic or determinate
development. Either because it was challenging or because it was so succinctly stated, the concrescence theory soon was put
to the test. The use of experiments to test a
theory of development was a novel idea at
that time and represents an early phase in
the paradigm shift to experimental embryology.
The first person to test the concrescence
theory was Kastschenko (1888), who op-
297
erated on the embryos of the catshark Scyliorhinus. He carried out three sets of experiments but did not illustrate his results
(Kastschenko, 1888, pg. 456). In the first,
he cut the germ ring on one side of a Stage
7, lancet-shaped embryo. A normal embryo
developed. In another experiment, he destroyed the posterior end of the blastoderm
but not the germ ring. The anterior half of
the embryo developed normally, but the
posterior half did not. If His's theory were
correct, the posterior half of the embryo
should have developed because the tissue
for its formation was intact in the germ
ring. In the third experiment, the entire embryo at Stage 7 was separated at its two
sides from the blastoderm, severing its connection to the germ ring. A normal embryo,
with both sides of the body intact, continued to develop to the three somite or tailbud
stage. According to His, a defective, partial
embryo should have been produced because
the cellular material needed to complete
embryo formation remained in the germ
ring.
Kastschenko's pioneer research was soon
followed by more extensive studies by
Morgan (1895) and Kopsch (1896, 1898),
who tested the concrescence theory in both
chondrichthyans and teleosts. Kopsch's results differed somewhat from those of
Kastschenko (Kopsch, 1950). Morgan's research is interesting not only because of its
bearing on the concrescence theory, but
also because of his experiments on early
blastomeres. Morgan (1895) carried out two
sets of experiments on the eggs of Fundulus. In the first set, he completely removed
one of the first two blastomeres. The remaining blastomere divided, and a somewhat smaller blastoderm was produced.
From it a single, normal embryo formed,
albeit somewhat smaller than usual. In the
second set, Morgan cut the germ ring on
one side of an early embryo. The embryo
continued to develop normally and underwent posterior elongation even though the
germ ring remained intact only on the unoperated side. The results contradict His's
theory. In retrospect, His's concrescence
theory was an excessively rigid statement
of the process of confluence of germ-ring
cells and their incorporation into the em-
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JOHN P. WOURMS
bryo (Solnica-Krezel et al., 1995; Ballard
et al., 1993). Before leaving the subject of
concrescence, I wish to point out that His
restated the theory in 1894 (His, 1894), but,
more importantly, in that paper he introduced physical models as a means of illustrating the mechanical basis of morphogenesis.
Other approaches to the study of developmental mechanisms were undertaken
during this period. Following his pioneering
application of cell lineage analysis to the
development of Clepsine, Whitman (Agassiz and Whitman, 1884) attempted to take
a similar approach to teleosts. Both his attempts and those of Wilson (1891) were
premature and doomed to failure because
there was no sure way of identifying individual blastomeres and their progeny and
tracing their fate during development.
The study of the physiology of embryonic nutrition is the last topic to be considered. This aspect of piscine development
had its origin in the study of viviparous
fishes. In many such fishes, there is a
marked difference between the small size of
the egg and the large size of the full-term
embryo (Wourms et al., 1988). How is this
accomplished? The problem of embryonic
nutrition in viviparous fishes was recognized as early as 1624 when Schoenveld
postulated that embryos of Zoarces ingested ovarian fluid as a source of nutrients. It
was not until the nineteenth century that
physiological and chemical studies of embryonic nutrition were attempted. Davy
(1834) appears to be the first to have undertaken this line of investigation. He compared the mean weight of the egg (182
grains) of the electric ray Torpedo with that
of the full-term embryo (479 grains). On
the basis of the increase in weight, he concluded that the embryo received additional
maternal nutrients during gestation, a correct conclusion but based on faulty data collection. Modern studies, reviewed in
Wourms et al. (1988), show that there is
actually a loss of organic weight during development of this fish. The apparent discrepancy arose because wet weight instead
of dry or organic weight was measured.
Teleosts also were studied. In 1867, Blake
analyzed the ovarian fluid of a surfperch
and found that proteins and lipids were the
major organic constituents and hence the
source of nutrition. Analysis of ovarian fluid was next carried out on Zoarces by
Stuhlmann (1887) and Kolster (1905), who
found it to be protein- and lipid-rich. Interest in this area of inquiry waned until it was
revived by Ranzi (1932) and attracted the
attention of Needham (1942).
EPILOGUE
I have attempted to trace the history of
fish embryology from its inception with Aristotle through its rise in the nineteenth century and its continuation into the twentieth
century. Over that span of time, knowledge
has progressed from a series of fragmentary
observations on a few elasmobranchs and
teleosts to a coherent body of information
on the development of each of the eleven
major groups of fishes. This growth of
knowledge, similar to that of other animals,
started primarily as a descriptive science
and subsequently expanded to incorporate
comparative, experimental, and analytical
approaches. At the descriptive and experimental levels, the achievements in elucidating fish development paralleled and exceeded those of most other vertebrates, except the domestic fowl, some amphibians,
and some mammals.
Fishes, as research subjects, were major
components of nineteenth century embryology. Many researchers, e.g., von Baer,
Harrison, His, Morgan, Miiller, and Whitman, were part of the mainstream of embryology. They influenced the course of
embryological research and, in turn, thenresearch on fishes was influenced by the research and conceptual advances of others.
In this respect, one should not overlook the
motivation for embryological research on
fishes. For the most part, investigators
chose to study fishes because of the intellectual challenge posed by some aspect of
their development and the opportunity to
investigate an interesting problem in an organism amenable to its study. The economic importance of fisheries was another factor. On the other hand, some investigators
found the study of fishes to be personally
satisfying and preferred the ambiance of
marine laboratories.
RISE OF FISH EMBRYOLOGY
The rise of fish embryology in the nineteenth century can best be appreciated by
examining factors that influenced the choice
of organisms, provided the conceptual basis
for initiating the study and interpreting the
results, and determined the technology
used. Some factors are unique to the study
of fishes, while others are applicable to embryology in general. Most of these factors
have already been discussed in the text. The
five principal factors that influence the
study of fish development are now gathered
together and listed in summary form: (1)
the properties of the material, i.e., transparency or size of the eggs or characteristics
of the embryos or larvae; (2) availability of
study material, i.e., routine aquatic collections, artificial fertilization, access to fish
hatcheries, expeditions; (3) a place to work,
i.e., the rise of university-based research
laboratories and marine stations; (4) changing technology, i.e., the development of embryological microtechnique and high resolution microscopy as well as the elaboration
of aquarium technology; and (5) conceptual
and disciplinary interactions, i.e., the rise of
evolutionary and cellular theory and the
formalization of embryology, physiology,
marine biology and experimental zoology
as discrete branches of biology with a set
of objectives and methodologies.
Having enjoyed prominence in the nineteenth and early twentieth centuries, the
study of fish embryology entered a period
(1914-1960) during which it was temporarily overshadowed by developmental
studies of other organisms. A number of
factors, namely, conceptual, cultural, logistical, and technological affected the study
of fish embryology (Wourms and Whitt,
1981).
First, there was a paradigm shift in embryology away from descriptive and comparative studies and to analytical studies,
especially experimental embryology (reviewed in Maienschein, 1991). A mechanistic explanation of development was
sought and the experimental method was
the means to that end. The effort to transform embryology into a rigorous experimental science was accompanied by an
overreaction to the speculative excesses of
proponents of the biogenetic law. Evolution
299
was considered irrelevant to the study of
development. As a result of these attitudes,
many embryologists lost sight of the fundamental link between development and
evolution.
The paradigm shift affected both the
course of embryological research in general
and also the course of research in fish embryology. One consequence of the shift was
that fishes lost their privileged position
among research organisms. The diversity of
body form and of style no longer mattered
because descriptive embryology was considered passe. Secondly, their position as
the basal group of vertebrates and the opportunities that this presented for comparative-evolutionary studies was no longer an
asset because the role of embryology in
evolutionary studies was now considered irrelevant. In addition, comparative studies of
"primitive" fishes had run their course and
were at a temporary impasse. The basic descriptions of their embryonic development
were in place. In the absence of modern
evolutionary-developmental concepts, there
was no way to further analyze the available
data. New morphological studies would not
be forthcoming until new techniques appeared, especially electron microscopy.
Many of the more interesting primitive fishes did not reproduce in captivity and required field research in exotic locales. This
was inconvenient, expensive and, during
the two world wars, impossible. Finally, the
developmental process, e.g., induction, or
the event, e.g., eye formation, became the
primary research objective, and the organism on which the research was conducted
was of little consequence.
Cursory examination of the research in
experimental embryology during the period
1914-1960 reveals that a few major themes
were dominant, for example: (1) cell lineage studies; (2) mosaic vs. regulative development; (3) morphogenetic movements
and fate maps; (4) primary embryonic induction; and (5) secondary inductive interactions involved in organ formation and tissue differentiation. Research was carried
out on a relatively limited number of organisms, in particular, eggs and larvae of
amphibians, the chicken, sea urchins, tunicates, some marine annelids, and molluscs
300
JOHN P. WOURMS
(Willier et ah, 1955). These organisms were
favored because they were convenient, had
desirable characteristics, posed few major
technical problems, and did not require
long-term culture (Rugh, 1962). The successful use of these organisms and the establishment of a data base for them in the
scientific literature biased subsequent investigators in their favor. They became "model
systems" (Bolker, 1995). There was also a
"founder effect." Students tended to use
the organisms that their mentor taught them
to use and avoided organisms that their
mentor considered unsuitable.
Restricting the discussion to vertebrates,
two factors, logistical and technological,
that affect the choice of experimental organisms merit detailed examination. The logistics of time-based experimental studies
of living embryos differ radically from
those associated with descriptive or comparative studies of living or fixed embryos.
Experimental research requires an ample
supply of embryos at the correct stage of
development. Supply must be such that experiments on the same embryonic stage can
be repeated over a period of days or weeks.
Given the chronic shortage of time, space,
and facilities in the academic world, investigators were forced to focus more on the
experiments and less on the maintenance of
the organism. Amphibians and the domestic
chicken fit the supply requirements nicely.
Fertilized chicken eggs can be obtained
year-round, in large numbers from farms
and maintained in simple, commercial incubators. Amphibians or amphibian eggs
can be collected in the wild or obtained
from commercial suppliers. As long as they
are kept cold, which is relatively easy to do,
they require little maintenance. Their chief
disadvantage is that they are not easy to
breed in the laboratory, so research with
amphibians originally was a springtime
venture. The use of pituitary and gonadotrophin treatments solved that problem.
Thus, amphibians and fowls provided a dependable and convenient source of experimental material.
Fishes, in contrast, posed a variety of
problems of convenience and dependability
of supply. During the period 1914-1960,
fish hatcheries and other sources of fertil-
ized eggs were uncommon. Some investigators had access to hatcheries, especially
for trout eggs. The most dependable method
of obtaining research material involved the
artificial fertilization of wild-caught specimens or breeding domesticated stock. Much
of the Fundulus research and research with
marine species was done with wild-caught
stock. Generally, this approach involved little in the way of long-term culture of the
adult fish. It is the other approach, the use
of domesticated stock, wherein the problems lies. James Atz (personal communication, also cited in the Wourms and Whitt,
1981) suggested that the most important
reason that relatively few fishes had been
studied in the past was that they were considered difficult to keep alive in captivity
and even more difficult to breed. For many
species, these supposed difficulties were
more perceived than real. Successful use of
fishes in developmental studies depends on
the ability to maintain them in long-term
culture. To do so requires a facility with
controlled conditions of temperature and
light. During the early and mid-twentieth
century, such facilities were not generally
available at most academic institutions Colony maintenance also requires a long-term
commitment of time on the part of the investigator or the assistants. Prior to the era
of governmental research grants, the assistance of a qualified technician was not generally available.
The technical advantages or disadvantages of fish embryos also need to be considered. Amphibian and chicken embryos are
large enough for free-hand microsurgery,
e.g., grafting, deletions, marking experiments, even on the part of relatively unskilled workers. It was easy to get the embryos out of their egg envelopes. Maintenance of experimentally treated material
was relatively simple. Development proceeded rapidly. Specimens were relatively
easy to section. In contrast, fish embryos,
especially those of teleosts, were small and
were difficult subjects for microsurgical
manipulations. The large eggs of elasmobranchs were hard to acquire in quantity
and at the desired stage of development.
Moreover, they developed too slowly, i.e.,
3—12 months. It was difficult to remove fish
RISE OF FISH EMBRYOLOGY
eggs from their investing envelopes. Prior
to the use of plastic polymers, teleost eggs
were difficult to section. Although ideally
suited for in vitro microscopy, the repertoire of light microscopic techniques that
could be brought to bear was soon exhausted. Teleost eggs and embryos would come
into their own again with the development
of new forms of light microscopy, e.g., differential interference, computer-enhanced
video microscopy, and fluorescence microscopy. Their small size would also make teleost eggs and embryos ideal candidates for
electron microscopy. Thus, no one single
factor, logistical or technical, was an insurmountable barrier to the use of fishes. Rather, it was a combination of these factors that
made amphibians and the chicken the experimental organisms of choice.
The study of fish embryology did not decline during the period 1914-1960. Although the number of individuals and the
amount of research done with other organisms increased considerably, the number of
investigators of fish embryology tended to
remain relatively constant. The field itself
was quite vigorous. My impressions are that
the first portion of the era was a time of
experimentation and exploration, and the
second part, i.e., 1950 to the present is a
time of exponential growth and maturation.
Significant contributions were made in a
number of areas, such as: (1) fertilization
(Yamamoto, 1961; (2) experimental embryology (Oppenheimer, 1937, 1947, 1979);
(3) morphogenetic movements (Ballard,
1981; Devillers, 1961; Trinkaus, 1951,
1984a,b, 1990); (4) physiology of development (Smith, 1957); (5) evolutionary and
ecological adaptations (Soin, 1971); and (6)
viviparity (Ranzi, 1932; Wourms et al.,
1988). (An account of this period lies outside of the scope of this paper. The citations, although far from inclusive, provide
access to the literature and indicate research
trends. Whitt and Wourms, 1981, afford a
view of the field early in its growth phase.)
Much of the research during this period was
done with Fundulus, the medaka (Oryzias
latipes), and the trout (Oncorhynchus mykiss = Salmo gairdneri).
Although the subject is more contemporary than nineteenth century, consideration
301
of the zebrafish Danio rerio is warranted
because it sheds light on trends in the study
of fish embryology. Currently, the zebrafish
is one of the favored "models" for molecular-genetic studies of vertebrate development. It is also important in other areas
such as the study of morphogenetic movements, the determination of embryonic fate,
and developmental neurobiology (Weinberg, 1992; Westerfield, 1995; Wylie,
1996). Its rise to prominence coincides with
the broad revival of interest in fish development and is driven by many of the same
factors responsible for the revival as well as
several factors that are specific to zebrafish.
The zebrafish is a small freshwater fish
that was discovered in the rivers of northeast India early in the nineteenth century.
Live specimens were first imported into
Germany and introduced into the tropical
fish hobby in the late nineteenth or early
twentieth centuries. Popular accounts of its
breeding soon appeared (Zimmermann,
1904; Young, 1913). Because it was easily
bred in captivity by amateur and commercial breeders, the zebrafish soon became a
staple of the tropical fish trade.
The first developmental studies were
done in the 1930s (Goodrich and Nichols,
1931; Roosen-Runge, 1936). Roosen-Runge and Lewis seem to have been the first to
recognize that the transparency of the egg
and absence of oil droplets presented a
unique opportunity for the study of morphogenetic movements (Roosen-Runge,
1936; Lewis and Roosen-Runge, 1944). A
series of normal stages was published by
Hisaoka and Battle (1958). A widely distributed, color motion picture with animated sequences of fate maps and morphogenetic movements helped popularize the zebrafish (Durden and Berrill, 1961). The zebrafish began to be used for fertilization
(Hart, 1970); oogenesis (Korfsmeier, 1966);
neurobiological (Rahmann and Korfsmeier,
1965); and fisheries-pollution studies (Laale, 1977). Much of the current interest in
zebrafish stems from pioneering work done
at the University of Oregon. Eaton and Farley (1974) established protocols for the production of zebrafish eggs in the laboratory
and subsequently used the embryos and larvae to study the development of Mauthner
302
JOHN P. WOURMS
neurons (Eaton and Farley, 1973). Kimmel
(1972) took advantage of the transparency
of the embryos to study the development of
Mauthner cell axons in living larvae. The
appearance of a new generation of fluorescent probes, especially non-diffusible cell
markers, the advent of epifluorescence and
confocal microscopy, and the development
of improved methods for intracellular injection of probes led to a series of studies of
cell lineage, fate maps, and morphogenetic
movements (Kimmel and Warga, 1988).
Perhaps the most significant advantage of
zebrafish derives from the development of
an effective genetic system for this species.
Previously, the potential of genetics in fish
development had been demonstrated with
the platyfish Xiphophorus (Kallman, 1975).
Platyfish, however, had limitations. Internal
fertilization and viviparity prevented access
to eggs and developing embryos. This was
not the case in zebrafish. George Streisinger
deserves credit for establishing the genetic
basis of zebrafish research (Streisinger, et
al, 1981; Grunwald and Streisinger, 1992;
Walker and Streisinger, 1983). The period
1980 to the present has witnessed an exponential expansion in research on the zebrafish embryo.
Returning to the theme of choice of research organisms, it is easy to see how the
zebrafish has acquired its current popularity. They afford many advantages and relatively few disadvantages. Adults are easy to
maintain. Their small size allows large colonies to be set up. Colony maintenance can
be semi-automated. A short generation time
of 2-3 months makes genetic studies feasible. Mutations can be induced by a variety
of physical and chemical agents. Captive
populations reproduce throughout the year.
Zebrafish are oviparous and fertilization is
external. Spawning is synchronized by
light, so large numbers of eggs at similar
stages of development are available and
easily collected. The eggs and embryos are
exquisitely transparent, thus facilitating observation. Eggs lack the oil droplets that
characterize the eggs of Fundulus and Oryzias and interfere with observation. The
eggs are relatively easy to dechorionate.
The small size of the egg and embryo
makes them ideal subjects for high resolu-
tion light microscopy of living material.
With the advent of new methods of micromanipulation and microinjection, small size
is no longer a disadvantage. Embryonic and
larval development are rapid so that a miniature fish is produced within days of fertilization.
Having extolled the virtues of zebrafish
and in the midst of the celebration of the
zebrafish as a "model system," some introspection is in order (Bolker, 1995). Are teleosts, such as the zebrafish, valid models
for understanding the development of tetrapod vertebrates? Probably not: teleostean
development, especially gastrulation and
neurulation, is highly specialized and evolutionarily derived. Teleosts evolved within
a different lineage than the tetrapods (Fig.
1) and make their evolutionary appearance
after the radiation of the tetrapods. Is information about the details of zebrafish development generally applicable to other teleosts and, if so, then to what extent? Teleosts
are the most speciose group of vertebrates
with a great diversity of reproductive styles.
Already problems associated with the diversity of teleostean developmental patterns
are coming to the fore (Trinkaus, 1996). Finally, the opportunities for research presented by the evolutionarily diverse developmental patterns of the other ten groups of
fishes should not be overlooked or ignored.
In retrospect, the interest in zebrafish development has been beneficial to the field.
Experimental studies of zebrafish and other
teleosts, coupled with a revival of interest
in comparative and evolutionary studies,
have revitalized the study of fish embryology.
ACKNOWLEDGMENTS
This article is dedicated to the memory
of Jane Oppenheimer. Although I was neither a student nor an associate of hers,
nonetheless her research in fish embryology
and her scholarly activities in the history of
embryology have had a profound effect on
me. I am greatly indebted to James Atz for
his enthusiastic support and mentorship
over a period of many years. Without access to the Dean Library of Ichthyology at
the American Museum of Natural History
and the library of Harvard's Museum of
RISE OF FISH EMBRYOLOGY
Comparative Zoology, it would have been
impossible to write this paper. I am grateful
to William Bemis and Edward Ruppert for
reading the original manuscript and offering
many detailed suggestions for its improvement. William Bemis also suggested the inclusion of a cladogram and kindly gave permission to use one of his. The editorial contributions of two anonymous reviewers are
acknowledged with thanks. Preparation of
this paper and my research have been supported by a National Science Foundation
grant, IBN-9507358.
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