AMER. ZOOL., 23:639-654 (1983)
Early Development of the Hagfish Pituitary Gland:
Evidence for the Endodermal Origin
of the Adenohypophysis1
AUBREY GORBMAN
Department of Zoology, University of Washington,
Seattle, Washington 98195
SYNOPSIS. The adenohypophysis in early head fold stage hagfish embryos is at first a
distinct differentiated thickening of the archenteric roof where it contacts the developing
infundibular portion of the brain. This portion of the archenteron eventually becomes
the dorsal epithelium of the nasopharyngeal duct. The later development of the adenohypophysis involves formation of multiple acinar outgrowths of the dorsal nasopharyngeal
epithelium which together form a layered mass of follicular tissue adjacent to the neurohypophysis. This mode of origin of the adenohypophysis by endodermal polyinvagination and delamination differs from all other vertebrates, including the lampreys. The
pertinence of this new information for considerations of monophyletic versus diphyletic
evolutionary origins of the modern cyclostome groups is pointed out. The unusual development of the hagfish adenohypophysis presents some new and unanticipated puzzles
within the general question of pituitary evolution.
stouti, has served for many years as the basis
There has been only one study of devel- for speculation about hypophyseal development of the pituitary gland of hagfish, opment. Kupffer considered the entire
and this was based on serial sections of a nasohypophyseal duct, projecting postesingle advanced (4.5 cm long) specimen riorly from the developing olfactory organ,
(Fernholm, 1968). The remarkable impli- to be the adenohypophyseal anlage
cation from Fernholm's study was that the although he realized that this duct extends
adenohypophysis in his specimen of Myxine posteriorly beyond the definitive hypophglutinosa appears to be derived from the yseal region. Fernholm's limited study that
epithelial tissue of the nasohypophyseal appears to show that the adenohypophysis
duct adjacent to the infundibulum by a forms from only a small part of the dorsal
horizontal layering-off process (delamina- surface of the nasohypophyseal duct clearly
tion) rather than by the ingrowth of a solid is at odds with Kupffer's interpretation.
Recently I had the opportunity to study
or hollow bar of epithelial tissue as seen in
all other vertebrates. If confirmed, this about 34 additional sets of serial sections
mode of origin of the hagfish adenohy- of embryos of Eptatretus stouti. This has
pophysis differs also from that of the lam- resulted in the present description of
prey. In several lamprey species studied a hypophyseal development from a pre-head
more or less typical cylindrical Rathke's fold stage to the later embryonic period
pouch-like ingrowth extends from the which Fernholm's specimen represents.
nasohypophyseal placode on the head sur- Although the single specimen of Myxine
face inward under the infundibulum (Kupf- studied by Fernholm suggests that the adenohypophysis forms by delamination, this
fer, 1894, 1906; Leach, 1934).
meager evidence probably cannot be conAlthough it is not specifically concerned sidered definitive. The further evidence
w:th the pituitary gland, Kupffer's 1899 provided by our series of younger embryos
study of formation of head structures of of Eptatretus fully confirms Fernholm's
the Pacific hagfish, Eptatretus (Bdellostoma)
conclusion.
A further important difference in devel1
From the Symposium on Evolution of Endocrine opment of the hagfish adenohypophysis
Systems m Lower Vertebrates, A Symposium Honoring Professor Aubrey Gorbman presented at the Annual Meet- revealed by this study, which separates it
ing of the American Society of Zoologists, 27-30 from all other vertebrates, is its origin from
December 1982, at Louisville, Kentucky.
endoderm. Evidence for this is given below.
INTRODUCTION
639
340
AUBREY GORBMAN
KuFig 3. 75 1.
19
FIG. 1. Six summary sketches by C. von Kupffer (1899) of sagittal sections of hagfish embryos. They are
arranged (top to bottom) in order of relative age. An arrow has been placed by me to indicate on Ku Figure
3 (top) the structure which is interpreted by me (see Figs. 8 and 11) to be the adenohypophyseal placode.
Furthermore, to be noted in Ku Figure 3 is that only the roof of the archenteron is drawn. The archenteric
floor, over the yolk, is not shown. In Ku 3 successive structures indicated in the archenteric roof (posterior
to anterior) are: the notochord, cd, the adenohypophysis (unlabeled), the archenteric roof, labeled en, and
the oral plate, or "pharyngeal membrane," rh. In Ku 4 the archenteric floor is shown and an unlabeled dotted
line (next to label 11, for olfactory organ) is shown but is not explained. This is presumably where Kupffer
considered the oral membrane to have perforated. The oral membrane reappears, according to Kupffer, in
later embryos as shown in Ku 6 and Ku 1. Ku 5 is a parasagittal drawing ot the same stage as Ku 4. Also
added b\ me to this figure are pairs of lines together with numbers indicating the corresponding planes of
section of several figures in the present paper. The numbers indicate the figures that are photographs of
embrvos studied in preparation of this report.
HAGFISH PITUITARY DEVELOPMENT
641
of relative stages of development in Kupffer's 1899 descriptions (Fig. 1).
All of the embryos described here were
Some comment must be made concerncollected in 1898 at Monterey, California ing the general state of preservation of the
through the efforts of Professor Bashford embryos examined, since this conceivably
Dean of Columbia University (Dean, 1898, might influence certain conclusions drawn
1899). They are labeled Bdellostoma stouti from this study and earlier ones. There was
and at this time they are part of the per- considerable variation in the apparent rate
manent collection of the Warren Anatom- or time of fixation of the embryos relative
ical Museum at Harvard University. Dean's to the time of death, particularly the younown work with the embryos he collected ger stages (see lengthy discussion in Kupfwas limited to description of whole mounts fer, 1899). Thus, among the embryos stud(Dean, 1899). However, about 1928 he ied #2336, an early head fold stage, is
made available some of his already sec- outstanding in terms of mitotic activity seen
tioned embryos as well as about 30 alcohol- and in general crispness of tissue boundpreserved embryos to Professor J. L. Conel aries (Figs. 12-15). Others were so poorly
of Boston University with the stipulation preserved that only the grossest of details
that they be placed eventually in the ana- could be distinguished reliably. Therefore,
tomical museum at Harvard "where they of more than 34 embryos studied, in whole
would be well cared for and be readily or in part, only 21 served as the basis for
accessible to investigators" (Conel, 1929). conclusions and photographs presented
Conel supervised the preparation of serial here. Another common feature of the hagsections of all of the alcohol-preserved fish embryo material was the obvious dorspecimens he received and he described soventral compression of tissues, resulting
development of the hagfish brain from in a flattening of the entire embryo, and
them (Conel, 1929, 1931). At the present especially its dorsal surface. This is seen in
time the Dean-Conel E. stouti sectioned one of the youngest embryos (#233 1) which
specimens are to my knowledge the only is otherwise well-fixed, as well as in some
hagfish embryos available for study.
older ones (e.g., #2346). Previous workers
One unfortunate feature of the Dean- (see Kupffer, 1899 and Conel, 1929) have
Conel embryos is that no information is attributed this flattening to the "tension"
available concerning their total length or exerted by the encompassing egg-shell,
relative age. In most instances the thick- trapping the embryos against the yolk. It
ness of the serial sections is indicated upon seems quite likely that the distortion was
the slides: if it is assumed that the serial created artifactually by use of hypotonic
sections are quite complete, then rough fixatives without puncture, or sufficient
calculations can be made of distances puncture, of the shell. In those few
between the anterior end of the embryo instances in which the embryo was
obviously dissected free of the shell before
and a particular structure.
fixation (e.g., #2336) the embryo and its
Study of the sections was done mostly by tubular organs (neural tube, gut) are
ordinary light microscopy. In a few serials rounded in profile. It is likely that this repin which fading of the stain was severe, the resents the normal shape rather than the
use of phase contrast optics was helpful. flattened profiles seen in many of the specRecording of observations was by photo- imens. According to Kupffer (1899) it was
micrography.
virtually impossible to dissect younger
A total of 34 sets of serial sections were embryos free of the shell once they were
examined. All but three sets are trans- fixed. He states that attempts to perform
versely cut. Two are cut sagitally and one such post-fixation dissections resulted in
frontally. The only practical way to assess "maceration" of the embryos. Generally,
relative ages of the embryos was by com- it was not considered possible to successparisons of apparent relative structural fully section the embryos until they were
development within the series. Less satis- isolated from the shell, although Conel
factory was comparison with the drawings
MATERIALS STUDIED
642
AUBREY GORBMAN
Fie. 2 X82 and Fie. 3X325. Cross section of embryo
#2375 ("youngest" specimen) through the shallow
infundibulum, the ventral outpocketing of the brain.
Under the brain is a single layer of endoderm, En,
which appears further undifferentiated. Other labels:
///, third ventricle of infundibulum; Y, yolk.
(1931) has published photomicrographs of
one sectioned embryo in which the shell is
included in the sections.
OBSERVATIONS
Pre-head fold embryo
Embryo #2375 is the least advanced
embryo that was studied and is cut in transverse sections. It is considerably younger
than the least developed embryo described
by Kupffer (1899). Judging from details of
its simple brain structure, it is similar to
the specimen that is referred to as "Embryo
219" by Conel (1929) who states that it
"represents one of the earliest stages of
development to be found in the collection." This specimen has been dissected
free of the yolk and appears to be reasonably well fixed. However, it may have been
injured during the dissection, since there
is an indefinite quality about cellular and
cell-layer boundaries (see Figs. 2, 3).
Nevertheless, this embryo clearly permits
certain conclusions.
The neural folds form a tubular brain in
embryo #2375 but are not yet completely
fused at the diencephalic level (Fig. 2). The
brain, as shown in Conel's (1929) figure of
a wax reconstruction model of his specimen 219, is divided into fore-, mid-, and
hindbrain by indentations. The floor of the
forebrain is pouched out as a shallow
"infundibulum." Underlying, and in direct
contact with the brain, is the single layer
of yolk sac endoderm (Fig. 3). The yolk sac
endoderm under the brain consists of fairly
uniform cuboidal cells. Only at the level of
the hindbrain and further posterior is this
juxta-neural endoderm layer further differentiated into larger cells which are in
the process of separating from this layer
as notochord (Fig. 26a). Conel's (1929)
reconstruction of this embryo's brain (or
of a similar embryo) shows the notochord
beginning at the level of the midbrainhindbrain boundary and extending posteriorly.
Thus, there is no indication in the infundibular region of local differentiation of a
pre-hypophysial structure in an embryo of
this stage. There is in this specimen no
ectodermal modification other than neural
tube formation. Since there is no head fold,
there is no point where surface ectoderm
in any way approaches the ventral side of
the brain or the infundibular (future neurohypophyseal) region.
Embryo #2331 is transversely sectioned
and is the next youngest specimen available
for study. It is strongly flattened dorsally
(Figs. 4, 5, and 6). Presumably this is an
artifact of the conditions at the time of
fixation, so that the brain has an almost
planar dorsal profile, no doubt dictated by
the shape of the overlying shell (which has
been removed). There is little or no head
fold. That is, the most anterior sections do
not project free over the yolk. The most
anterior sections contain only brain, covered dorsally by ectoderm, and the neural
folds are closed. About 150 p behind the
anterior extremity of the brain the first
head endoderm is encountered in the form
of a broad cellular sheet below the brain
(Fig. 4). This part of the head is marked
off on each side by lateral body folds, and
the brain and endoderm (foregut) are separated from the yolk below by the heart.
In comparison with Kupffer's illustrations,
HAGFISH PITUITARY DEVELOPMENT
this embryo would probably fall between
his first and second stages (Fig. 1, Ku 3 and
Ku 4). However, unlike Kupffer's embryo,
#2331 has no opening between the gut and
a subcephalic ectodermal pocket (as indicated in Kupffer's Fig. 4), there being no
such pocket and the gut having no lumen
at its anterior end. Although a slit-like
lumen is visible in Figure 4, the foregut
lumen about 60 n behind this section, as
shown in Figure 5, is much clearer. Thus
the gut in these anterior sections is a forward blind projection over the heart from
the simple endodermal layer seen further
back, and it is separated from the yolk sac
by the heart. Figure 6 represents a cross
section 290 ju behind the anterior extremity. The brain is narrower here in the diencephalic region. Ventrally a rather large
hypothalamic or infundibular pouch-like
structure is collapsed or flattened so that
it appears to consist of two double-layered
wings of cells. Below the brain are three
parallel rows of cells. The uppermost rows
are the dorsal and ventral walls of the pharynx, with their intervening lumen
obscured, possibly by the vertical pressure
referred to previously. However, laterally
the endoderm is much thicker and contains
an apparent pharyngeal pouch. Immediately below the double walled mid-pharynx
the third row of cells is the yolk sac endoderm, overlying the yolk itself.
In Figure 7, representing the level 410
p behind the anterior tip of the embryo,
endodermal structure is simpler, there now
being in the midline only two endodermal
layers visible: 1) a dorsal layer composed
of large cells forming the roof of the
archenteron and pressed medially to the
infundibulum of the brain; 2) a more ventral much thinner layer covers the yolk itself
(see also Fig. 10). Lateral to the infundibulum the more dorsal endodermal layer
forms two dorsally projecting wings. No
gut lumen is clearly apparent except within
the two projections lateral to the infundibulum. It may be noted that at this level the
neural folds are open dorsally. Figure 8
represents a section only 20 n posterior to
Figure 7. At this level an important change
has occurred in the endoderm immediately
ventral to the infundibulum: In place of
643
the single layer of larger cells now there is
a medial thickening about four cells thick.
This is the adenohypophyseal placode. It
is readily and consistently identified and
traced through the later stages of development as a thickened endodermal structure limited to the small area below the
ventral posterior tip of the infundibulum.
Figures 10 and 11 represent higher magnifications of the infundibular regions just
anterior to the adenohypophysis and at the
level of the adenohypophysis. The adenohypophyseal placode is only about 80 n
in length in this embryo. Figure 9 represents the cross sectional structure at 580 fj.
behind the anterior extremity. The juxtaneural endoderm is again thin and single
layered and the infundibular expansion of
the brain floor has almost disappeared. At
about 650 fi behind the anterior extremity
another more rounded structure begins to
be seen separating dorsally in the midline
from the archenteric roof, the notochord
(no illustration). It is readily distinguishable from the adenohypophysis because it
is histologically different and because it is
not associated with the infundibulum. Furthermore, unlike the adenohypophysis it is
extremely elongated, extending posteriorly for most of the length of the embryo.
Embryo #2331 has been described in
some detail because it establishes the
apparent germ layer origin of the hagfish
adenohypophysis and is the earliest stage
in which this structure was identifiable.
These observations are summarized in diagrammatic form in Figure 26b.
Only the hypophyseal area of the older
embryos will be described below in some
detail. Questions concerning the development of the olfactory, pharyngeal, stomodeal, and other head structures will be
dealt with more extensively in a later study.
It should be noted in Kupffer's Figure 3
(see Ku 3 in Fig. 1) that the adenohypophyseal thickening is clearly drawn in its proper
place next to the infundibulum, but it is
not labeled or referred to in his text. Furthermore, it is not mentioned by any later
reviewer who has evaluated Kupffer's writings. Kupffer himself eliminates this structure in later versions of drawings of this
stage (e.g., see Kupffer, 1906).
644
AUBREY GORBMAN
INF "•
f
*
10 —,
FIGS. 4-11. Selected cross sections of embryo #2331. Figures 4-9 (X86) are arranged in their anteriorposterior order, with Figure 4 being the most anterior, about 150 nm behind the rostral end. In all figures
a white X (Figs. 4 and 5) or a black asterisk has been placed in the most dorsal endodermal layer. Above this
layer the brain may be seen. Figure 10 (X342) is a section posterior to the level of Figure 7 but anterior to
Figure 8. Figure 11 (X342) is within several sections of Figure 8. Further discussion of these figures is in the
text. Labels: A, archenteric cavity; AD, adenohypophyseal placode; H, heart: /, infundibular cavity: IXF,
infundibulum: P, lateral pharyngeal space, possible pharyngeal pouch; Y, yolk.
HAGFISH PITUITARY DEVELOPMENT
645
Fics. 12-15. Selected cross sections through embryo #2336. Figures 12, 13, and 14 X130. Figure 15 X520.
In Figure 12 the attachment of the short head fold to the blastoderm is visible, with ectoderm at this point
completely encircling the head. The head contains mostly forebrain, but the blind anterior tip of the nasopharyngeal duct (A'P) first appears at this level below the brain. FIG. 13. Posterior to the attachment of the
head to the yolk sac the differentiation of the archenteric space into a dorsal nasopharyngeal duct and a
ventral pharynx (two slits extending ventrolaterally, with communication between them) is seen. Fie. 14.
Further posterior, the infundibular portion of the diencephalon projects posteriorly and is now cut off from
the brain above. The third ventricle (III) in it is labeled. Below the infundibulum is the blind posterior end
of the nasopharyngeal duct. Its dorsal wall is multilayered and applied to the infundibulum. Its ventral wall
is thin and difficult to distinguish. Fie. 15. is a higher magnification of the adenohypophyseal portion (AD)
of the nasohypophyseal duct.
Short head fold embnos
Insofar as general development of the
head structures is concerned, what is
referred to here as a "short head fold
embryo" is roughly comparable to Kupffer's Figure 6 (see Fig. 1) or slightly earlier
(see also Fig. 26c). It should be emphasized
that in no material examined by me has
any continuity been seen, as shown in Kupffer's Figures 4 and 5, between the subcephalic ectoderm and the lumen of the
primitive gut. The possibility that Kupffer's interpretation of such a continuity is
in error will be discussed further below.
In embryos of this stage of development
the anterior extension of the foregut ends
blindly in the future olfactory area (Fig.
12), ventral to the brain and posteriorly it
is undergoing a separation into two chambers (Fig. 13). The more dorsal of the two
chambers is the nasohypophyseal canal: the
more ventral is the cleft-like stomodeum
which extends posteriorly to the pharynx.
In embryo #2336 shortly anterior to the
point where the infundibulum has formed
a separate ventral chamber of the diencephalon (Fig. 13) the nasopharyngeal canal
above is undergoing separation from the
broad pharyngeal cavity below by growth
toward the midline of two lateral masses
or processes of tissue. Posterior to this level
the nasopharyngeal canal again ends
blindly, still in contact with the infundibulum (Fig. 14). At higher magnification (Fig.
15) it is clear that the upper wall of the
nasopharyngeal canal where it touches the
646
AUBREY GORBMAN
FIG. 16. Sagittal section, embryo #2377 (X38). Anterior to the right. The arrow below points to the posterior
end of the subcephalic pocket. The spaces at the anterior end of the head: the nasopharyngeal cavity XP is
labeled. Above and to the right the large space is the forebrain ventricle. The slit-like space below and left
HAGFISH PITUITARY DEVELOPMENT
infundibulum is thickened as an adenohypophysis. These observations are summarized diagrammatically in Figure 26c.
The two sagitally cut embryos, #2377
and #2378 (slightly more advanced than
#2377), are both very instructive though
not well fixed. Both appear slightly older
than #2336 in that the head fold has grown
further over the yolk, and in each the separation of nasopharyngeal duct above from
stomodeum and pharynx below is complete. Figures 16, 17, and 18 show that the
nasopharyngeal duct now has grown further posteriorly slightly beyond the infundibulum and adenohypophyseal area, but
ends blindly against the dorsal pharynx.
Even at low magnification it is clear that
the dorsal wall of the nasopharyngeal duct
is thickened forming the adenohypophyseal placode as it passes the infundibulum.
These relationships are clearer at higher
magnification (Fig. 17). At this stage the
adenohypophyseal placode is longer in
median section than it was earlier. It is more
clearly differentiated posteriorly from the
rest of the nasohypophyseal ductal epithelium. In the anterior direction the transition from proliferated adenohypophyseal
epithelium to "normal" epithelium is more
gradual. There is yet no separation of the
adenohypophyseal cells as a layer parallel
to the ductal epithelium as there is in still
older embryos. In Figure 18, however, the
adenohypophyseal placode appears to be
developing proliferative outgrowths.
Older embryos with a long head
fold over the yolk
Most of the specimens in the Dean-Conel
collection of Eptatretus embryos are rela-
647
tively well advanced in development. Their
head development is fairly well represented in Kupffer's Figure 1. It should be
noted, however, that this figure omits representing the adenohypophysis. I have
found by this stage that the adenohypophysis is a well-defined and clearly delimited
structure in all embryos, located between
the postero-ventral surface of the infundibulum and the dorsal surface of the nasopharyngeal canal (Figs. 19 and 20). The
canal itself at this stage continues posteriorly and opens to the pharynx through
a newly established communicating aperture (see frontal section, Fig. 22). In earlier
embryos of this stage the openings of the
olfactory organ and stomodeum remain
sealed (Kupffer's Fig. 1, Figs. 19, 23, and
26d). However, at later stages, eventually
perforations form among the tentacles that
come to surround the two external openings (nasal and buccal).
At first the adenohypophysis forms a follicular or acinar mass, some of the acini
having lumens that remain open to the
nasopharyngeal duct (Fig. 20). In later
stages the solid mass of adenohypophysis
is marked off from the reconstituted epithelium of the nasopharyngeal duct, and
the boundary between the two tissues is
indistinct only laterally (Fig. 21). In still
later embryos (Figs. 23 and 24) the acinar
adenohypophysis is separated distinctly
from the adjacent thin epithelium of the
nasopharyngeal duct that is adjacent to it.
This is the condition in the late embryo of
Myxine described by Fernholm (1968). The
further cytodifferentiation of the adenohypophysis and adjacent neurohypophysis
was not followed because this material is
of it is the stomodeal part of the pharynx. The more posterior part of the pharynx is labeled P. There is no
longer any communication (as in Fig. 13) between the nasopharyngeal and pharyngeal (stomodeal) cavities.
For a simplified diagrammatic interpretation of this photograph, see Figure 26d.
Fie. 17. Higher magnification of an adjacent sagittal section (XI40) of embryo #2377. The nasopharyngeal
cavity .Vf can be traced posteriorly to its blind ending above the pharynx P. The dorsal wall of this cavity,
where it is applied to the infundibulum (third ventricle, III, of the infundibulum is labeled) is thickened as
the adenohypophyseal placode. The slit-like stomodeum S is labeled.
FIG. 18. Sagittal section of slightly older embryo #2378 (X38). Anterior to the left. Arrow, as above, points
to the attachment of the head fold to the blastoderm. Continuity from the stomodeum 5 to the gill pouch
region of the pharynx P is seen. The nasopharyngeal space S'P is further differentiated. Anteriorly the folds
of the olfactory organ are forming. The dorsal wall of the nasopharyngeal duct, as it passes the infundibulum
above it is thickened and thrown into folds (the adenohypophyseal placode).
648
AUBREY GORBMAN
INF
np
NP
<£
19
.20
Fie;. 19. Cross section (X27), embryo #2343 through the eye (£), midportion of the nasopharyngeal duct
(n/j) and hindbrain (above np). The solid mass of tissue below the nj> is the septum between the nasopharyngeal
cavity anterior to this level and the stomodeum posterior to it. To orient this illustration in its antero-posterior
position, see Figure 1.
FIG. 20. Higher magnification (X98) of the nasopharyngeal duct (XP) region of Figure 19. The posterior
tip of the infundibulum, IXF (=neurohypophysis) is visible as well as the differentiated adenohypophysis AD.
The adenohypophysis consists of acini that are still connected to the epithelium of the dorsal wall of the
nasohypophyseal duct.
FIG. 21. Embryo #2357 cross section (X98) of the adenohypophyseal level of the nasopharyngeal duct XP.
Here the acini of the adenohypophysis are connected to the duct epithelium only laterally. Medially a thin
new dorsal epithelium of the duct has formed. Other labels: ///, third ventricle in infundibulum (=neurohypophysis): P, pharynx (stomodeal portion).
Fu.. 22. Embryo #2270. Horizontal section (X27) to show the full length of the infundibulum (III) from the
diencephalon to its posterior neurohypophyseal extremity, at which the adenohypophysis (arrow) is applied.
At this dorsal level the adenohypophysis is small. This section also shows the posterior communication of the
nasohypophyseal duct, njj, with the pharynx, [/. More ventrally the two seemingly separate parts of the pharynx
are continuous. To orient this photograph in its dorso-ventral position, see Figure 1.
HAGFISH PITUITARY DEVELOPMENT
649
25
FIGS. 23 and 24. Embryo #2356. Low magnification (X29) and higher magnification (XI06) of cross sections
of a later embryo at the adenohypophyseal (AD) level. At this level the hindbrain is above the infundibulum
and adenohypophysis and the nasopharyngeal duct (np) and stomodeal part of the pharynx (S) are below
them. The adenohypophysis is almost completely free of the nasopharyngeal duct epithelium. The label III
is in the lumen of the neurohypophysis. To orient Figure 23 in the antero-posterior position, see Figure 1.
FIG. 25. Embryo #2392 (XI06). One of the latest embryos studied. Skeletal and other structures mark this
embryo as much more advanced in development than the preceding one (Figs. 23 and 24). The horny teeth
are forming in the stomodeal pharynx (s) below the broad nasopharyngeal canal (NP)- The adenohypophysis
(AD) is completely separate from the epithelium below it. There is a connective tissue septum between the
adenohypophysis and the neurohypophysis (III) above it.
not favorable for such study and special
approaches are required for evaluating the
pertinent questions of differentiation of
hormone secretory properties.
The development of the Eptatretus neurohypophysis is relatively straightforward,
although it has certain unusual aspects.
Almost the entire infundibulum is destined
to become neurohypophysis. The infundibulum itself develops precociously, and
for a while is one of the largest structural
components of the brain. However, in older
embryos differential growth of the rest of
the brain reduces the relative size of the
infundibulum (e.g., compare Figs. 16 and
24). In the oldest embryos the infundibulum-neurohypophysis is the relatively small
flattened structure (Figs. 24 and 25) that
it is finally in the fully developed stage.
DISCUSSION
Although the precise number of hagfish
embryos studied by Kupffer (1899) is
unknown, if we mayjudge from statements
650
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7 6 54
l O O O O O O O O O
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HAGFISH PITUITARY DEVELOPMENT
made by Kupffer himself, and by Conel
(1929), the number was small. Furthermore, Kupffer's (1899) descriptions of the
difficulty of freeing embryos from the shell
prior to embedding and sectioning and his
references to the "maceration" of many
embryos during their preparation for sectioning lead one to suspect the possibility
of artifactual features in his sectioned
material. Conel (1929) refers to the fact
that Bashford Dean experienced so much
difficulty in preparing sectioned hagfish
embryo material that he eventually ceased
his efforts toward this end. Although Dean
collected almost all of the known embryos
of Eptatretus, his only descriptions of hagfish development were limited to study of
whole mounts. Thirty years after collecting about 150 embryos he was able to give
Conel about 30 embryo specimens remaining in his collection, still preserved in alcohol (Conel, 1929). On the other hand, we
may feel fortunate that as many as 21
embryos are today sufficiently intact to permit study of early development of the pituitary gland. Even in this material we must
remain alert to the possibility or probability that certain features that can be
described, or even photographed, are not
representative of the normal or natural
state of development. The strong distortion and flattening of the more dorsal
structures in the sectioned embryos must
651
be considered an artifact of this type. There
is a clear need for freshly and properly
fixed material.
Since Kupffer's 1899 article has
remained for more than 80 yr virtually the
only authoritative source of information,
another unfortunate matter must be raised.
The only recording of Kupffer's observations was in the form of five stylized drawings that are highly interpretive and poor
in detail (Fig. 1). Although photomicrography was unknown in 1898, some remarkably fine drawings of microscopic structure
were made at this time, and it is regrettable
that hagfish embryology was not the subject of such artistic effort.
Even so, careful examination of Kupffer's drawings of the early head fold embryo
(Kupffer's Fig. 3 in Fig. 1) clearly shows an
unlabeled triangular adenohypophyseal
thickening in the archenteric roof (labeled
en) just posterior and ventral to the infundibular outpocketing in the floor of the
brain. It is interesting that although he
includes the adenohypophysis in his drawing of the youngest embryo, he fails to do
so in the older embryos in which it is more
obvious!
In his 1899 paper and in lafer publications Kupffer displays an interesting inconsistency in reasoning that must be dealt
with at this point. Referring to the late
embryo illustrated in his Figure 1 (Ku 1 in
FIG. 26. a, b, and c are semi-diagrammatic sagittal reconstructions based on study of serial cross-sections; d
is a similar approximation based on the sagittal section illustrated photographically in Figure 16. All are
drawn to the same approximate size, but their different approximate magnifications are indicated next to
each drawing. Superficial ectoderm is represented as solid black. Widely stippled areas are brain tissue. The
notochord is marked with vertical lines. Endoderm and gut structures are stippled with small dots, but the
adenohypophyseal region is densely stippled. Yolk is represented by circles. Mesodermal areas, especially in
c and d are not shaded, a is based on embryo #2375; b is based on embryo #2331; c is based on embryo
#2336; d is based on embryo #2377. The numbers and long vertical lines indicate figures that illustrate these
levels in corresponding photographic figures elsewhere in this paper. Particularly to be noted is the progression
of endodermal changes. In a the endoderm is a single layer over the yolk, with only the notochord separating
from it posteriorly. In b a horizontal split and anterior growth of the most dorsal endoderm forms an
archenteric cavity under the forebrain, anterior to the adenohypophyseal rudiment. In c the horizontal split
has progressed in a posterior direction forming a (future) nasopharyngeal space below the adenohypophysis,
a narrow stomodeal space below and posterior to the nasopharyngeal space, leading to the pharynx (ph).
Proliferation of endoderm, and investment of endoderm by mesoderm now has greatly thickened some parts
of these foregut structures. In d the shelf of tissue first seen in c below the adenohypophysis has separated
the nasopharyngeal space (np) from the slit-like stomodeum (s). The nasopharyngeal space ends blindly both
anteriorly and posteriorly. The posterior end of the nasopharyngeal space (canal) beyond the adenohypophysis
will open to the pharynx (ph) which now is below it (see Fig 22 and Ku 1 in Fig. 1). Other labels: in,
infundibulum; hb, hindbrain.
652
AUBREY GORBMAN
Fig. 1 reproduced here) he points out that
the external openings of both the nasopharyngeal duct and the stomodeum are
sealed off by membranes (each labeled rh').
He continues the analysis as follows (my
translation of the German).
From these facts several questions
stand out. First of all is the question
whether the epithelial layers which close
off both canals from the exterior are primary structures or whether they are secondary closures of earlier openings. In
the first case the stomodeal closure plate
would be formed from pharyngeal epithelium and that of the nasohypophyseal
canal would be of the same type. Then
the epithelium of both canals would be
endodermal and this would lead to the
paradoxical conclusion that the epithelium of the olfactory organ also is exclusively endodermal. It would be much
simpler to assume that both canals have
closed secondarily. [My underlining]
as proposed by Kupffer, in which there is
an initial closure between two spaces (Kupffer's Fig. 3) followed by an opening (Kupffer's Fig. 4), followed by a re-closure (Kupffer's Figs. 6 and 1), and then a final
reopening.
The evidence for an archenteric origin
of the hagfish adenohypophysis presented
here is readily verifiable and apparently not
subject to interpretation in terms other
than the obvious ones. These observations
are summarized in Figure 26. If we accept
this evidence, then a number of basic questions arise; at this time there are no clear
answers to these. First, how can we explain
so fundamental a difference from the general vertebrate pattern in origin of the hagfish adenohypophysis and other head
structures and a difference from its closest
relative, the lamprey? For all other vertebrates the adenohypophysis forms from an
ingrowth of an ectodermal anlage from the
exterior surface of the embryo. In the hagfish it appears to form from an internal
endodermal or archenteric surface. At the
time of its formation the hagfish adenohypophysis is in a "typical" anatomical
position, next to the infundibular outpouching of the brain. Thus, the differences in this development between the
hagfish and all other vertebrates precede
the actual appearance of the adenohypophyseal anlage. In all other vertebrates,
including the lamprey, the tissue layer in
immediate contact with the forebrain and
infundibular region is superficial ectoderm. In the hagfish this part of the brain
is in direct contact with the archenteric
roof, far from any contact with ectoderm.
So basic a difference in distribution of
embryonic tissue layers argues for a long
established divergence from the petromyzontids and can be taken as further evidence of a diphyletic evolutionary origin
of the two cyclostome types.
Kupffer's "paradox" applies as well to
the adenohypophysis as to the olfactory
organ, since it occupies a position well posterior to the olfactory organ and is continuous with it through the nasopharyngeal
canal. It is important to emphasize that in
none of the head fold embryos I examined
have I seen an opening between the ectodermal subcephalic pocket and the
"endoderm" lined foregut extension from
which the stomodeum and nasopharyngeal
canal form. Thus, it is quite possible that
the apparent ecto-endodermal continuity
in the embryo illustrated in Kupffer's Figure 4 (in Fig. 1) was created artificially in
dissecting and manipulating it at the time
of fixation and/or subsequently. Second,
if the adenohypophysis is already identifiable as an archenteric structure adjacent
to the infundibulum (Kupffer's Fig. 3 in
Fig. 1) before the (presumed) opening to
the subcephalic area of the youngest
Since the adenohypophysis can form
embryo, then it already is endodermal at from juxta-infundibular tissues of disthis early stage. Even if later an opening tinctly different origins, is this evidence that
were formed, as claimed, rostral to the ade- the infundibulum is able to "induce" adenohypophysis, this would not alter its nohypophysis from any adjacent epithelial
already established archenteric derivation. tissue? Such an inductive propert\ was proThird, it seems difficult to imagine a com- posed by A. B. Burch (1945), R. F. Blount
plex developmental sequence in this region, (1945),R. M. Eakin( 1950), and others who
HAGFISH PITUITARY DEVELOPMENT
found that experimental interruption of the
contact between developing adenohypophysis and infundibulum led to various
degrees of deficiency of differentiation of
the adenohypophysis of amphibian
embryos.
The derivation of the hagfish adenohypophysis from the mid-dorsal archenteron
clearly makes it non-ectodermal, but does
it necessarily require that it be considered
endodermal? We have characterized the
germ layer origin of the hagfish adenohypophysis as endodermal only for convenience. The very similar cellular origin
of the notochord is quite obvious and it
required careful examination to differentiate notochordal from adenohypophyseal
development. Notochordal cells were
clearly different histologically, and markedly larger than the rest of the archenteric
roof; furthermore, they already have separated from the archenteric roof in the
earliest embryo studied (#2375). On the
other hand, the adenohypophyseal cells are
not readily distinguishable from the
archenteric epithelium; they remain part
of the archenteric roof until the embryo is
well advanced into head fold formation.
When adenohypophysis separates from the
archenteric epithelium it is by polyinvagination and formation of an acinar layer
parallel to the archenteric roof. Thus, adenohypophysis and notochord are readily
distinguished from each other even in the
earliest stages. However, the similarities in
their origin (rather than their differences)
lead to the question of how to designate
their respective germ layer origins. There
has always been a terminological difficulty
in referring to the germ layer origin of the
notochord as endoderm, or chordamesoderm, or even other alternatives. The same
difficulties apply to designation of the germ
layer origin of the hagfish adenohypophysis. The convenience of reference to this
origin as endodermal has been taken here,
with no commitment in respect to the more
general problems of the germ layer concept.
653
Fic. 27. Part of a figure from A. Gorbman and H.
A. Bern Textbook of Comparative Endocrinology, J o h n
Wiley and Sons, New York, 1962. This figure illustrates a hypothetical sequence of evolutionary events
(c to e) in which the mucoprotein secreting nasal sac
epithelium adjacent to the infundibulum is converted
into a glycoprotein hormone secreting adenohypophysis. This is remarkably similar to the sequence of
changes in the hagfish embryo when a part of the
nasopharyngeal ductal epithelium adjacent to the
infundibulum forms acinar proliferations that leave
the epithelium as a layer and form a neurohypophysisadjacent adenohypophysis.
ence from all other vertebrates (note especially Figs. 20, 21, and 24). This recalls a
purely speculative proposal made in 1962
(Gorbman and Bern, 1962) of a possible
pattern of evolution of the vertebrate adenohypophysis from the brain-adjacent
mucous membrane (Fig. 27). This proposal
was made in part because both mucoproteins and adenohypophyseal hormones are
glycoprotein in nature. It is easy to imagine
an evolutionary scenario in which glandular acinar tissue derived from the mucous
membrane of the nasal epithelium began
to produce a hormone-like substance. Hormone production by tissues derived from
the mucous membrane elsewhere along the
gut surface (stomach, intestine, pancreas,
liver) is consistent with this. If this were
indeed the case, then the further adaptive
advantage to the system of linking it to
nervous regulation by the nearby brain
The differentiation of the hagfish ade- could lead to the development of a closer
nohypophysis from the nasopharyngeal brain-gut epithelium relationship such as
epithelium by multifollicular ingrowths that is seen in the fully differentiated pituitary
fuse in a laminar pattern is a basic differ- gland. It is of interest that Figure 18 or
654
AUBREY GORBMAN
Figure 20 could have been used to illustrate the 1962 speculation almost as well
as the purely conceptual drawings.
ACKNOWLEDGMENTS
This study was aided by grant #PCM
8141393 from the National Science Foundation. Special thanks must be given to the
Anatomy Department, Harvard University for making available to me the slides
used in this study and for provision of laboratory facilities for their initial study. Particularly I must thank David Gunner, curator of the Warren Anatomical Museum,
for his time and trouble in rediscovering
the Dean-Conel hagfish embryo slides in
the museum collection and Richard Cloney
for advice during preparation of the manuscript and drawings.
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