/ . Embryol. exp. Morph. Vol. 63, pp. 1-16, 1981
Printed in Great Britain © Company of Biologists Limited 1981
Amphibian pronephric duct morphogenesis:
segregation, cell rearrangement and directed
migration of the Ambystoma duct rudiment
By T. J. POOLE 1 AND M. S. STEINBERG 2
From the Department of Biology, Princeton University, U.S.A.
SUMMARY
The axolotl pronephric duct rudiment is readily accessible to both SEM observation and
surgical manipulation. The rudiment segregates from the dorsal part of the lateral mesoderm
and then extends caudally along the ventrolateral border of the segmenting somites, eventually
contacting the cloacal wall. The marked thinning of the rudiment which accompanies this
migration is paralleled by a corresponding reduction in cell number across the duct's diameter
and by caudad translocation and elongation of vital dye marks applied to the duct mesoderm.
Duct extension thus involves appreciable cell rearrangement. The morphology of duct mesoderm and its substratum (somite and lateral mesoderm) suggests that active locomotion of
cells near its tip marshals the duct's caudad elongation. Filopodia and small focal are$s of
intercellular contact may mediate the adhesions between duct cells which must be broken and
reformed as the cells rearrange.
INTRODUCTION
The amphibian pronephric duct during its early morphogenetic phase provides an example of directed tissue migration that is especially well suited, for
experimental analysis. Scanning electron microscopy of normal embryos fixed at
various stages, vital dye marking and simple surgical deletions or blockages have
shown the events of duct formation to be very similar in Ambystoma maculatum
(Poole & Steinberg, 1977) and, in the present work, in the axolotl A. mexicanum.
In these embryos the pronephric duct rudiment segregates from the mesoderm
as an ovoid, solid tissue mass five to six somites long and then by cell rearrangement extends to more than twice its original length along the ventrolateral
margin of the somites to join with the cloaca. Thus, the salamander duct forms
by the caudal extension of a solid stream of cells along a predetermined and
easily identifiable path readily accessible to scanning electron microscopic (SjEM)
observation and surgical manipulation.
The mode and mechanisms of outgrowth of the amphibian pronephric duct
have been subjects of some controversy since the turn of the century (reviewed
1
Author's present address: Department of Surgery, Harvard Medical School, Children's
Hospital Medical Center, 300 Longwood Avenue, Boston, MA 02115, U.S.A.
2
Author's address (for reprints): Department of Biology, Princeton University, Princeton,
New Jersey 08544, U.S.A.
2
T. J. POOLE AND M. S. STEINBERG
by Burns, 1955; Fox, 1963; Poole & Steinberg, 1977). According to one view, the
duct forms by progressive recruitment of cells in situ, while another view holds
that it forms by caudal extension of an anterior rudiment. The latter view, which
has come to be generally accepted (exceptions: Shin-Ike, 1955; Fox & Hamilton,
1964; see Poole & Steinberg, 1977 for species differences), has been supported by
experiments utilizing localized vital dye staining, surgical deletion, blockage or
reorientation of the duct tip, and explantation. Since elongating Ambystoma
pronephric duct rudiments do not have a higher mitotic rate than surrounding
tissues (Overton, 1959), their extension seems to be due to cell migration. The
migratory propensity of duct rudiment fragments has previously been demonstrated by outgrowth in plasma clots (Overton, 1959), by ablation of a major
part of the duct rudiment (Nieuwkoop, 1947) and by transplantation of young
duct rudiments to virgin 'duct paths' of older hosts (Gipouloux & Cambar,
1961; Cambar & Gipouloux, 1970).
What are the nature and specificity of the environmental factors determining
the duct's course? Holtfreter and others addressed this question by confronting
the advancing duct primordium with surgically produced foreign tissue terrains.
Holtfreter (1944) found that the (urodele) duct could be deviated, by a wound,
ventrally onto the surface of the lateral mesoderm. From this position it was able
in several exceptional cases to return to its normal path and complete its migration. In the same year, Tung & Ku (1944), working with anuran embryos, found
that the duct rudiment resisted extension at right angles to this path. Bijtel
(1948) observed a deviation of the duct from its normal path to a laterally
implanted secondary cloaca.
Thus, although there is much suggestive evidence, the manner in which the
cells of the duct rudiment migrate and the environmental factors that guide them
are not yet understood. Because yolkiness of amphibian embryos during duct
migration makes paraffin sectioning at this stage difficult, the results of surgical
operations have usually been assessed on embryos fixed at later stages, after
much of the yolk has been digested. Thus the consequences of microsurgical
procedures were first observed only after the duct rudiment had completed
its caudal migration, when secondary influences might have deviated the duct
from its originally chosen path. We therefore chose to make our observations by
scanning electron microscopy, which not only permits observations to be made
at any time but also reveals the appearance of individual cells and cellular
processes during elongation of the duct rudiment.
MATERIALS AND METHODS
Axolotl {Ambystoma mexicanum) embryos were obtained from spawnings of
our colony and that of Indiana University. Embryos were staged according to
Schreckenberg & Jacobson, 1975 (S & J) and manually demembranated with fine
watchmaker's forceps in full-strength Steinberg's solution (see Discussion with
Reviewers in Poole & Steinberg, 1977).
Amphibian pronephric duct morphogenesis
1 mm
(c)
(d)
"
Fig. 1. Scanning electron micrographs of Ambystoma mexicanum embryos fixed before peeling of ectoderm from the right side. Arrows indicate pronephric duct's caudal
tip. Duct rudiment's extension is accompanied by the segmentation of additional
somites and straightening of the embryonic axis, {a) Stage 22, (b) Stage 24, (c) Stage
28, id) Stage 32.
Experimental manipulations were carried out under aseptic conditions irt fullstrength Steinberg's solution using standard microsurgical procedures (Jacobson,
1967). Embryos were vitally stained with Nile blue sulphate-dyed agar slivers by
a procedure similar to that described by Keller (1975).
Embryos were generally fixed at room temperature with modified Karnovsky's
(1965) fixative (2-5% glutaraldehyde, 2-5 % paraformaldehyde and 5 mM calcium
chloride in 0-1 M-sodium cacodylate buffer, pH 7-4). After | - 1 h fixation,
the ectoderm was manually peeled off with fine watchmaker's forcep$ and
tungsten needles under a dissecting microscope. Peeled embryos were transferred
to fresh fixative and usually left at 4 °C overnight. Samples were then finsed
T. J. POOLE AND M. S. STEINBERG
(a)
(6)
Fig. 2. Camera-lucida tracings of vitally stained embryos, (a) Distal segment of
pronephric duct stained with Nile blue sulfate at stage 22 has moved caudad and
elongated markedly by stage 32. (b) Proximal segment of duct stained at stage 26 has
moved caudad and elongated to a lesser extent by stage 32. A stained section of the
duct's path is obscured as the duct passes over it.
in several changes of 0-15 M sodium cacodylate buffer and postfixed in sodium
cacodylate-buffered 1 % osmium tetroxide for 1-3 h at 4 °C.
Embryos for scanning electron microscopy were dehydrated in ethanol and
critical-point dried from liquid CO2. Dried embryos were affixed to stubs with a
low-resistance contact cement (Fullam) or with silver paint and sputter coated
with gold-palladium (60:40). Specimens were examined at 15-25 kV in a JEOL
JSM-35 scanning electron microscope. For transmission electron microscopy,
dehydrated samples were embedded in Epon 812. Transverse sections, 1-2 fim
thick, were cut with glass knives, mounted on slides and stained with methylene
blue and azure II. Ultrathin sections (50-70 nm) were then cut from selected
regions, mounted on grids, double stained with 2 % uranyl acetate and lead
citrate and examined with a JEOL 100C electron microscope operated at 80 kV.
The dimensions of embryos were measured directly from SEM negatives with
a Zeiss MOP-3 image analyzer.
RESULTS
Segregation and elongation of the duct rudiment
The axolotl pronephric duct segregates between the levels of trunk somites 2
and 7 as a solid, ovoid or tear-shaped body of cells at S. & J. stages 22 and 23
Amphibian pronephric duct morphogenesis
Utf
Fig. 3. Two axolotl embryos were split at stage 22 by a dorsal incision and peeled after
fixation at stage 32. (a) A deep cut has blocked duct migration, (b) A more shallow
cut has permitted some extension of the duct rudiment below the wound.
(Fig. 1 a). With development it extends along the ventrolateral border of the
somites while narrowing markedly (Figs. 1 b-d). The level of origin and extent of
migration have been confirmed by vital dye marking. A mark placed to include
the caudal limit of the rudiment and adjacent somite mesoderm at stage 23
(Fig. 2 a) shows a pronounced translocation and spreading of stained duct cells
over 24 h at 24 °C. Marks made at older stages when rearrangement is already in
progress and placed further caudally and cranially show reduced spreading and
caudal translocation (Fig. 2 b). Marked cells behind the duct tip are not incorporated into the advancing duct. These results are consistent with expectations
based upon the morphology observed in low-power SEM micrographs (Fig. 1)
and suggest that duct rudiment elements tend to remain near their original
neighbours during the rearrangement accompanying extension. Finally, the level
of origin and propensity for extension of the duct rudiment are clearly shown by
surgical intervention. Most simply, a deep transection of the axial tissues caudal
to the duct rudiment's tip (i.e. posterior to trunk somite 7, see Fig. 2a), in, all 18
cases halted duct progression at the level of the incision (Fig. 3 a). This confirms
that the rudiment extends over 5 somite widths at stage 22. Following a; more
shallow incision, the duct rudiment has, in eight cases, detoured ventrolaterally
a short distance across lateral mesoderm and returned to its normal path Caudal
to the incision (Fig. 3 b).
Duct rudiment elongation occurs by cell rearrangement
How do the pronephric duct shape changes come about? The cellular basis
of the reduction in the duct's diameter can be appreciated by comparing SEM
micrographs at a given level (beneath trunk somite 6) at various stages of development. In the sequence shown in Fig. 4, the duct narrows from about eight cell
widths at stage 23 to two cell widths at stage 32. The duct cells themselves Change
T. J. POOLE AND M. S. STEINBERG
•
i''jfcfjMZT^s^y'
Amphibian pronephric duct morphogenesis
i
7
little if at all in size or shape. The marked thinning of the rudiment, accompanied by a decrease in the number of cells across its diameter, is also seen in
cross-sectional views. Figure 5 shows two views produced by fracturing transversely through trunk somite 6 of critical-point-dried embryos. Thinning of the
rudiment reduces the number of cells spanning the duct's width from six to eight
at stage 24 (Fig. 5 a) to two to three at stage 28 (Fig. 56). The same reduction
can be seen in 2 /tm Epon sections. All of these observations indicate that cell
rearrangements, and not proliferation or cell-shape changes, are primarily
responsible for Ambystoma pronephric duct extension.
In Fig. 6 the developmental changes in several parameters of duct outgrowth
are summarized graphically. This illustrates several significant points. Despite
the increase in total embryo length (straight line head to tail), the length of the
duct path surprisingly remains nearly constant. Inspection of the tracings in Fig.
7 reveals that the embryo's elongation between stages 24 and 32 results from the
gradual straightening of the embryonic axis and the lifting and extension of the
head. The boundary between presumptive somite and lateral mesoderm ^vhich
defines the duct's path is quite curved at stage 22 and merely straightens out as
the embryo 'elongates'. Duct extension is closely correlated with somite segmentation; during elongation, the caudal tip of the duct rudiment maintains a
position two somite widths behind the most caudally developing somite fissure.
Finally, both the increase in duct length and the decrease in duct diameter are
linear with time and have similar slopes (Fig. 6).
The substratum for duct migration
As seen in the transverse fractures (Fig. 5), the duct rudiment is botfdered
medially by somite mesoderm, ventrally by lateral mesoderm and (lorsolaterally by ectoderm. As reported previously (Poole & Steinberg, 1977), the cells
of exposed Ambystoma duct rudiments (mesoderm viewed en face) are attached
via lobopodia, lamellipodia and many fine filopodia both to each other at the
surface of the rudiment and to adjacent somite and lateral mesoderm cells at its
edge (see Fig. 8 and Fig. 9). On the surface of the somites with which the duct
makes attachments are localized webs of 50-100 nm fibres (apparently extracellular collagen fibres) as well as numerous fine, interdigitating cell extensions.
The inner ectodermal surface is seen in the SEM to be partially covered by $. basal
Fig. 4. The duct rudiment, seen here below trunk somite 6, thins markedly by £ell
rearrangement as it elongates, (a) Stage 22, six to eight cells wide; (b) Stage 26, five
to six cells wide; (c) Stage 28, about four cells wide; (d) Stage 32, two to three cells
wide.
Fig. 5. Decrease in cell number in transverse sections of the pronephric duct
(arrows) is apparent in critical-point-dried embryos fractured through the level of
trunk somite 6. (a) A. maculatum (essentially like A. mexicanum), Stage 24; (b) A.
mexicanum, Stage 28.
T. J. POOLE AND M. S. STEINBERG
.
10
15
No. of somites segmented
'••••... DDX10
20
Fig. 6. Dimensional changes during axolotl pronephric duct extension. Embryo
length measurements (EL; • ) are recorded as the straight-line distance from tip of
head to tip of tail. Total path length (PL; O) and duct length (DL; • ) measurements are curvilinear. Duct diameter (DD; • ) is the linear distance from somite
to lateral mesoderm across the duct at the level of trunk somite 6 as seen in Fig. 4.
Dimensions taken from scanning electron micrographs.
lamina which obscures cell boundaries. Some 50-100 nm fibres are also seen, but
there are no apparent features which might guide the duct's migration. The duct
and adjacent mesoderm adhere weakly if at all to the inner surface of the ectoderm. This is evident when the ectoderm is removed. After fixation, it can
usually be easily peeled from the mesoderm with little evidence of damage to the
latter. It can also be peeled from living embryos with little sign of firm adhesions
to, distortion of, or damage to duct or adjacent mesoderm. Finally, chance
fractures of dried embryos in which the duct mesoderm remained next to the
ectoderm showed few and tenuous associations of duct cells with the inner
Amphibian pronephric duct morphogenesis
Fig. 7. Tracings of scanning electron micrographs of partially peeled axolotl embryos,
showing the caudad progression of the pronephric duct, (a) Stage 22, (6) Stage 23,
(c) Stage 32.
Fig. 8. Higher magnification view of posterior portion of stage-27 axolotl pronepHric
duct rudiment (PD), somites (S) and lateral mesoderm (L).
10
.•«
T. J. POOLE AND M. S. STEINBERG
•
(c)
Fig. 9. Region of tip of stage-31 axolotl pronephric duct rudiment, (a) Posterior third
of duct rudiment, (b) Cells near the tip overlap in the manner offish scales, (c) Enlargement of the area indicated in Fig. 9 b. Overlapping cells extend filopodia which
contact underlying cells within the duct rudiment, (d) Meshwork of fibers approximately 0-2 (ivci in diameter, seen as occasional small patches on cell surfaces at
this stage.
surface of the ectoderm. It thus appears that the ventral edge of the somites and
the subjacent lateral mesoderm comprise the substratum for the duct's migration.
The morphology of duct cells and their contacts
Cells near the duct's tip show some anteroposterior elongation (Fig. 8) and
tend to overlap in the manner offish scales (see also Fig. 9 b). Back from the tip
(as in Fig. 4), the duct's cells are in a more 'relaxed' configuration. Cell-to-cell
adhesion near the duct's tip occurs byflat,overlapping cell processes from which
arise numerous adherent filopodia roughly 200 nm in diameter and averaging
about 10 fim in length (Fig. 9c, Fig. \0b-d). The large lobopodial processes
Amphibian pronephric duct morphogenesis
11
\c)
Fig. 10. Cell contacts visible in transverse fractures behind trunk somite 7 of a stage32 axolotl pronephric duct rudiment, (a) Low magnification overview showing neutfal
tube, notochord, somites, epidermis, endoderm, pronephric duct and lateral mesoderm. (b) At higher magnification the ectoderm is seen to be bilaminar (bracket) and
the cells of the pronephric duct rudiment (arrows) are seen to be in the process of
adopting a radial arrangement. A fibrous network resembling collagen covers the
exposed intersomitic surface (S). (c) The wedge shape of duct cells at this stage is
apparent here. The cell depicted has a broad base at the duct's outer surface
(arrows) and an apex (asterisk) centrally where the duct's lumen will form. Several
blunt processes (small arrows) extend between cells, (d) An area near the center of the
duct rudiment (triangle). Filopodia extend along the cell surface (small arrows). The
adjoining cells are also connected by shorter, blunt processes (large arrow). 1
extending out toward somite and lateral mesoderm are much rarer back from
the tip, especially at later stages of migration. Vital dye marking (Fig. 2) and the
thinning visible in Figs. 4c-dshow that these cells are still rearranging. Whether
they are all engaged in active locomotion like a stream of Fundulus de$p cells
(Trinkaus, 1973) or whether the force causing their rearrangement arises from
the locomotory activity of cells at the leading edge remains to be determined.
T. J. POOLE AND M. S. STEINBERG
Nu
Fig. 11. Transmission electron micrographs of sections through the pronephric duct
rudiment of a stage-26 axolotl embryo, (a) An area of contact between two duct
rudiment cells several somites anterior to the duct's caudal tip. (b) Interdigitating
filopodia (arrows) of duct cells show close contacts with opposing cell surfaces,
suggesting that they mediate cell-cell adhesions, (c) Cells near the advancing tip of
the duct rudiment possess long, flattened lamellipodia and are separated by more
extracellular space. Yolk platelets (YO), lipid droplets (LD), nuclei (Nu) and mitochondria (Mi) are indicated.
Transmission electron microscope studies of cell shapes and junctions provide
a structural basis for the rearrangements and give clues to the type of locomotory
activity involved. Figure 11 shows electron micrographs of sections taken
through a stage-26 axolotl embryo several somite widths anterior to the caudal
tip of the duct primordium. Electron-dense yolk platelets (YO), lipid-filled
droplets or vesicles (LD), nuclei (Nu) and mitochondria (Mi) are visible. There
are large gaps between cells, close apposition of cell membranes occurring in
discrete areas (Fig. 11; several examples circled in Fig. 11 a). Frequently, close
apposition occurs where a process of one cell touches the body of another
(arrows in Fig. 11 b). Further caudally there is even more intercellular space and
duct cell surfaces show fewer complex processes. In addition, long lamellipodial
Amphibian pronephric duct morphogenesis
13
processes are seen at the ventromedial edge of the duct near its tip (Fig. 1 he).
Such structures may be important in caudal translocation of these cells durjng
duct extension. The morphology and contacts observed as well as the scarcity of
specialized junctions call to mind the observations of Nakatsuji (1975, 1976)
on the motile cells of urodele and anuran gastrulae, and those of Hogan a^id
Trinkaus (1977) and Trinkaus and Lentz (1967) on the migratory deep cells of
the Fundulus gastrula.
DISCUSSION
Scanning electron microscopic observations of the outer mesodermal surface
of normal and surgically modified embryos have provided new insights into the
mechanisms directing the caudad extension of the amphibian pronephric duct.
Even at low magnifications in the SEM, the Ambystoma pronephric rudiment
can be seen to segregate out as a solid mass from the dorsal portion of the hortiogeneous lateral plate mesoderm ventral to trunk somites 2 through 7, as previously inferred by O'Connor (1938) and Holtfreter (1944) from studies utilising
vital staining and transplantation. Our own staining and surgical procedures confirm their results. The Ambystoma duct clearly forms by the caudal extension of
an anterior rudiment.
Although a variety of cellular mechanisms (such as cell shape change, cell
reorientation, individual cell movement, cell proliferation) might in principle
cause the observed transformation from a short, thick cord to a long thin One,
our observations have shown that this solid mesodermal cylinder of nearconstant volume extends itself by cell rearrangement. The rudiment's marked
thinning during elongation is accompanied by the redistribution of a nearly Constant number of constituent cells most of which remain in 'relaxed', polygonal
shapes throughout the process. The force guiding this cellular rearrangement is
not made obvious by ultrastructural observations. Although the morphology
and distribution of cell processes, contacts and junctions suggest that the
caudal-most cells are actively pulling out the duct rudiment, it is difficult to
reconstruct with certainty the process of duct extension from the static images
obtainable with electron microscopy. We have been able to approach this
problem experimentally by surgical rearrangements of the salamander mesoderm. The results will be discussed in subsequent papers.
'
Cell rearrangement as a morphogenetic mechanism
Morphogenetic movements can be classified according to whether the cells
migrate as individuals or as part of a cell group. Translocations of individual
cells such as germ cells, neural crest cells and processes of neurons toward
specific destinations require guidance by specific environmental clues. $EM
observations by a number of authors have implicated cellular and extracellular
fibers in such guidance (Bancroft & Bellairs, 1976; Ebendal, 1976, 1977;
14
T. J. POOLE AND M. S. STEINBERG
Lofberg & Ahlfors, 1978; Tosney, 1978; Wylie, Heasman, Swan & Anderton
1979). The morphogenesis of cell groups, however, may be guided by other
control mechanisms mediated by cell interactions such as adhesion, contact
inhibition or changes in the shapes of firmly associated, individual cells (Phillips
Steinberg & Lipton, 1977). One of us has recently divided tissue movements into
two broad categories. When cells remain firmly affixed to their neighbours,
individual cell shape changes (such as apical constriction or cell elongation) can
summate to produce tensions which result in the expansion, contraction or
folding of the cell sheet, which behaves as a deformable solid. Tissues may also
flow in the manner of a viscous liquid, a process that has sometimes been overlooked. In such cases cells may retain their original shapes but move past one
another, changing cell neighbours (Phillips et al. 1977; Phillips & Steinberg,
1978). These movements of cells relative to one another have been termed 'cell
shear' (Jacobson & Gordon, 1976), 'cell slippage' (Phillips et al. 1977; Phillips
& Steinberg, 1978) and 'cell rearrangement' (Fristrom, 1976). Such fluid rearrangements of cells occur during pronephric duct rudiment extension. Cell
slippage might result either from active cell movements or from passive relaxation of tensions externally imposed on a tissue mass during morphogenesis.
Recent detailed studies of cell movements in embryos, made possible at least in
part by advances in SEM techniques (see review by Poole & Steinberg, 1977), have
made it increasingly clear that movement of cells in groups or streams is more
common than previously realized (Trinkaus, 1976). Unfortunately, the mechanisms and forces mediating and directing such morphogenetic movements in
vivo have remained obscure. Elsewhere we present evidence that the same
thermodynamic principles which govern adhesion-mediated cell sorting and
tissue spreading in vitro (reviewed by Steinberg, 1978 a, b) also operate within
embryos to direct cell migrations and stabilize anatomically 'correct' cell
associations (Poole & Steinberg, 1978, 1981).
We thank Edward Kennedy, Doris White, Pam Knab-Mclntyre and Dorothy Spero for
their technical assistance. This study was supported by research grants PCM76-84588 from
the National Science Foundation and CA136O5 from the National Cancer Institute, and by
PHS training grant CA9167 from the National Cancer Institute. The electron microscopy was
carried out in Department of Biology facilities supported by the Whitehall Foundation. From
a dissertation submitted by T. J.P. to the Department of Biology, Princeton University, in
partial fulfillment of the requirements for the Ph.D. degree.
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{Received 3 November 1980, revised 9 January 1981)