Amphibian pronephric duct morphogenesis: …dev.biologists.org/content/develop/63/1/1.full.pdfAmphibian pronephric duct morphogenesis: segregation, cell rearrangement and directed

/ . Embryol. exp. Morph. Vol. 63, pp. 1-16, 1981Printed in Great Britain © Company of Biologists Limited 1981

Amphibian pronephric duct morphogenesis:segregation, cell rearrangement and directedmigration of the Ambystoma duct rudiment

By T. J. POOLE1 AND M. S. STEINBERG2

From the Department of Biology, Princeton University, U.S.A.

SUMMARY

The axolotl pronephric duct rudiment is readily accessible to both SEM observation andsurgical manipulation. The rudiment segregates from the dorsal part of the lateral mesodermand then extends caudally along the ventrolateral border of the segmenting somites, eventuallycontacting the cloacal wall. The marked thinning of the rudiment which accompanies thismigration is paralleled by a corresponding reduction in cell number across the duct's diameterand 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 meso-derm and its substratum (somite and lateral mesoderm) suggests that active locomotion ofcells near its tip marshals the duct's caudad elongation. Filopodia and small focal are$s ofintercellular contact may mediate the adhesions between duct cells which must be broken andreformed as the cells rearrange.

INTRODUCTION

The amphibian pronephric duct during its early morphogenetic phase pro-vides an example of directed tissue migration that is especially well suited, forexperimental analysis. Scanning electron microscopy of normal embryos fixed atvarious stages, vital dye marking and simple surgical deletions or blockages haveshown 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 mesodermas an ovoid, solid tissue mass five to six somites long and then by cell rearrange-ment extends to more than twice its original length along the ventrolateralmargin of the somites to join with the cloaca. Thus, the salamander duct formsby the caudal extension of a solid stream of cells along a predetermined andeasily identifiable path readily accessible to scanning electron microscopic (SjEM)observation and surgical manipulation.

The mode and mechanisms of outgrowth of the amphibian pronephric ducthave been subjects of some controversy since the turn of the century (reviewed

1 Author's present address: Department of Surgery, Harvard Medical School, Children'sHospital 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, theduct forms by progressive recruitment of cells in situ, while another view holdsthat it forms by caudal extension of an anterior rudiment. The latter view, whichhas come to be generally accepted (exceptions: Shin-Ike, 1955; Fox & Hamilton,1964; see Poole & Steinberg, 1977 for species differences), has been supported byexperiments utilizing localized vital dye staining, surgical deletion, blockage orreorientation of the duct tip, and explantation. Since elongating Ambystomapronephric duct rudiments do not have a higher mitotic rate than surroundingtissues (Overton, 1959), their extension seems to be due to cell migration. Themigratory propensity of duct rudiment fragments has previously been demon-strated by outgrowth in plasma clots (Overton, 1959), by ablation of a majorpart of the duct rudiment (Nieuwkoop, 1947) and by transplantation of youngduct rudiments to virgin 'duct paths' of older hosts (Gipouloux & Cambar,1961; Cambar & Gipouloux, 1970).

What are the nature and specificity of the environmental factors determiningthe duct's course? Holtfreter and others addressed this question by confrontingthe 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 ablein several exceptional cases to return to its normal path and complete its migra-tion. In the same year, Tung & Ku (1944), working with anuran embryos, foundthat 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 laterallyimplanted secondary cloaca.

Thus, although there is much suggestive evidence, the manner in which thecells of the duct rudiment migrate and the environmental factors that guide themare not yet understood. Because yolkiness of amphibian embryos during ductmigration makes paraffin sectioning at this stage difficult, the results of surgicaloperations have usually been assessed on embryos fixed at later stages, aftermuch of the yolk has been digested. Thus the consequences of microsurgicalprocedures were first observed only after the duct rudiment had completedits caudal migration, when secondary influences might have deviated the ductfrom its originally chosen path. We therefore chose to make our observations byscanning electron microscopy, which not only permits observations to be madeat any time but also reveals the appearance of individual cells and cellularprocesses during elongation of the duct rudiment.

MATERIALS AND METHODS

Axolotl {Ambystoma mexicanum) embryos were obtained from spawnings ofour colony and that of Indiana University. Embryos were staged according toSchreckenberg & Jacobson, 1975 (S & J) and manually demembranated with finewatchmaker's forceps in full-strength Steinberg's solution (see Discussion withReviewers in Poole & Steinberg, 1977).

Amphibian pronephric duct morphogenesis

1 mm(c) (d) "

Fig. 1. Scanning electron micrographs of Ambystoma mexicanum embryos fixed be-fore peeling of ectoderm from the right side. Arrows indicate pronephric duct's caudaltip. Duct rudiment's extension is accompanied by the segmentation of additionalsomites and straightening of the embryonic axis, {a) Stage 22, (b) Stage 24, (c) Stage28, id) Stage 32.

Experimental manipulations were carried out under aseptic conditions irt full-strength Steinberg's solution using standard microsurgical procedures (Jacobson,1967). Embryos were vitally stained with Nile blue sulphate-dyed agar slivers bya 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 calciumchloride 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$ andtungsten needles under a dissecting microscope. Peeled embryos were transferredto 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 ofpronephric duct stained with Nile blue sulfate at stage 22 has moved caudad andelongated markedly by stage 32. (b) Proximal segment of duct stained at stage 26 hasmoved caudad and elongated to a lesser extent by stage 32. A stained section of theduct's path is obscured as the duct passes over it.

in several changes of 0-15 M sodium cacodylate buffer and postfixed in sodiumcacodylate-buffered 1 % osmium tetroxide for 1-3 h at 4 °C.

Embryos for scanning electron microscopy were dehydrated in ethanol andcritical-point dried from liquid CO2. Dried embryos were affixed to stubs with alow-resistance contact cement (Fullam) or with silver paint and sputter coatedwith gold-palladium (60:40). Specimens were examined at 15-25 kV in a JEOLJSM-35 scanning electron microscope. For transmission electron microscopy,dehydrated samples were embedded in Epon 812. Transverse sections, 1-2 fimthick, were cut with glass knives, mounted on slides and stained with methyleneblue and azure II. Ultrathin sections (50-70 nm) were then cut from selectedregions, mounted on grids, double stained with 2 % uranyl acetate and leadcitrate and examined with a JEOL 100C electron microscope operated at 80 kV.

The dimensions of embryos were measured directly from SEM negatives witha Zeiss MOP-3 image analyzer.

RESULTS

Segregation and elongation of the duct rudiment

The axolotl pronephric duct segregates between the levels of trunk somites 2and 7 as a solid, ovoid or tear-shaped body of cells at S. & J. stages 22 and 23


UtfFig. 3. Two axolotl embryos were split at stage 22 by a dorsal incision and peeled afterfixation at stage 32. (a) A deep cut has blocked duct migration, (b) A more shallowcut has permitted some extension of the duct rudiment below the wound.

(Fig. 1 a). With development it extends along the ventrolateral border of thesomites while narrowing markedly (Figs. 1 b-d). The level of origin and extent ofmigration have been confirmed by vital dye marking. A mark placed to includethe caudal limit of the rudiment and adjacent somite mesoderm at stage 23(Fig. 2 a) shows a pronounced translocation and spreading of stained duct cellsover 24 h at 24 °C. Marks made at older stages when rearrangement is already inprogress and placed further caudally and cranially show reduced spreading andcaudal translocation (Fig. 2 b). Marked cells behind the duct tip are not in-corporated into the advancing duct. These results are consistent with expectationsbased upon the morphology observed in low-power SEM micrographs (Fig. 1)and suggest that duct rudiment elements tend to remain near their originalneighbours during the rearrangement accompanying extension. Finally, the levelof origin and propensity for extension of the duct rudiment are clearly shown bysurgical intervention. Most simply, a deep transection of the axial tissues caudalto the duct rudiment's tip (i.e. posterior to trunk somite 7, see Fig. 2a), in, all 18cases halted duct progression at the level of the incision (Fig. 3 a). This confirmsthat the rudiment extends over 5 somite widths at stage 22. Following a; moreshallow incision, the duct rudiment has, in eight cases, detoured ventrolaterallya short distance across lateral mesoderm and returned to its normal path Caudalto the incision (Fig. 3 b).

Duct rudiment elongation occurs by cell rearrangement

How do the pronephric duct shape changes come about? The cellular basisof the reduction in the duct's diameter can be appreciated by comparing SEMmicrographs at a given level (beneath trunk somite 6) at various stages of develop-ment. In the sequence shown in Fig. 4, the duct narrows from about eight cellwidths at stage 23 to two cell widths at stage 32. The duct cells themselves Change


• i''jfcfjMZT^s^y'

Amphibian pronephric duct morphogenesis i 7

little if at all in size or shape. The marked thinning of the rudiment, accom-panied by a decrease in the number of cells across its diameter, is also seen incross-sectional views. Figure 5 shows two views produced by fracturing trans-versely through trunk somite 6 of critical-point-dried embryos. Thinning of therudiment reduces the number of cells spanning the duct's width from six to eightat stage 24 (Fig. 5 a) to two to three at stage 28 (Fig. 56). The same reductioncan be seen in 2 /tm Epon sections. All of these observations indicate that cellrearrangements, and not proliferation or cell-shape changes, are primarilyresponsible for Ambystoma pronephric duct extension.

In Fig. 6 the developmental changes in several parameters of duct outgrowthare summarized graphically. This illustrates several significant points. Despitethe increase in total embryo length (straight line head to tail), the length of theduct 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 thegradual straightening of the embryonic axis and the lifting and extension of thehead. The boundary between presumptive somite and lateral mesoderm ^vhichdefines the duct's path is quite curved at stage 22 and merely straightens out asthe embryo 'elongates'. Duct extension is closely correlated with somite seg-mentation; during elongation, the caudal tip of the duct rudiment maintains aposition two somite widths behind the most caudally developing somite fissure.Finally, both the increase in duct length and the decrease in duct diameter arelinear 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 botfderedmedially by somite mesoderm, ventrally by lateral mesoderm and (lorso-laterally by ectoderm. As reported previously (Poole & Steinberg, 1977), the cellsof exposed Ambystoma duct rudiments (mesoderm viewed en face) are attachedvia lobopodia, lamellipodia and many fine filopodia both to each other at thesurface of the rudiment and to adjacent somite and lateral mesoderm cells at itsedge (see Fig. 8 and Fig. 9). On the surface of the somites with which the ductmakes attachments are localized webs of 50-100 nm fibres (apparently extra-cellular 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 £ellrearrangement as it elongates, (a) Stage 22, six to eight cells wide; (b) Stage 26, fiveto six cells wide; (c) Stage 28, about four cells wide; (d) Stage 32, two to three cellswide.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 oftrunk somite 6. (a) A. maculatum (essentially like A. mexicanum), Stage 24; (b) A.mexicanum, Stage 28.


. '••••... DDX10

10 15No. of somites segmented

20

Fig. 6. Dimensional changes during axolotl pronephric duct extension. Embryolength measurements (EL; • ) are recorded as the straight-line distance from tip ofhead to tip of tail. Total path length (PL; O) and duct length (DL; • ) measure-ments are curvilinear. Duct diameter (DD; • ) is the linear distance from somiteto 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, butthere are no apparent features which might guide the duct's migration. The ductand adjacent mesoderm adhere weakly if at all to the inner surface of the ecto-derm. This is evident when the ectoderm is removed. After fixation, it canusually be easily peeled from the mesoderm with little evidence of damage to thelatter. It can also be peeled from living embryos with little sign of firm adhesionsto, distortion of, or damage to duct or adjacent mesoderm. Finally, chancefractures of dried embryos in which the duct mesoderm remained next to theectoderm showed few and tenuous associations of duct cells with the inner


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 pronepHricduct rudiment (PD), somites (S) and lateral mesoderm (L).


. • « •

(c)

Fig. 9. Region of tip of stage-31 axolotl pronephric duct rudiment, (a) Posterior thirdof duct rudiment, (b) Cells near the tip overlap in the manner offish scales, (c) En-largement of the area indicated in Fig. 9 b. Overlapping cells extend filopodia whichcontact underlying cells within the duct rudiment, (d) Meshwork of fibers approxi-mately 0-2 (ivci in diameter, seen as occasional small patches on cell surfaces atthis stage.

surface of the ectoderm. It thus appears that the ventral edge of the somites andthe subjacent lateral mesoderm comprise the substratum for the duct's migra-tion.

The morphology of duct cells and their contacts

Cells near the duct's tip show some anteroposterior elongation (Fig. 8) andtend 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-celladhesion near the duct's tip occurs by flat, overlapping cell processes from whicharise numerous adherent filopodia roughly 200 nm in diameter and averagingabout 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 stage-32 axolotl pronephric duct rudiment, (a) Low magnification overview showing neutfaltube, notochord, somites, epidermis, endoderm, pronephric duct and lateral meso-derm. (b) At higher magnification the ectoderm is seen to be bilaminar (bracket) andthe cells of the pronephric duct rudiment (arrows) are seen to be in the process ofadopting a radial arrangement. A fibrous network resembling collagen covers theexposed intersomitic surface (S). (c) The wedge shape of duct cells at this stage isapparent 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. Severalblunt processes (small arrows) extend between cells, (d) An area near the center of theduct rudiment (triangle). Filopodia extend along the cell surface (small arrows). Theadjoining cells are also connected by shorter, blunt processes (large arrow). 1

extending out toward somite and lateral mesoderm are much rarer back fromthe tip, especially at later stages of migration. Vital dye marking (Fig. 2) and thethinning visible in Figs. 4c-dshow that these cells are still rearranging. Whetherthey are all engaged in active locomotion like a stream of Fundulus de$p cells(Trinkaus, 1973) or whether the force causing their rearrangement arises fromthe locomotory activity of cells at the leading edge remains to be determined.


Nu

Fig. 11. Transmission electron micrographs of sections through the pronephric ductrudiment of a stage-26 axolotl embryo, (a) An area of contact between two ductrudiment cells several somites anterior to the duct's caudal tip. (b) Interdigitatingfilopodia (arrows) of duct cells show close contacts with opposing cell surfaces,suggesting that they mediate cell-cell adhesions, (c) Cells near the advancing tip ofthe duct rudiment possess long, flattened lamellipodia and are separated by moreextracellular space. Yolk platelets (YO), lipid droplets (LD), nuclei (Nu) and mito-chondria (Mi) are indicated.

Transmission electron microscope studies of cell shapes and junctions providea structural basis for the rearrangements and give clues to the type of locomotoryactivity involved. Figure 11 shows electron micrographs of sections takenthrough a stage-26 axolotl embryo several somite widths anterior to the caudaltip of the duct primordium. Electron-dense yolk platelets (YO), lipid-filleddroplets or vesicles (LD), nuclei (Nu) and mitochondria (Mi) are visible. Thereare large gaps between cells, close apposition of cell membranes occurring indiscrete areas (Fig. 11; several examples circled in Fig. 11 a). Frequently, closeapposition 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 andduct cell surfaces show fewer complex processes. In addition, long lamellipodial


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 durjngduct extension. The morphology and contacts observed as well as the scarcity ofspecialized junctions call to mind the observations of Nakatsuji (1975, 1976)on the motile cells of urodele and anuran gastrulae, and those of Hogan a idTrinkaus (1977) and Trinkaus and Lentz (1967) on the migratory deep cells ofthe Fundulus gastrula.

DISCUSSION

Scanning electron microscopic observations of the outer mesodermal surfaceof normal and surgically modified embryos have provided new insights into themechanisms directing the caudad extension of the amphibian pronephric duct.Even at low magnifications in the SEM, the Ambystoma pronephric rudimentcan be seen to segregate out as a solid mass from the dorsal portion of the hortio-geneous lateral plate mesoderm ventral to trunk somites 2 through 7, as pre-viously inferred by O'Connor (1938) and Holtfreter (1944) from studies utilisingvital staining and transplantation. Our own staining and surgical procedures con-firm their results. The Ambystoma duct clearly forms by the caudal extension ofan anterior rudiment.

Although a variety of cellular mechanisms (such as cell shape change, cellreorientation, individual cell movement, cell proliferation) might in principlecause the observed transformation from a short, thick cord to a long thin One,our observations have shown that this solid mesodermal cylinder of near-constant volume extends itself by cell rearrangement. The rudiment's markedthinning during elongation is accompanied by the redistribution of a nearly Con-stant number of constituent cells most of which remain in 'relaxed', polygonalshapes throughout the process. The force guiding this cellular rearrangement isnot made obvious by ultrastructural observations. Although the morphologyand distribution of cell processes, contacts and junctions suggest that thecaudal-most cells are actively pulling out the duct rudiment, it is difficult toreconstruct with certainty the process of duct extension from the static imagesobtainable with electron microscopy. We have been able to approach thisproblem experimentally by surgical rearrangements of the salamander meso-derm. The results will be discussed in subsequent papers. '

Cell rearrangement as a morphogenetic mechanism

Morphogenetic movements can be classified according to whether the cellsmigrate as individuals or as part of a cell group. Translocations of individualcells such as germ cells, neural crest cells and processes of neurons towardspecific destinations require guidance by specific environmental clues. $EMobservations by a number of authors have implicated cellular and extracellularfibers in such guidance (Bancroft & Bellairs, 1976; Ebendal, 1976, 1977;


Lofberg & Ahlfors, 1978; Tosney, 1978; Wylie, Heasman, Swan & Anderton1979). The morphogenesis of cell groups, however, may be guided by othercontrol mechanisms mediated by cell interactions such as adhesion, contactinhibition or changes in the shapes of firmly associated, individual cells (PhillipsSteinberg & Lipton, 1977). One of us has recently divided tissue movements intotwo broad categories. When cells remain firmly affixed to their neighbours,individual cell shape changes (such as apical constriction or cell elongation) cansummate to produce tensions which result in the expansion, contraction orfolding of the cell sheet, which behaves as a deformable solid. Tissues may alsoflow in the manner of a viscous liquid, a process that has sometimes been over-looked. In such cases cells may retain their original shapes but move past oneanother, changing cell neighbours (Phillips et al. 1977; Phillips & Steinberg,1978). These movements of cells relative to one another have been termed 'cellshear' (Jacobson & Gordon, 1976), 'cell slippage' (Phillips et al. 1977; Phillips& Steinberg, 1978) and 'cell rearrangement' (Fristrom, 1976). Such fluid re-arrangements of cells occur during pronephric duct rudiment extension. Cellslippage might result either from active cell movements or from passive relaxa-tion of tensions externally imposed on a tissue mass during morphogenesis.Recent detailed studies of cell movements in embryos, made possible at least inpart by advances in SEM techniques (see review by Poole & Steinberg, 1977), havemade it increasingly clear that movement of cells in groups or streams is morecommon than previously realized (Trinkaus, 1976). Unfortunately, the mech-anisms and forces mediating and directing such morphogenetic movements invivo have remained obscure. Elsewhere we present evidence that the samethermodynamic principles which govern adhesion-mediated cell sorting andtissue spreading in vitro (reviewed by Steinberg, 1978 a, b) also operate withinembryos to direct cell migrations and stabilize anatomically 'correct' cellassociations (Poole & Steinberg, 1978, 1981).

We thank Edward Kennedy, Doris White, Pam Knab-Mclntyre and Dorothy Spero fortheir technical assistance. This study was supported by research grants PCM76-84588 fromthe National Science Foundation and CA136O5 from the National Cancer Institute, and byPHS training grant CA9167 from the National Cancer Institute. The electron microscopy wascarried out in Department of Biology facilities supported by the Whitehall Foundation. Froma dissertation submitted by T. J.P. to the Department of Biology, Princeton University, inpartial fulfillment of the requirements for the Ph.D. degree.

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{Received 3 November 1980, revised 9 January 1981)

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Amphibian pronephric duct morphogenesis: …dev.biologists.org/content/develop/63/1/1.full.pdfAmphibian pronephric duct morphogenesis: segregation, cell rearrangement and directed