an sem analysis of neural crest migration in the mouse · thick sections were re-embedded in...

/. Embryol. exp. Morph. 74, 97-118 (1983) 9 7Printed in Great Britain © The Company of Biologists Limited 1983

An SEM analysis of neural crest migration in the

mouse

By C. A. ERICKSON1 AND J. A. WESTON2

From the Department of Zoology, University of California and Department ofBiology, University of Oregon

SUMMARY

The cellular morphology and migratory pathways of the trunk neural crest are described innormal mouse embryos, and in embryos homozygous for Patch in which neural crestderivatives develop abnormally. Trunk neural crest cells initially appear in 85-day embryos asa unique cell population on the dorsal neural tube surface and are relatively rounded. Oncethey begin to migrate the cells flatten and orient somewhat tangentially to the neural tube, andadvance ventrad between the somites and neural tube. At the onset of migration neural crestcells extend lamellipodia onto the surface of the tube while detaching their trailing processesfrom the lumenal surface. The basal lamina on the dorsal neural tube is discontinuous whencell migration begins in this region. As development proceeds, the basal lamina graduallybecomes continuous from a lateral to dorsal direction and neural crest emigration isprogressively confined to the narrowing region of discontinuous basal lamina. Cell separationfrom the neural tube ceases concomitant with completion of a continuous basement mem-brane. Preliminary observations of the mutant embryos reveal that abnormal extracellularspaces appear and patterns of crest migration are subsequently altered. We conclude that theextracellular matrix, extracellular spaces and basement membranes may delimit crest migra-tion in the mouse.

INTRODUCTION

The neural crest in the trunk region of the vertebrate embryo first appears asa strand of cells on the dorsal surface of the neural tube along the lines of fusionof the neural folds (Bancroft & Bellairs, 1976; Tosney, 1978,1982). When thesecells begin to migrate they flatten on the neural tube and soon thereafter departalong two pathways: (1) ventrally between the neural tube and somites and (2)laterally over the somites to populate the epidermal ectoderm (Horstadius, 1950;Weston, 1963, 1970; Tosney, 1978; Lofberg & Ahlfors, 1978; Thiery, Duband& Delouvee, 1982; Duband & Thiery, 1982).

The processes by which neural crest cells separate from the neural epitheliumof the neural tube and initiate migration are not known. Indeed, little is under-stood concerning the initiation of migration of any population of embryonic cells

1 Author's address: Department of Zoology, University of California, Davis, California95616, U.S.A.

2 Author's address: Department of Biology, University of Oregon, Eugene, Oregon 97403,U.S.A.

98 C. A. ERICKSON AND J. A. WESTON

(see Trinkaus, 1976), although at least three factors may be relevant: (1) theseparation of cells from the epithelium (Newgreen & Gibbins, 1982), (2) theactivation of locomotor activity, and (3) the availability of spaces and substratumwhere cells can move. Hyaluronic acid has been hypothesized to promote theappearance of extracellular spaces that allow palate cells (Greene & Pratt, 1976),heart cells (Markwald, Fitzharris, Bank & Bernanke, 1978; Markwald, Fitzhar-ris, Bolender & Bernanke, 1979; Manasek, 1976), corneal stroma fibroblasts(Toole & Trelstad, 1971) cranial neural crest cells (Pratt, Larsen & Johnston,1975), and trunk neural crest cells (Derby, 1978; Pintar, 1978) to begin moving.The evidence concerning glycosaminoglycan involvement in the onset of migra-tion is correlative, however, and numerous other extracellular components arealso likely to be involved as well (Lofberg, Ahlfors & Fallstrom, 1980; Tosney,1978; Weston, 1982; Loring, Erickson & Weston, 1977; Mayer, Hay & Hynes,1981; Newgreen & Thiery, 1980; Duband & Thiery, 1982; Thiery et al. 1982;Weston, 1982).

Once migration has begun, the pathways taken by crest cells are restricted(Weston, 1963, 1982; LeDouarin & Teillet, 1974; Noden, 1975), and it is clearthat the environment through which the cells move is in large measure respons-ible for their precise distribution (Weston & Butler, 1966; Noden, 1978;LeDouarin, 1980; Thiery etal. 1982; Weston, 1982).

To establish causal relationships between environmental factors and morpho-genetic behaviour, analysis of perturbations of the process would be helpful. Avariety of mutants are available with altered crest cell morphogenetic behaviour(Weston, 1970; Weston, 1980). We therefore began a study of the crest migrationin normal and mutant mice which may provide more direct evidence for thefactors that might direct crest morphogenesis. We report here morphologicalstudies of crest migration in normal mice (see also Derby, 1978), paying par-ticular attention to the early events of trunk neural crest segregation from theneuroepithelium, and the structural constituents of the crest pathway which mayguide cell migration. We have established the normal events in early mouse crestmorphogenesis and present preliminary data from a mouse embryo homozygousfor Patch, in which neural crest derivatives develop abnormally.

MATERIALS AND METHODS

C57BL/6J mice were originally obtained from the Jackson Laboratory (BarHarbor, Maine) and further inbred in our facility. Patch homozygotes wereobtained from matings of Ph/+ heterozygotes maintained on a C57BL/6J back-ground and inbred for 25 generations. Embryos were obtained from timedmatings, where day 0 was defined as the day that vaginal plugs were discovered.Fertilization was assumed to have occurred 3 to 4 h after the midpoint of the darkperiod preceding discovery of the plug (see Green, 1966).

Neural crest migration in the mouse 99

Embryos between 85 and 10 days of gestation were dissected free from extra-embryonic tissue with watchmakers forceps in warm Hank's balanced saltsolution, washed 2x in fresh saline and placed immediately into fixative at 37 °C(see below).

SEM preparation

Embryos were fixed for at least 2 h in 2-5 % glutaraldehyde, 1 % paraformal-dehyde, 2-5 % DMSO in 0-1 M-Na cacodylate buffer, pH7-4. For some prepara-tions cetylpyridinium chloride (0-5 % CPC) was added to the fix to help retainglycosaminoglycans (GAG) in the tissue (see Pratt et al. 1975; Derby & Pintar,1978). After three changes of 0-1 M-Na cacodylate buffer, the embryos werepostfixed in 1 % OSO4 in 0-1 M-Na cacodylate buffer at 4 °C for \\ h. The embryoswere dehydrated in a graded ethanol series and critical-point dried (Technics)with CO2 according to Anderson (1951). For observation, the embryos weremounted on aluminium stubs with silver conducting paint (Ladd), sputter coatedwith platinum-gold (Technics evaporator) and observed in an AMR scanningelectron microscope at 20 kV.

For SEM analysis, embryos were dissected in the following ways: (1) Toobserve neural crest cells along the surface of the neural tube the ectoderm hadto be removed. The most successful method was to press Scotch tape gentlyagainst the critical-point-dried specimen and lift off the ectoderm. Since severalcells layers could be removed if too much pressure was applied, over 100 embryoswere examined to be confident of which embryos showed the least damage. (2)In order to observe presumptive crest cells within the neural tube, and migrationalong the ventral pathways which are obscured by the somites, embryos werefixed for approximately lOmin.and then cut perpendicular to the neural tube atthe appropriate axial levels with iridectomy scissors (see Tosney, 1978). Theywere then fixed Hh more, processed as above, and observed in cross section.

Light microscopy

To identify presence of glycosaminoglycans and basement membranes, mouseembryos were fixed for 2h in 10 % formalin in 0-1 M-Na cacodylate buffer with0-5 % cetylpyridinium chloride (CPC) and 0-25 % poly vinyl pyrrolidine (PVP)at pH7-4. The embryos were dehydrated in an ethanol series, embedded inParaplast and sectioned at 7/im. The sections were then hydrated, treated with5 M - H C L for 30 s and stained overnight with 1 % Alcian blue in 0-025 M-MgCb,pH2-6. Most of the stained material in the extracellular spaces represents GAG(see Derby & Pintar, 1978). No assessment of the specific types of GAG foundin certain regions was made since this has been reported in detail for the sameinbred line by Derby (1978).

TEM and thick section preparation

Embryos to be examined in plastic thick sections or the TEM were fixed and


dehydrated as for SEM, rinsed 3x in propylene oxide and embedded in Epon-Araldite. Embryos were serially sectioned at 1-5/im with glass knives on aReichert ultramicrotome, mounted on slides and stained with toluidine blue(1 %) on a 40°C hotplate. When the appropriate axial levels were located, thethick sections were re-embedded in Epon-Araldite (Schabtach & Parkening,1974), thin sectioned on a DuPont diamond knife, and stained with uranylacetate and lead citrate. The sections were viewed in a Phillips 400 microscope.

RESULTS

Formation and early migration of trunk crestAs in the chick (Weston & Butler, 1966; Bancroft & Bellairs, 1976; Tosney,

1978) and the amphibian (Lofberg & Ahlfors, 1978; Lofberg etal. 1980), develop-ment of the mouse trunk neural crest varies temporally along the embryonic axis,so that developmentally older stages of crest morphogenesis occur at more ante-rior axial levels, while younger stages are found at more posterior levels in the sameembryo. Thus, between 9 and 10 days of gestation, a variety of stages of crest ap-pearance and early migration may be observed in any single embryo. The variousstages of crest morphogenesis in embryos from day 8! to day 10 are summarized inTable 1. For efficiency and clarity, the analysis of crest appearance and behaviourwill be discussed as they occur only in a 9|-day mouse embryo.

Immediately after closure of the neural tube in the trunk, the crest cannot bereadily distinguished as a separate population of cells distinct from the neuro-epithelium. Rather the dorsal neural tube surface has many observable cell

Table 1. Distribution of neural crest cells along the embryonic axis

8% days (8-12 s)10 somites

Neural tubefusion

Crest cellappearance

Cell migration

Advanced migrationCells betweensomitesCells clearedfrom dorsal NT

"16"

2-3

None

i

None

9 days (13-20 s)20 somites

"24"

"21"-17

17-16

15

10

Wi days (21-29 s)24 somites

~ "29"

"26"-24

24-23

21

16

10 days (30-34 s)30 somites

"32"-27

26

25

22

For each of the above stages, phases of crest migration were correlated with axial level byexamining specimens in the SEM whose ectoderm had been removed or in Epon cross sections(Fig. 3). The numbers represent somite number and are a convenient axial level marker. Somitesin quotation marks represent future positions of those somites in the nonsegmented mesodermand are based on equivalent somitelengths. Somites form from unsegmented mesoderm atapproximately 1 somite/h of development.


outlines with some space in between (Fig. IB, C). At developmentally moreadvanced axial levels (anterior) in the same embryo, the spaces between thedorsal neural tube cells increase and long finger-like processes (perhapsequivalent to filopodia) protrude into the extracellular space above the tube. Incontrast, only an occasional broad flat lamellipodium extends from the tube (Fig.ID, E; see below) approximately six 'somite lengths' posterior to the last(developmentally youngest) somite of a 9|-day embryo. Two to four somitelengths posterior to the last somite, a few whole cells appear on the neural tubesurface (Fig. ID).

At the level of the last somite of a 9|-day embryo the number of NC cells ontop of the neural tube has increased. These cells are multilayered, relativelyrounded and do not appear oriented in any consistent direction (Fig. 2B, C).

Soon after their appearance as a group of cells distinct from the neuralepithelium, some of the multilayered cells begin to migrate laterally over the sideof the tube. The cells move together and a common front reaches the dorsalborder of the somite within one-somite length (approximately 1 h of develop-ment). Unlike avian crest cells (see Bancroft & Bellairs, 1976; Tosney, 1978)mouse crest cells flatten only slightly as they begin to migrate, and many, but notall, of these cells are oriented tangential to the longitudinal axis of the neuraltube (Fig. 2B, D; cf. Lofberg etal. 1980).

In 9- to 10-day embryos no crest cells are observed laterally between theectoderm and somites (see also, Derby, 1978), unlike the case in chick (Tosney,1978; Bancroft & Bellairs, 1976) and axolotl (Lofberg & Ahlfors, 1978). Insteadlarge groups of cells accumulate in the cleft above and between the somites (Fig.2D, Fig. 3D). In contrast, the migration between the somites and neural tubeappears to be uniform, and not initially segmented, as suggested by Weston inthe chick embryo (1963). However, once crest cells have migrated halfway downthe neural tube they can no longer be easily distinguished from dispersingsclerotome cells (see Fig. 3). It is not possible from these observations to sayconclusively whether neural crest migrate in the intersomitic spaces as well asmedial to, or into, the somites. Recent evidence from chick-quail chimaerasreveals that neural crest cells do not invade the somite (Duband & Thiery, 1982;Thiery etal. 1982).

Segregation of crest cells from the neural epithelium

Immediately after neural fold fusion, the neural tube appears as a pseudo-stratified epithelium (Fig. 3B). Soon after fusion (posterior to the level of the lastsomite in a 9|-day embryo), spaces begin to develop between the cells in thedorsal neural tube. As the extracellular spaces increase, the curvature of the topof the tube decreases (broadens) considerably (Fig. 3C). At the same time a largeextracellular space appears above the neural tube and a few crest cells haveclearly separated from the tube (Fig. 3C). The means by which crest cellsseparate from the neuroepithelium to become a distinct population of cells is

Neural crest migration in the mouse 103Fig. 1. (A) Low-magnification SEM of the posterior trunk region of a 9|-day mouseembryo from which the ectoderm (e) has been partially removed. The posteriorneuropore (pn) is visible to the right and the neural tube at this axial level is borderedby unsegmented mesoderm (m). xl63. (B) High magnification of box B in Fig. 1A.Soon after neural tube fusion neural crest cells are not distinguishable as a separatecell population. Note the distinct cell outlines of the dorsal neural tube cells and thelarge spaces between them. X1610. (C) Detail from Fig. IB. While some extra-cellular material is seen there is clearly no complete basement membrane, since celloutlines are so distinct. X7800. (D) High magnification of box D in Fig. 1A. At thismore anterior axial region, broad, flattened lamellipodia now extend from the neuraltube, while their trailing ends are still contained within the tube. Some cells havecompletely separated from the neural tube and are observed as single cells (blackarrowheads). X1610. (E) Detail of cell marked with white arrowhead from Fig. IDshowing a lamellipodium with filopodial-like processes (arrowheads) extending fromit. Some extracellular matrix is also observed which is frequently hard to distinguishfrom the filopodia. X7700. Scale bar = 100ptm, A; = 10/im, B, D; = l̂ um, C, E.

Fig. 2. (A) Low-magnification SEM showing the trunk region of a day-9-5 mouseembryo, with the tail extending anterior to posterior from the bottom to the top ofthe micrograph. x55. (B) Higher magnification of Fig. 2A. The epithelium has beenremoved, exposing the neural crest cells on the dorsal surface of the neural tube. Theearliest developmental stages of NC morphogenesis are found at posterior axiallevels, while later stages of crest migration are found more anteriorly. X360. (C)High magnification of box C from Fig. 2B. The neural crest cells have only recentlyappeared as a separate cell population and are rounded and multilayered. The cellsalso appear to be randomly oriented, x 1470. (D) High magnification of box D fromFig. 2B. At this axial level neural crest cells have begun their ventral migration andhave reached the level of the somites. Note that the cells are only slightly flattenedand elongated compared to their premigratory appearance. X1470. (E) High mag-nification of box E from Fig. 2B. Most of the neural crest cells have cleared the topof the neural tube and accumulated in clefts between the somites. Some of the neuralcrest cells are oriented tangential to the direction of migration. The basement mem-brane of the dorsal neural tube is not yet complete, as evidenced by a few obviouscell borders. X1570. Scale bar = 100/zm, A, B; = lOjum, C, D, E.Fig. 3. Micrographs of toluidine-blue-stained Epon thin sections of a day-9-5 mouseneural tube. (A) Cross section of neural folds at the moment they meet and fuse. Theepithelium and somites (s) are closely apposed to the neural tube. (B) Cross sectionsof mouse neural tube just after fusion of the neural folds. The neural tube cells arearranged in a tight pseudostratified epithelium. Note that the somites and epitheliumare tightly apposed to the NT, with little extracellular matrix. (C) Cross section twosomite lengths posterior to the last somite. Extracellular spaces now separate thedorsal neural tube cells and the dorsal neural tube has widened. The neural tube isseparated from the overlying ectoderm and surrounding somites by extracellularmatrix-filled spaces. A few neural crest cells have now emigrated from the neuraltube and appear as a distinct population of cells in the space above the neural tube.Note, that the epithelium is still tightly apposed to the somites in a 'cleft' and noneural crest cells have moved laterally over the somites. (D) Cross section foursomites anterior to the last somite. Neural crest cells (nc) have spread ventrally in thespace between the neural tube and somites and have also collected in the cleftbetween adjacent somites. None have spread between the ectoderm and somites.Arrowhead indicates an intersomitic blood vessel. x350. Scale bar = 50 fim.

C. A. ERICKSON AND J. A. WESTON

Fig. 2. For legend see p. 103.


r -v

1

\

Fig. 3. For legend see p. 103.

elucidated further in SEM cross sections of the trunk region. At early develop-mental stages, just after neural fold fusion, the cells in the dorsal neural tube arearranged in an epithelium, apparently connected to the luminal surface, withsmall spaces between the cells. A few short filopodia-like processes are seen toextend from these cells and a number of cells have begun blebbing activity (Fig.4C). Shortly thereafter these filopodia lengthen (Fig. 4B, D) and subsequently


V1*

v *.

!\ '*

Figs 4A-D. For legend see p. 108.

Neural crest migration in the mouse

H

Figs 4E-H. For legend see p. 108.


cells with lamellipodia extend onto the surface of the tube while their trailingprocesses still remain within the neuroectoderm (Fig. 4E, G). Many such cellshave been observed in early stages and it seems clear that crest cells somehowdetach from the wall of the neurocoel and extend out onto the neural tubesurface. After neural crest cells have all departed the neural tube, the cells of thedorsal NT are once again tightly apposed and cell lamellipodia and filopodia areno longer seen (Fig. 4F, H).

Fig. 4. (A) Low-magnification SEM of a cross section of a neural tube shortly afterfusion of the neural folds. Some spaces have formed between the presumptive neuralcrest cells (nc) and the dorsal neural tube has expanded (see Fig. 3B at the equivalentstage). x480. (B) Low-magnification SEM of a cross section of a neural tube4-somite lengths posterior to the last somite of a day-9-5 embryo. A portion of theepithelium (e) has been removed to reveal the dorsal surface of the tube as well.xl540. (C) High magnification of the specimen in Fig. 4A. At this stage no neuralcrest cells have extended processes above the surface of the neural tube. Note thatthe dorsal neural tube cells have numerous short filopodia and some of the cells areblebbing (arrowheads). x3600. (D) High magnification of the specimen in Fig. 4B.Spaces have appeared between the prospective crest cells in the dorsal neural tube.Note the long filopodia extending from many of these cells and the absence of suchprocesses from the more lateral region of the neural tube. X3400. (E) Low-magnification SEM of a cross section of a neural tube two somite lengths posteriorto the last somite of a day-9-5 embryo. The epithelium has been removed and revealsneural crest cells beginning to collect on the dorsal neural tube (nt) surface. x370.(F) Low-magnification SEM of a cross section of a neural tube at somite 7 in a day-9 • 5 embryo. Neural crest cells are no longer migrating outoftheneuraltube(nt)andare cleared from the dorsal neural tube surface. x770. (G) High magnification of thespecimen in Fig. 4E. One neural crest cell (*) has extended a process above the dorsalneural tube surface while its trailing edge remains within the tube (arrowhead).X3700. (H) High magnification of the dorsal neural tube in Fig. 4F. Note that thetube cells are tightly apposed in parallel array and no cell process or filopodia areobserved. Some neural crest cells still remain on top of the neural tube (arrowhead).X2240. Scale bar = 50jum in A, E; = 10|UminB, C, D, F, G, H.

Fig. 5. (A) A toluidine-blue-stained Epon thick section through somite 20 of a day-9-5 mouse embryo. Adjacent thin sections were examined in the electron microscopeand boxed areas enlarged in Figs 5B-E. x350. (B) An electron micrograph of boxB in 5A. A neural crest cell (nc) is escaping the neural tube (nt) and entering theextracellular space beneath the epidermal ectoderm (e). The basal lamina is discon-tinuous over the dorsal neural tube (black arrowhead) and is largely absent fromsome areas where presumptive neural crest cells are leaving the tube (whitearrowhead). The basal lamina beneath the epidermal ectoderm is intact (see also5D). x6000. (C) An electron micrograph of box C in 5A. A neural crest (nc) is inthe extracellular space adjacent to the dorsolateral neural tube (nt). Neural crestcells do not leave the neural tube at this level and the basal lamina is continuous, evenover adjacent cells (arrowhead), x 10000. (D) An electron micrograph of box D in5A. This higher magnification micrograph demonstrates the dense intact basallamina (arrowheads) beneath the epidermal ectoderm (e), and the patchydistribution of matrix material on the surface of a neural crest cell. x21500. (E) Ahigh magnification of box E in 5A revealing the dense, continuous basal lamina overthe ventral neural tuhe. x21500. (F) An electron micrograph through somite 6 of aday-9-5 embryo showing two adjacent dorsal neural tube cells. Note the continuousbasal lamina even spanning intercellular spaces, x 10 000.


EMB74


Boundaries, morphology and composition of the crest migratory pathway

Basal lamina on the dorsal neural tube. As neural crest cells separate from theneuroepithelium, the ectoderm is bounded by an intact basal lamina (Fig. 5B, D;cf. Hay, 1968; Bancroft & Bellairs, 1976; Meade & Norr, 1977) whereas the basallamina on the surface of the dorsal neural tube is incomplete (Fig. 5B). This isalso seen in the SEM of 9i-day embryos where distinct cell boundaries can beobserved along the dorsal surface of the tube unobscured by basement mem-brane material (Fig. IB, D). When the crest begins separating from the neuraltube, the basal lamina is discontinuous coincident with the region from which thecrest migrates (Fig. 5B, C; see also Fig. 4).

At axial levels where crest cell separation from the neural tube has ceased (10somites lengths anterior to the last somite in 9|-day embryos) the basal laminacompletely envelops the neural tube (Fig. 5F; see also Fig. 4H).

Extracellular matrix. Just prior to the separation of the neural crest cells fromthe neural tube, the space between the ectoderm and neural tube is restricted(Fig. 3B). Alcian-blue-staining material is present in dense bands along the basalsurface of the ectoderm, somites and ventral neural tube and presumablyrepresents the basement membranes. Observable stain is absent between thepresumptive neural crest cells in the dorsal portion of the neural tube (Fig. 6A).

As crest migration begins an Alcian-blue-staining material appears in theinterstitial space above the neural tube (Fig. 6B; cf. Derby, 1978). This spacecontinues to expand laterally as crest migration proceeds (Fig. 6C; see also Fig.3). As neural crest cells fill this space they are surrounded by Alcian-blue-staining material, but even at the height of migration no observable Alcian-bluematerial is found in the dorsal neural tube, surrounding individual presumptivecrest cells in the neuroepithelium.

When migration has ceased the dense-staining basement membrane aroundthe neural tube is complete, although it is difficult to determine from only paraf-fin sections if the basement membrane around the dorsal neural tube isassociated with the neural tube, the overlying epithelium or both (Fig. 6D). Thepresence of intact basement membrane was confirmed, however, in transmissionelectron micrographs (see Fig. 5).

Mutant embryos

Adult heterozygotes of the Patch (Ph) mutant reveal a characteristic pigmentpattern and slightly altered facial characteristics (Griineberg & Truslove, 1960).The mutant is a recessive lethal, so that homozygous embryos die between day 8and day 14 of gestation. Patch mutant embryos that survive to the later stages ex-hibit a number of obvious morphological abnormalities, including enlarged heart,cleft palate, and spina bifida. Homozygotes can be recognized at early develop-mental stages, corresponding to the time of onset of neural crest cell migration, by


Fig. 6. (A) Alcian-blue-stained section of a day-9-5 mouse embryo just after fusionof the neural folds. The dense staining along the basal surface of the somites (s),ectoderm (e), and ventral neural tube (nt) probably represent basement membranes.Note the tight apposition of the ectoderm to the neural tube and somites. Remnantsof extraembryonic membranes (ex) are closely apposed to the ectoderm. (B) Alcian-:blue-stained section of a day-9-5 embryo two somite lengths posterior to the lastsomite. Neural crest cells have now populated the space above the neural tube, asrevealed in phase contrast image of this same section. Neural crest cells that havemigrated laterally (black arrowheads) are surrounded by matrix material while moremedial cells (white arrowheads) that have most recently separated from the tubehave stained material only at their dorsal surface. There is no discernible differencein Alcian blue staining between presumptive crest and ventral neural tube cells. (C)Alcian-blue-stained section of a day-9-5 embryo two somites anterior to the lastsomite. Neural crest cells have now entered the ventral pathway between the neuraltube and somite (curved arrow). As the basement membrane becomes complete overthe dorsal neural tube (cf. Fig. 5F), Alcian blue staining becomes more intense(white arrowheads). Again only neural crest cells which have escaped from the tubeare surrounded by Alcian blue staining. (D) Cross section of a day-9-5 embryo eightsomites anterior to the last somite. Neural crest cells have now migrated ventrallybetween the neural tube and somite and many have collected in the cleft betweensomites (curved arrow). Note the tight apposition which still exists between thesomite and overlying ectoderm. Neural crest cells are no longer separating from theneural tube and the dorsal neural tube is covered by a dense staining layer(arrowhead). x275.


o


l£2s>

Fig. 8. Toluidine-blue-stained Epon thick sections through two blebs of a day-9-5Ph/Ph animal. Both blebs have matrix material along the basal surface of theepithelium. Note particularly in Fig. 8B the migration of what appear to be neuralcrest cells over the lateral surface of the somite and along the basal surface of theectoderm, as well as between the somite and neural tube. x350.

Fig. 7. (A) Low-magnification SEM of a Ph/Ph embryo from a litter 9-5 days old.Its development is retarded compared to its littermates and has the characteristicblisters beneath the ectoderm (arrowheads). x55. (B) Higher magnification of theposterior bleb which has been broken open for observation. The neural crest has notyet begun its migration at this axial level (arrowhead). Note the presence of strandsof matrix material in the blebs. s = somites; e = epidermal ectoderm;m = unsegmented mesoderm. x550. (C) Higher magnification of the anterior bleb.Some neural crest cells (nc) have appeared and have migrated along the basal surfaceof the blister. Somite(s) development is very abnormal in these regions. x550. (D)The same bleb as in Fig. 7C viewed from its posterior edge. The continuity of neuralcrest (nc) migration into the bleb can be clearly seen (black arrowhead). Note theneural crest cell marked by the white arrowhead which appears to have a rufflinglamellipodium. x550. Scale bar = 100 ̂ m, A; = 10/im, B, C, D.


the failure of neural tube closure and the appearance of fluid-filled blebs, whichproduce abnormally enlarged interstitial spaces lateral to the NT (Fig. 7A-D).

Microscopic analyses of these embryos reveal that the spaces between somiteand overlying epithelium, which are abnormally large (Figs 7C, D; 8), containwhat appear to be crest cells. This contrasts with the situation in normal embryoswhere crest migration over the somites occurs several days later (see Derby,1978). The blebs into which the crest cells move are bounded on their basalsurfaces by toluidine-blue- (and Alcian-blue-) staining material (Fig. 8). Thecentres of these blebs are frequently devoid of any observable matrix.

DISCUSSION

The morphology of the neural crest cells and the surrounding embryonicstructures as observed in the SEM provide indirect information on how environ-mental cues may affect their migration. Our evidence helps elucidate: (1) whatfactors initiate crest migration, and (2) what defines the pathway of this migra-tion.

What initiates migration?

In contrast to the situation at cranial levels (Nichols, 1981), the neural crestcells in the trunk region of the mouse embryo do not begin to migrate until wellafter the neural folds have fused. When the cells initially separate from the neuraltube they are rounded and appear to lack migratory organelles such aslamellipodia or filopodia. Soon after their separation (within one somite length,or approximately l h of development) they elongate, flatten and extendprocesses, which suggest that the cells have become migratory. The crest mayacquire motility and escape from the neural tube due to a variety of stimuli.

(1) It has been suggested that the crest cells produce high levels of HA priorto migration which helps to create the spaces into which they move (Derby, 1978;Pintar, 1978; Weston, Derby & Pintar, 1978; Pratt etal. 1975). Indeed spaces doappear between presumptive crest cells just prior to their separation from thetube, and between the neural tube and overlying ectoderm. Since little or noAlcian-blue-stained material is seen between the presumptive crest cells, how-ever, it seems unlikely that the HA production is solely responsible for the spacesbetween the cells and the initiation of migration. Furthermore, Toole, Under-hill, Mikuni-Takagashi & Orkin (1980) suggest that HA alone is probably noteven sufficient for disruption of an epithelium. It is likely, therefore, that the HAis responsible for forming the space above the cells, but may not be sufficient tostimulate migration.

(2) Presumptive crest cells may lose adhesions between each other, as eviden-ced by loss of contact between the cells. This may allow them to detach easilyfrom the neuroectoderm (see also Newgreen & Gibbins, 1982).

Neural crest migration in the mouse 115(3) Neural crest cells may begin migration because previously latent locomo-

tory capabilities are activated. In a variety of systems, cells begin locomotion atspecific times during development (see Trinkaus, 1976). The initiation oflocomotor ability in other embryonic tissues is accompanied initially by blebbingactivity of cells and only later by the extension of pseudopods (Trinkaus, 1973;Gustafson & Wolpert, 1967). Just prior to crest migration similar blebbing activ-ity is seen in presumptive crest cells in the dorsal neural tube which may indicatethat some internal signal permits cells to begin migration.

(4) After the neural folds fuse to form a tube, epidermal ectoderm separatesfrom the neural ectoderm at which time the basal lamina on the dorsal surfaceof the neural tube is incomplete. It is possible that discontinuities in this extra-cellular matrix structure then permit cells of the neuroepithelium to emigrate.The cessation of migration coincides with the completion of basement membraneover the neural tube.

While all of these processes may have a role in initiation of crest migration, thedisruption of the basal lamina appears to be the event of greatest consequencesince: (1) in a variety of systems this structure acts as a barrier to migration(Marchesi, 1970; Loitta, Lee & Morakis, 1980) and (2) since in this study initia-tion and containment of neural crest migration are correlated with discontinuousand complete basal laminae respectively. It remains to be investigated if a break-down in the basal lamina allows the cells to escape, or if its discontinuity issymptomatic of other more basic disruptions in the neuroepithelium, such as lossof cell adhesions.

Several possibilities could account for the establishment of a complete basallamina over the dorsal surface of the neural tube. As the crest cells leave, thetube may manufacture or deposit new extracellular matrix elements. Alter-natively, as the crest migration depletes the number of cells within the dorsalneural tube, the lateral borders of the neural tube will be drawn together so thatthe edges of the basal lamina which are intact at all stages over the lateral andventral surfaces of the neural tube will eventually meet. This latter idea is suppor-ted by the observation that the top of the tube broadens as crest cells firstmigrate, but narrows and flattens during escape of the crest cells (see Fig. 3; Fig.4E, F).

What defines the pathway of migration?

The pathways occupied by the migrating neural crest cells appear to be limitedto spaces bounded by basal lamina. Crest cells move initially only into interstitialspaces lateral to the NT. When the space between the ectoderm and somite isobstructed by the apposition of ectoderm and somite, crest cell migration isblocked (see Fig. 3, and Newgreen & Gibbins, 1982; Derby, 1978). Moreover,crest cells are found in the somite mesenchyme only after the epithelial somitebreaks down to form the sclerotome (see Weston et al. 1980; Weston, 1963;Erickson, Tosney & Weston, 1980; Bronner-Fraser & Cohen, 1980).


When spaces open up in aberrant locations or at inappropriate times, such asin the mouse mutant Patch, the crest cells can apparently move into these regionsby migrating along the borders of the somites and the ectoderm. While we do notknow yet the amount or composition of extracellular matrix in these blebs, itseems to be unlikely that this material differentially attracts crest cells sinceneural crest cells appear to migrate everywhere in the chick embryo if they areexperimentally placed there (e.g. Erickson etal. 1980; Bronner-Fraser & Cohen,1980). In addition, neural crest cells appear to be able to use many differentcombinations of macromolecules to support their migration in vitro (Maxwell,1976; Greenberg, Seppa, Seppa & Hewitt, 1981; Newgreen & Thiery, 1980;Newgreen et al. 1982; Erickson & Turley, unpublished data). Thus it appearsthat the availability of space is the more important parameter for crest migration,and that they are unable to cross the basement membrane of their associatedepithelia or pass between two epithelial sheets if these are in tight association,such as between the dermatome and the overlying ectoderm.

We would like to thank J.-P. Thiery and P. B. Armstrong for critical reading of themanuscript. This research was supported by NIH postdoctoral fellowship DE05080, NSFGrant PCM-8004524, and NIH Grant DE05630-01 to C.A.E.; and DE-04316 to J.A.W.Additional support was provided by USPHS Biomedical Support Grant RR07080 to the U.ofO.

REFERENCESABERCROMBIE, M. (1966). The locomotory behaviour of cells. In Cells and Tissues in Culture

(ed. E. Willmer), pp. 177-202. New York: Academic Press.ABERCROMBIE, M. &HEAYSMAN, J.E. M. (1954). Observations on the social behaviour of cells

in tissue culture. II. 'Monolayering' of fibroblasts. Expl Cell Res. 6, 293-306.ANDERSON, T. F. (1951). Techniques for the preservation of three-dimensional structure in

preparing specimens for the electron microscope. Trans. N.Y. Acad. ScL, Ser. II, 13,130-134.

BANCROFT, M. & BELLAIRS, R. (1976). The neural crest cells of the trunk region of the chickembryo studied by SEM and TEM. Zoon 4, 73-85.

BRONNER-FRASER, M. & COHEN, A. M. (1980). Analysis of the neural crest ventral pathwayusing injected tracer cells. Devi Biol. 77, 130-141.

CARTER, S. (1967). Haptotaxis and the mechanisms of cell motility. Nature 213, 256-260.DAVIS, E. M. & TRINKAUS, J. P. (1981). Significance of cell-to-cell contacts for the directional

movement of neural crest cells within a hydrated collagen lattice. J. Embryol. exp. Morph.63, 29-51.

DERBY, M. A. (1978). Analysis of glycosaminoglycans within the extracellular environmentsencountered by migrating neural crest cells. Devi Biol. 66, 321-336.

DERBY, M. A. & PINTAR, J. E. (1978). The histochemical specificity of Streptomyceshyaluronidase and chondroitinase ABC. Histochemical J. 10, 529-547.

DUBAND, J. L. & THIERY, J.-P. (1982). Distribution of fibronectin in the early phase of aviancephalic neural crest cell migration. Devi Biol. 93, 308-323.

ERICKSON, C. A. (1978). Analysis of the formation of parallel arrays by BHK cells in vitro.Expl Cell Res. 115, 303-315.

ERICKSON, C. A., TOSNEY, K. W. & WESTON, J. A. (1980). Analysis of migratory behavior ofneural crest and fibroblastic cells in embryonic tissues. Devi Biol. 77, 142-156.

GREEN, E. L. (1966). Biology of the Laboratory Mouse. New York: McGraw-Hill.

Neural crest migration in the mouse 111GREENBERG, J. H., SEPPA, S., SEPPA, H. & HEWITT, A. T. (1981). Role of collagen and

fibronectin in neural crest cell adhesion and migration. Devi Biol. 87, 259-266.GREENE, R. M. & PRATT, R. M. (1976). Developmental aspects of secondary palate formation.

/. Embryol. exp. Morph. 36, 225-245.GRUNEBERG, H. & TRUSLOVE, G. M. (1960). Two closely linked genes in the mouse. Genet.

Res. 1, 69.GUSTAFSON, T. & WOLPERT, L. (1967). Cellular movement and contact in sea urchin morpho-

genesis. Biol. Rev. (Cambridge Philos. Soc.) 42, 442-498.HAY, E. D. (1968). Organization and fine structure of epithelium and mesenchyme in the

developing chick embryo. In Epithelial-Mesenchymal Interactions (eds R. Fleischmajer &R. E. Billingham), pp. 31-65. Baltimore: The Williams and Wilkins Co.

HORSTADIUS, S. (1950). The Neural Crest. New York and London: Oxford Univ. Press.LE DOUARIN, N. M. (1980). Migration and differentiation of neural crest. In Currental Topics

in Developmental Biology, 16, 31-85.LE DOUARIN, N. M. & TEILLET, M. A. (1974). Experimental analysis of the migration and

differentiation of neuroblasts of the autonomic nervous system and of neuroectodermalmesenchymal derivites, using a biological cell marking technique. Devi Biol. 41, 162-184.

LIOTTA, L. A., LEE, C. W. & MORAKIS, D. J. (1980). New method for preparing large surfacesof intact human basement membrane for tumor invasion studies. Cancer Letters 11,141-152.

LOFBERG, J., AHLFORS, K. & FALLSTROM, C. (1980). Neural crest cell migration in relation toextracellular matrix organization in the embryonic axolotl trunk. Devi Biol. 75, 148-167.

LOFBERG, J. &AHLFORS, K. (1978). Extracellular matrix organization and early neural crestcell migration in the axolotl embryo. Zoon 6, 87-107.

LORING, J., ERICKSON, C. & WESTON, J. (1977). Surface proteins of neural crest, crest-derivedand somite cells in vitro. J. Cell Biol. 75, 71a.

MANASEK, F. J. (1976). Macromolecules of the extracellular compartment of embryonic andmature hearts. Circulation Res. 38, 331-337.

MARCHESI, V. T. (1970). Mechanisms of blood cell migration across blood vessel walls. InRegulation of Hematopoiesis (ed. A. S. Gordon), pp. 943-958. New York: Appleton-Century-Crofts.

MARKWALD, R. R., FITZHARRIS, T. P., BANK, H. & BERNANKE, D. H. (1978). Structuralanalysis of the matrical organization of glycosaminoglycans in developing endocardialcushions. Devi Biol. 62, 292-316.

MARKWALD, R. R., FITZHARRIS, T. P., BOLENDER, D. L. & BERNANKE, D. H. (1979). Struc-tural analysis of cell: matrix association during the morphogenesis of atrioventricularcushion tissue. Devi Biol. 69, 634-654.

MAXWELL, G. D. (1976). Substrate dependence of cell migration from explanted neural tubesin vitro. Cell Tissue Res. 172, 325-330.

MAYER, B. W., HAY, E. D. & HYNES, R. O. (1981). Immunocytochemical localization offibronectin in embryonic chick trunk and area vasculosa. Devi Biol. 82, 267-286.

MEADE, P. A. & NORR, S. C. (1977). The onset of neural crest cell migration: the basallaminae. J. Cell Biol. 75, 48a.

NEWGREEN, D. & GIBBINS, I. (1982). Factors controlling the time of onset of the migration ofneural crest cells in the fowl embryo. Cell Tissue Res. 224, 145-160.

NEWGREEN, D. F., GIBBINS, I. L., SAUTER, J., WALLENFELS, B. & WUTZ, R. (1982). Ultra-structural and tissue culture studies on the role of fibronectin, collagen and glycosamino-glycons in the migration of fowl embryo neural crest cells. Cell Tissue Res. 221, 521-549.

NEWGREEN, D. & THIERY, J.-P. (1980). Fibronectin in early avian embryos: synthesis anddistribution along the migration pathways of neural crest cells. Cell Tissue Res. 211,269-291.

NICHOLS, D. H. (1981). Neural crest formation in the head of the mouse embryo as observedusing a new histological technique. J. Embryol. exp. Morph. 64, 105-120.

NODEN, D. (1975). An analysis of the migratory behavior of avian cephalic neural crest cells.Devi Biol. 42, 106-130.

NODEN, D. M. (1978). Interactions directing the migration and cytodifferentiation of avian


neural crest cells. In The Specificity of Embryological Interactions (ed. D. Garrod), pp.5-49. London: Chapman & Hall.

PINTAR, J. E. (1978). Distribution and synthesis of glycosaminoglycans during quail neuralcrest morphogenesis. Devi Biol. 67, 444-464.

PRATT, R. M., LARSEN, M. A. & JOHNSTON, M. C. (1975). Migration of cranial neural crestcells in a cell-free hyaluronate-rich matrix. Devi Biol. 44, 298-305.

SCHABTACH, E. & PARKENING, T. S. (1974). A method for sequential high resolution light andelectron microscopy of selected areas of the same material. /. Cell Biol. 61, 261-264.

THIERY, J.-P., DUBAND, J. L. & DELOUV£E, A. (1982). Pathways and mechanisms of aviantrunk neural crest cell migration and localization. Devi Biol. 93, 324-343.

TICKLE, C. & TRINKAUS, J. P. (1976). Observations of nudging cells in culture. Nature 261,413-414.

TOOLE, B. P. & TRELSTAD, R. L. (1971). Hyaluronate production and removal during cornealdevelopment in the chick. Devi Biol. 26, 28-35.

TOOLE, B. P., UNDERHILL, C. B., MIKUNI-TAKAGASHI, Y. & ORKIN, R. W. (1980).Hyaluronate in morphogenesis. In Current Research Trends in Prenatal CraniofacialDevelopment (eds R. M. Pratt & R. L. Chistiansen), pp. 263-275. New York: ElsevierNorth Holland, Inc.

TOSNEY, K. W. (1978). The early migration of neural crest cells in the trunk region of the avianembryo: an SEM-TEM study. Devi Biol. 62, 317-333.

TOSNEY, K. W. (1982). The segregation and early migration of cranial neural crest cells in theavian embryo. Devi Biol. 89, 13-24.

TRINKAUS, J. P. (1973). Surface activity and locomotion of Fundulus deep cells during blastulaand gastrula stages. Devi Biol. 30, 68-103.

TRINKAUS, J. P. (1976). On the mechanism of metazoan cell movements. In The Cell Surfacein Animal Embryogenesis and Development (eds G. Poste & G. L. Nicolson), pp. 225-329.Amsterdam: Elsevier/North-Holland Biomedical Press.

TwiTTY, V. C. & Niu, M. C. (1948). Causal analysis of chromatophore migration. J. exp. Zool.108, 405-437.

WESTON, J. A. (1963). A radioautographic analysis of the migration and localization of trunkneural crest cells in the chick. Devi Biol. 6, 279-310.

WESTON, J. A. (1970). The migration and differentiation of neural crest cells. Adv. Morpho-genesis 8, 41-114.

WESTON, J. A. (1980). Role of the embryonic environment in neural crest morphogenesis. InCurrent Research Trends in Prenatal Craniofacial Development (eds R. M. Pratt & R. L.Christiansen), pp. 27-45. New York: Elsevier North Holland, Inc.

WESTON, J. A. (1982). Motile and social behavior of neural crest cells. In Cell Behaviour (edsR. Bellairs, A. Curtis & G. Dunn), pp. 429-470. Cambridge: Cambridge University Press.

WESTON, J. A. & BUTLER, S. L. (1966). Temporal factors affecting localization of neural crestcells in the chicken embryo. Devi Biol. 14, 246-266.

WESTON, J. A., DERBY, M. A. & PINTAR, J. E. (1978). Changes in the extracellular environ-ment of neural crest cells during their early migration. Zoon 6, 103-113.

(Accepted 8 November 1982)

an sem analysis of neural crest migration in the mouse · thick sections were re-embedded in...

Documents