The cellular morphology and migratory pathways of the trunk neural crest are described in normal mouse embryos, and in embryos homozygous for Patch in which neural crest derivatives develop abnormally. Trunk neural crest cells initially appear in -day embryos as a unique cell population on the dorsal neural tube surface and are relatively rounded. Once they begin to migrate the cells flatten and orient somewhat tangentially to the neural tube, and advance ventrad between the somites and neural tube. At the onset of migration neural crest cells extend lamellipodia onto the surface of the tube while detaching their trailing processes from the lumenal surface. The basal lamina on the dorsal neural tube is discontinuous when cell migration begins in this region. As development proceeds, the basal lamina gradually becomes continuous from a lateral to dorsal direction and neural crest emigration is progressively confined to the narrowing region of discontinuous basal lamina. Cell separation from the neural tube ceases concomitant with completion of a continuous basement membrane. Preliminary observations of the mutant embryos reveal that abnormal extracellular spaces appear and patterns of crest migration are subsequently altered. We conclude that the extracellular matrix, extracellular spaces and basement membranes may delimit crest migration in the mouse.

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

The processes by which neural crest cells separate from the neural epithelium of the neural tube and initiate migration are not known. Indeed, little is understood concerning the initiation of migration of any population of embryonic cells (see Trinkaus, 1976), although at least three factors may be relevant: (1) the separation of cells from the epithelium (Newgreen & Gibbins, 1982), (2) the activation of locomotor activity, and (3) the availability of spaces and substratum where cells can move. Hyaluronic acid has been hypothesized to promote the appearance 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 migration is correlative, however, and numerous other extracellular components are also likely to be involved as well (Löfberg, 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 clear that the environment through which the cells move is in large measure responsible for their precise distribution (Weston & Butler, 1966; Noden, 1978; LeDouarin, 1980; Thiery et al. 1982; Weston, 1982).

To establish causal relationships between environmental factors and morphogenetic behaviour, analysis of perturbations of the process would be helpful. A variety of mutants are available with altered crest cell morphogenetic behaviour (Weston, 1970; Weston, 1980). We therefore began a study of the crest migration in normal and mutant mice which may provide more direct evidence for the factors that might direct crest morphogenesis. We report here morphological studies of crest migration in normal mice (see also Derby, 1978), paying particular attention to the early events of trunk neural crest segregation from the neuroepithelium, and the structural constituents of the crest pathway which may guide cell migration. We have established the normal events in early mouse crest morphogenesis and present preliminary data from a mouse embryo homozygous for Patch, in which neural crest derivatives develop abnormally.

C57BL/6J mice were originally obtained from the Jackson Laboratory (Bar Harbor, Maine) and further inbred in our facility. Patch homozygotes were obtained from matings of Ph/ + heterozygotes maintained on a C57BL/6J background and inbred for 25 generations. Embryos were obtained from timed matings, 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 dark period preceding discovery of the plug (see Green, 1966).

Embryos between and 10 days of gestation were dissected free from extra-embryonic tissue with watchmakers forceps in warm Hank’s balanced salt solution, washed 2× 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 % paraformaldehyde, 2·5 % DMSO in 0·1 M-Na cacodylate buffer, pH 7·4. For some preparations cetylpyridinium chloride (0·5 % CPC) was added to the fix to help retain glycosaminoglycans (GAG) in the tissue (see Pratt et al. 1975; Derby & Pintar, 1978). After three changes of 0·1 M-Na cacodylate buffer, the embryos were postfixed in 1 % OsO4 in 0·1 M-Na cacodylate buffer at 4 °C for . The embryos were dehydrated in a graded ethanol series and critical-point dried (Technics) with CO2 according to Anderson (1951). For observation, the embryos were mounted on aluminium stubs with silver conducting paint (Ladd), sputter coated with platinum-gold (Technics evaporator) and observed in an AMR scanning electron microscope at 20 kV.

For SEM analysis, embryos were dissected in the following ways: (1) To observe neural crest cells along the surface of the neural tube the ectoderm had to be removed. The most successful method was to press Scotch tape gently against the critical-point-dried specimen and lift off the ectoderm. Since several cells layers could be removed if too much pressure was applied, over 100 embryos were examined to be confident of which embryos showed the least damage. (2) In order to observe presumptive crest cells within the neural tube, and migration along the ventral pathways which are obscured by the somites, embryos were fixed for approximately 10 min. and then cut perpendicular to the neural tube at the appropriate axial levels with iridectomy scissors (see Tosney, 1978). They were then fixed more, processed as above, and observed in cross section.

Light microscopy

To identify presence of glycosaminoglycans and basement membranes, mouse embryos were fixed for 2h in 10 % formalin in 0·1 M-Na cacodylate buffer with 0·5 % cetylpyridinium chloride (CPC) and 0·25 % polyvinyl pyrrolidine (PVP) at pH7·4. The embryos were dehydrated in an ethanol series, embedded in Paraplast and sectioned at 7 μm. The sections were then hydrated, treated with 5 M-HCL for 30 s and stained overnight with 1 % Alcian blue in 0·025 M-MgCl2, pH 2·6. Most of the stained material in the extracellular spaces represents GAG (see Derby & Pintar, 1978). No assessment of the specific types of GAG found in certain regions was made since this has been reported in detail for the same inbred 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 3× in propylene oxide and embedded in Epon-Araldite. Embryos were serially sectioned at 1–5μm with glass knives on a Reichert ultramicrotome, mounted on slides and stained with toluidine blue (1 %) on a 40°C hotplate. When the appropriate axial levels were located, the thick sections were re-embedded in Epon-Araldite (Schabtach & Parkening, 1974), thin sectioned on a DuPont diamond knife, and stained with uranyl acetate and lead citrate. The sections were viewed in a Phillips 400 microscope.

Formation and early migration of trunk crest

As in the chick (Weston & Butler, 1966; Bancroft & Bellairs, 1976; Tosney, 1978) and the amphibian (Löfberg & Ahlfors, 1978; Löfberg et al. 1980), development of the mouse trunk neural crest varies temporally along the embryonic axis, so that developmentally older stages of crest morphogenesis occur at more anterior axial levels, while younger stages are found at more posterior levels in the same embryo. Thus, between 9 and 10 days of gestation, a variety of stages of crest appearance and early migration may be observed in any single embryo. The various stages of crest morphogenesis in embryos from day to day 10 are summarized in Table 1. For efficiency and clarity, the analysis of crest appearance and behaviour will be discussed as they occur only in a -day mouse embryo.

Table 1.

Distribution of neural crest cells along the embryonic axis

Distribution of neural crest cells along the embryonic axis
Distribution of neural crest cells along the embryonic axis

Immediately after closure of the neural tube in the trunk, the crest cannot be readily distinguished as a separate population of cells distinct from the neuroepithelium. Rather the dorsal neural tube surface has many observable cell outlines with some space in between (Fig. IB, C). At developmentally more advanced axial levels (anterior) in the same embryo, the spaces between the dorsal neural tube cells increase and long finger-like processes (perhaps equivalent to filopodia) protrude into the extracellular space above the tube. In contrast, 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 -day embryo. Two to four somite lengths posterior to the last somite, a few whole cells appear on the neural tube surface (Fig. ID).

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

Fig. 1.

(A) Low-magnification SEM of the posterior trunk region of a 912-day mouse embryo from which the ectoderm (e) has been partially removed. The posterior neuropore (pn) is visible to the right and the neural tube at this axial level is bordered by unsegmented mesoderm (m). ×163. (B) High magnification of box B in Fig. 1A. Soon after neural tube fusion neural crest cells are not distinguishable as a separate cell population. Note the distinct cell outlines of the dorsal neural tube cells and the large spaces between them. ×1610. (C) Detail from Fig. IB. While some extracellular material is seen there is clearly no complete basement membrane, since cell outlines are so distinct. ×7800. (D) High magnification of box D in Fig. 1A. At this more anterior axial region, broad, flattened lamellipodia now extend from the neural tube, while their trailing ends are still contained within the tube. Some cells have completely separated from the neural tube and are observed as single cells (black arrowheads). ×1610. (E) Detail of cell marked with white arrowhead from Fig. ID showing a lamellipodium with filopodial-like processes (arrowheads) extending from it. Some extracellular matrix is also observed which is frequently hard to distinguish from the filopodia. ×7700. Scale bar = 100μm, A; = 10μm, B, D; = 1μm, C, E.

Fig. 1.

(A) Low-magnification SEM of the posterior trunk region of a 912-day mouse embryo from which the ectoderm (e) has been partially removed. The posterior neuropore (pn) is visible to the right and the neural tube at this axial level is bordered by unsegmented mesoderm (m). ×163. (B) High magnification of box B in Fig. 1A. Soon after neural tube fusion neural crest cells are not distinguishable as a separate cell population. Note the distinct cell outlines of the dorsal neural tube cells and the large spaces between them. ×1610. (C) Detail from Fig. IB. While some extracellular material is seen there is clearly no complete basement membrane, since cell outlines are so distinct. ×7800. (D) High magnification of box D in Fig. 1A. At this more anterior axial region, broad, flattened lamellipodia now extend from the neural tube, while their trailing ends are still contained within the tube. Some cells have completely separated from the neural tube and are observed as single cells (black arrowheads). ×1610. (E) Detail of cell marked with white arrowhead from Fig. ID showing a lamellipodium with filopodial-like processes (arrowheads) extending from it. Some extracellular matrix is also observed which is frequently hard to distinguish from the filopodia. ×7700. Scale bar = 100μm, A; = 10μm, B, D; = 1μm, C, E.

Fig. 2.

(A) Low-magnification SEM showing the trunk region of a day-9·5 mouse embryo, with the tail extending anterior to posterior from the bottom to the top of the micrograph. ×55. (B) Higher magnification of Fig. 2A. The epithelium has been removed, exposing the neural crest cells on the dorsal surface of the neural tube. The earliest developmental stages of NC morphogenesis are found at posterior axial levels, while later stages of crest migration are found more anteriorly. ×360. (C) High magnification of box C from Fig. 2B. The neural crest cells have only recently appeared as a separate cell population and are rounded and multilayered. The cells also appear to be randomly oriented, × 1470. (D) High magnification of box D from Fig. 2B. At this axial level neural crest cells have begun their ventral migration and have reached the level of the somites. Note that the cells are only slightly flattened and elongated compared to their premigratory appearance. ×1470. (E) High magnification of box E from Fig. 2B. Most of the neural crest cells have cleared the top of thé neural tube and accumulated in clefts between the somites. Some of the neural crest cells are oriented tangential to the direction of migration. The basement membrane of the dorsal neural tube is not yet complete, as evidenced by a few obvious cell borders. ×1570. Scale bar = 100μm, A, B; = 10μm, C, D, E.

Fig. 2.

(A) Low-magnification SEM showing the trunk region of a day-9·5 mouse embryo, with the tail extending anterior to posterior from the bottom to the top of the micrograph. ×55. (B) Higher magnification of Fig. 2A. The epithelium has been removed, exposing the neural crest cells on the dorsal surface of the neural tube. The earliest developmental stages of NC morphogenesis are found at posterior axial levels, while later stages of crest migration are found more anteriorly. ×360. (C) High magnification of box C from Fig. 2B. The neural crest cells have only recently appeared as a separate cell population and are rounded and multilayered. The cells also appear to be randomly oriented, × 1470. (D) High magnification of box D from Fig. 2B. At this axial level neural crest cells have begun their ventral migration and have reached the level of the somites. Note that the cells are only slightly flattened and elongated compared to their premigratory appearance. ×1470. (E) High magnification of box E from Fig. 2B. Most of the neural crest cells have cleared the top of thé neural tube and accumulated in clefts between the somites. Some of the neural crest cells are oriented tangential to the direction of migration. The basement membrane of the dorsal neural tube is not yet complete, as evidenced by a few obvious cell borders. ×1570. Scale bar = 100μm, A, B; = 10μm, C, D, E.

Soon after their appearance as a group of cells distinct from the neural epithelium, some of the multilayered cells begin to migrate laterally over the side of the tube. The cells move together and a common front reaches the dorsal border of the somite within one-somite length (approximately 1 h of development). Unlike avian crest cells (see Bancroft & Bellairs, 1976; Tosney, 1978) mouse crest cells flatten only slightly as they begin to migrate, and many, but not all, of these cells are oriented tangential to the longitudinal axis of the neural tube (Fig. 2B, D; cf. Löfberg et al. 1980).

In 9-to 10-day embryos no crest cells are observed laterally between the ectoderm and somites (see also, Derby, 1978), unlike the case in chick (Tosney, 1978; Bancroft & Bellairs, 1976) and axolotl (Löfberg & Ahlfors, 1978). Instead large 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 tube appears to be uniform, and not initially segmented, as suggested by Weston in the chick embryo (1963). However, once crest cells have migrated halfway down the neural tube they can no longer be easily distinguished from dispersing sclerotome cells (see Fig. 3). It is not possible from these observations to say conclusively whether neural crest migrate in the intersomitic spaces as well as medial to, or into, the somites. Recent evidence from chick-quail chimaeras reveals that neural crest cells do not invade the somite (Duband & Thiery, 1982; Thiery et al. 1982).

Fig. 3.

Micrographs of toluidine-blue-stained Epon thin sections of a day-9·5 mouse neural tube. (A) Cross section of neural folds at the moment they meet and fuse. The epithelium and somites (s) are closely apposed to the neural tube. (B) Cross sections of mouse neural tube just after fusion of the neural folds. The neural tube cells are arranged in a tight pseudostratified epithelium. Note that the somites and epithelium are tightly apposed to the NT, with little extracellular matrix. (C) Cross section two somite lengths posterior to the last somite. Extracellular spaces now separate the dorsal neural tube cells and the dorsal neural tube has widened. The neural tube is separated from the overlying ectoderm and surrounding somites by extracellular matrix-filled spaces. A few neural crest cells have now emigrated from the neural tube 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 no neural crest cells have moved laterally over the somites. (D) Cross section four somites anterior to the last somite. Neural crest cells (nc) have spread ventrally in the space between the neural tube and somites and have also collected in the cleft between adjacent somites. None have spread between the ectoderm and somites. Arrowhead indicates an intersomitic blood vessel. x350. Scale bar = 50μm.

Fig. 3.

Micrographs of toluidine-blue-stained Epon thin sections of a day-9·5 mouse neural tube. (A) Cross section of neural folds at the moment they meet and fuse. The epithelium and somites (s) are closely apposed to the neural tube. (B) Cross sections of mouse neural tube just after fusion of the neural folds. The neural tube cells are arranged in a tight pseudostratified epithelium. Note that the somites and epithelium are tightly apposed to the NT, with little extracellular matrix. (C) Cross section two somite lengths posterior to the last somite. Extracellular spaces now separate the dorsal neural tube cells and the dorsal neural tube has widened. The neural tube is separated from the overlying ectoderm and surrounding somites by extracellular matrix-filled spaces. A few neural crest cells have now emigrated from the neural tube 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 no neural crest cells have moved laterally over the somites. (D) Cross section four somites anterior to the last somite. Neural crest cells (nc) have spread ventrally in the space between the neural tube and somites and have also collected in the cleft between adjacent somites. None have spread between the ectoderm and somites. Arrowhead indicates an intersomitic blood vessel. x350. Scale bar = 50μm.

Segregation of crest cells from the neural epithelium

Immediately after neural fold fusion, the neural tube appears as a pseudostratified epithelium (Fig. 3B). Soon after fusion (posterior to the level of the last somite in a -day embryo), spaces begin to develop between the cells in the dorsal neural tube. As the extracellular spaces increase, the curvature of the top of the tube decreases (broadens) considerably (Fig. 3C). At the same time a large extracellular space appears above the neural tube and a few crest cells have clearly separated from the tube (Fig. 3C). The means by which crest cells separate from the neuroepithelium to become a distinct population of cells is elucidated further in SEM cross sections of the trunk region. At early developmental stages, just after neural fold fusion, the cells in the dorsal neural tube are arranged in an epithelium, apparently connected to the luminal surface, with small spaces between the cells. A few short filopodia-like processes are seen to extend from these cells and a number of cells have begun blebbing activity (Fig. 4C). Shortly thereafter these filopodia lengthen (Fig. 4B, D) and subsequently cells with lamellipodia extend onto the surface of the tube while their trailing processes still remain within the neuroectoderm (Fig. 4E, G). Many such cells have been observed in early stages and it seems clear that crest cells somehow detach from the wall of the neurocoel and extend out onto the neural tube surface. After neural crest cells have all departed the neural tube, the cells of the dorsal NT are once again tightly apposed and cell lamellipodia and filopodia are no longer seen (Fig. 4F, H).

Fig. 4.

(A) Low-magnification SEM of a cross section of a neural tube shortly after fusion of the neural folds. Some spaces have formed between the presumptive neural crest cells (nc) and the dorsal neural tube has expanded (see Fig. 3B at the equivalent stage). ·480. (B) Low-magnification SEM of a cross section of a neural tube 4-somite lengths posterior to the last somite of a day-9-5 embryo. A portion of the epithelium (e) has been removed to reveal the dorsal surface of the tube as well. ·1540. (C) High magnification of the specimen in Fig. 4A. At this stage no neural crest cells have extended processes above the surface of the neural tube. Note that the dorsal neural tube cells have numerous short filopodia and some of the cells are blebbing (arrowheads). ·3600. (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 such processes from the more lateral region of the neural tube. ·3400. (E) Low-magnification SEM of a cross section of a neural tube two somite lengths posterior to the last somite of a day-9-5 embryo. The epithelium has been removed and reveals neural crest cells beginning to collect on the dorsal neural tube (nt) surface. ×370. (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 out of the neural tube (nt) and are cleared from the dorsal neural tube surface. ×770. (G) High magnification of the specimen in Fig. 4E. One neural crest cell (*) has extended a process above the dorsal neural tube surface while its trailing edge remains within the tube (arrowhead). ·3700. (H) High magnification of the dorsal neural tube in Fig. 4F. Note that the tube cells are tightly apposed in parallel array and no cell process or filopodia are observed. Some neural crest cells still remain on top of the neural tube (arrowhead). ·2240. Scale bar = 50μm in A, E; = 10μm in B, C, D, F, G, H.

Fig. 4.

(A) Low-magnification SEM of a cross section of a neural tube shortly after fusion of the neural folds. Some spaces have formed between the presumptive neural crest cells (nc) and the dorsal neural tube has expanded (see Fig. 3B at the equivalent stage). ·480. (B) Low-magnification SEM of a cross section of a neural tube 4-somite lengths posterior to the last somite of a day-9-5 embryo. A portion of the epithelium (e) has been removed to reveal the dorsal surface of the tube as well. ·1540. (C) High magnification of the specimen in Fig. 4A. At this stage no neural crest cells have extended processes above the surface of the neural tube. Note that the dorsal neural tube cells have numerous short filopodia and some of the cells are blebbing (arrowheads). ·3600. (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 such processes from the more lateral region of the neural tube. ·3400. (E) Low-magnification SEM of a cross section of a neural tube two somite lengths posterior to the last somite of a day-9-5 embryo. The epithelium has been removed and reveals neural crest cells beginning to collect on the dorsal neural tube (nt) surface. ×370. (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 out of the neural tube (nt) and are cleared from the dorsal neural tube surface. ×770. (G) High magnification of the specimen in Fig. 4E. One neural crest cell (*) has extended a process above the dorsal neural tube surface while its trailing edge remains within the tube (arrowhead). ·3700. (H) High magnification of the dorsal neural tube in Fig. 4F. Note that the tube cells are tightly apposed in parallel array and no cell process or filopodia are observed. Some neural crest cells still remain on top of the neural tube (arrowhead). ·2240. Scale bar = 50μm in A, E; = 10μm in B, C, D, F, G, H.

Boundaries, morphology and composition of the crest migratory pathway

Basal lamina on the dorsal neural tube

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

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 microscope and boxed areas enlarged in Figs 5B-E. ×350. (B) An electron micrograph of box B in 5A. A neural crest cell (nc) is escaping the neural tube (nt) and entering the extracellular space beneath the epidermal ectoderm (e). The basal lamina is discontinuous over the dorsal neural tube (black arrowhead) and is largely absent from some areas where presumptive neural crest cells are leaving the tube (white arrowhead). The basal lamina beneath the epidermal ectoderm is intact (see also 5D). ×6000. (C) An electron micrograph of box C in 5A. A neural crest (nc) is in the extracellular space adjacent to the dorsolateral neural tube (nt). Neural crest cells do not leave the neural tube at this level and the basal lamina is continuous, even over adjacent cells (arrowhead), × 10000. (D) An electron micrograph of box D in 5A. This higher magnification micrograph demonstrates the dense intact basal lamina (arrowheads) beneath the epidermal ectoderm (e), and the patchy distribution of matrix material on the surface of a neural crest cell. ×21500. (E) A high magnification of box E in 5A revealing the dense, continuous basal lamina over the ventral neural tube. ×21500. (F) An electron micrograph through somite 6 of a day-9-5 embryo showing two adjacent dorsal neural tube cells. Note the continuous basal lamina even spanning intercellular spaces, × 10 000.

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 microscope and boxed areas enlarged in Figs 5B-E. ×350. (B) An electron micrograph of box B in 5A. A neural crest cell (nc) is escaping the neural tube (nt) and entering the extracellular space beneath the epidermal ectoderm (e). The basal lamina is discontinuous over the dorsal neural tube (black arrowhead) and is largely absent from some areas where presumptive neural crest cells are leaving the tube (white arrowhead). The basal lamina beneath the epidermal ectoderm is intact (see also 5D). ×6000. (C) An electron micrograph of box C in 5A. A neural crest (nc) is in the extracellular space adjacent to the dorsolateral neural tube (nt). Neural crest cells do not leave the neural tube at this level and the basal lamina is continuous, even over adjacent cells (arrowhead), × 10000. (D) An electron micrograph of box D in 5A. This higher magnification micrograph demonstrates the dense intact basal lamina (arrowheads) beneath the epidermal ectoderm (e), and the patchy distribution of matrix material on the surface of a neural crest cell. ×21500. (E) A high magnification of box E in 5A revealing the dense, continuous basal lamina over the ventral neural tube. ×21500. (F) An electron micrograph through somite 6 of a day-9-5 embryo showing two adjacent dorsal neural tube cells. Note the continuous basal lamina even spanning intercellular spaces, × 10 000.

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

Extracellular matrix

Just prior to the separation of the neural crest cells from the 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 basal surface of the ectoderm, somites and ventral neural tube and presumably represents the basement membranes. Observable stain is absent between the presumptive neural crest cells in the dorsal portion of the neural tube (Fig. 6A).

Fig. 6.

(A) Alcian-blue-stained section of a day-9-5 mouse embryo just after fusion of 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. Remnants of 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 last somite. Neural crest cells have now populated the space above the neural tube, as revealed in phase contrast image of this same section. Neural crest cells that have migrated laterally (black arrowheads) are surrounded by matrix material while more medial cells (white arrowheads) that have most recently separated from the tube have stained material only at their dorsal surface. There is no discernible difference in 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 last somite. Neural crest cells have now entered the ventral pathway between the neural tube and somite (curved arrow). As the basement membrane becomes complete over the dorsal neural tube (cf. Fig. 5F), Alcian blue staining becomes more intense (white arrowheads). Again only neural crest cells which have escaped from the tube are surrounded by Alcian blue staining. (D) Cross section of a day-9·5 embryo eight somites anterior to the last somite. Neural crest cells have now migrated ventrally between the neural tube and somite and many have collected in the cleft between somites (curved arrow). Note the tight apposition which still exists between the somite and overlying ectoderm. Neural crest cells are no longer separating from the neural tube and the dorsal neural tube is covered by a dense staining layer (arrowhead). ×275.

Fig. 6.

(A) Alcian-blue-stained section of a day-9-5 mouse embryo just after fusion of 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. Remnants of 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 last somite. Neural crest cells have now populated the space above the neural tube, as revealed in phase contrast image of this same section. Neural crest cells that have migrated laterally (black arrowheads) are surrounded by matrix material while more medial cells (white arrowheads) that have most recently separated from the tube have stained material only at their dorsal surface. There is no discernible difference in 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 last somite. Neural crest cells have now entered the ventral pathway between the neural tube and somite (curved arrow). As the basement membrane becomes complete over the dorsal neural tube (cf. Fig. 5F), Alcian blue staining becomes more intense (white arrowheads). Again only neural crest cells which have escaped from the tube are surrounded by Alcian blue staining. (D) Cross section of a day-9·5 embryo eight somites anterior to the last somite. Neural crest cells have now migrated ventrally between the neural tube and somite and many have collected in the cleft between somites (curved arrow). Note the tight apposition which still exists between the somite and overlying ectoderm. Neural crest cells are no longer separating from the neural tube and the dorsal neural tube is covered by a dense staining layer (arrowhead). ×275.

As crest migration begins an Alcian-blue-staining material appears in the interstitial space above the neural tube (Fig. 6B; cf. Derby, 1978). This space continues 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-blue material is found in the dorsal neural tube, surrounding individual presumptive crest cells in the neuroepithelium.

When migration has ceased the dense-staining basement membrane around the neural tube is complete, although it is difficult to determine from only paraffin sections if the basement membrane around the dorsal neural tube is associated with the neural tube, the overlying epithelium or both (Fig. 6D). The presence of intact basement membrane was confirmed, however, in transmission electron micrographs (see Fig. 5).

Mutant embryos

Adult heterozygotes of the Patch (Ph) mutant reveal a characteristic pigment pattern and slightly altered facial characteristics (Grüneberg & Truslove, 1960). The mutant is a recessive lethal, so that homozygous embryos die between day 8 and day 14 of gestation. Patch mutant embryos that survive to the later stages exhibit a number of obvious morphological abnormalities, including enlarged heart, cleft palate, and spina bifida. Homozygotes can be recognized at early developmental stages, corresponding to the time of onset of neural crest cell migration, by the failure of neural tube closure and the appearance of fluid-filled blebs, which produce abnormally enlarged interstitial spaces lateral to the NT (Fig. 7A-D).

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 characteristic blisters beneath the ectoderm (arrowheads). ×55. (B) Higher magnification of the posterior bleb which has been broken open for observation. The neural crest has not yet begun its migration at this axial level (arrowhead). Note the presence of strands of matrix material in the blebs. s = somites; e = epidermal ectoderm; m = unsegmented mesoderm. ×550. (C) Higher magnification of the anterior bleb. Some neural crest cells (nc) have appeared and have migrated along the basal surface of the blister. Somite(s) development is very abnormal in these regions. ×550. (D) The same bleb as in Fig. 7C viewed from its posterior edge. The continuity of neural crest (nc) migration into the bleb can be clearly seen (black arrowhead). Note the neural crest cell marked by the white arrowhead which appears to have a ruffling lamellipodium. ×550. Scale bar = 100μm, A; = 10μm, B, C, D.

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 characteristic blisters beneath the ectoderm (arrowheads). ×55. (B) Higher magnification of the posterior bleb which has been broken open for observation. The neural crest has not yet begun its migration at this axial level (arrowhead). Note the presence of strands of matrix material in the blebs. s = somites; e = epidermal ectoderm; m = unsegmented mesoderm. ×550. (C) Higher magnification of the anterior bleb. Some neural crest cells (nc) have appeared and have migrated along the basal surface of the blister. Somite(s) development is very abnormal in these regions. ×550. (D) The same bleb as in Fig. 7C viewed from its posterior edge. The continuity of neural crest (nc) migration into the bleb can be clearly seen (black arrowhead). Note the neural crest cell marked by the white arrowhead which appears to have a ruffling lamellipodium. ×550. Scale bar = 100μm, A; = 10μm, B, C, D.

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

Fig. 8.

Toluidine-blue-stained Epon thick sections through two blebs of a day-9·5 Ph/Ph animal. Both blebs have matrix material along the basal surface of the epithelium. Note particularly in Fig. 8B the migration of what appear to be neural crest cells over the lateral surface of the somite and along the basal surface of the ectoderm, as well as between the somite and neural tube. ×350.

Fig. 8.

Toluidine-blue-stained Epon thick sections through two blebs of a day-9·5 Ph/Ph animal. Both blebs have matrix material along the basal surface of the epithelium. Note particularly in Fig. 8B the migration of what appear to be neural crest cells over the lateral surface of the somite and along the basal surface of the ectoderm, as well as between the somite and neural tube. ×350.

The morphology of the neural crest cells and the surrounding embryonic structures as observed in the SEM provide indirect information on how environmental cues may affect their migration. Our evidence helps elucidate: (1) what factors initiate crest migration, and (2) what defines the pathway of this migration.

What initiates migration?

In contrast to the situation at cranial levels (Nichols, 1981), the neural crest cells in the trunk region of the mouse embryo do not begin to migrate until well after the neural folds have fused. When the cells initially separate from the neural tube they are rounded and appear to lack migratory organelles such as lamellipodia or filopodia. Soon after their separation (within one somite length, or approximately 1 h of development) they elongate, flatten and extend processes, which suggest that the cells have become migratory. The crest may acquire 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 prior to migration which helps to create the spaces into which they move (Derby, 1978; Pintar, 1978; Weston, Derby & Pintar, 1978; Pratt et al. 1975). Indeed spaces do appear between presumptive crest cells just prior to their separation from the tube, and between the neural tube and overlying ectoderm. Since little or no Alcian-blue-stained material is seen between the presumptive crest cells, however, it seems unlikely that the HA production is solely responsible for the spaces between the cells and the initiation of migration. Furthermore, Toole, Underhill, Mikuni-Takagashi & Orkin (1980) suggest that HA alone is probably not even sufficient for disruption of an epithelium. It is likely, therefore, that the HA is responsible for forming the space above the cells, but may not be sufficient to stimulate migration.

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

  3. Neural crest cells may begin migration because previously latent locomotory capabilities are activated. In a variety of systems, cells begin locomotion at specific times during development (see Trinkaus, 1976). The initiation of locomotor ability in other embryonic tissues is accompanied initially by blebbing activity of cells and only later by the extension of pseudopods (Trinkaus, 1973; Gustafson & Wolpert, 1967). Just prior to crest migration similar blebbing activity is seen in presumptive crest cells in the dorsal neural tube which may indicate that some internal signal permits cells to begin migration.

  4. After the neural folds fuse to form a tube, epidermal ectoderm separates from the neural ectoderm at which time the basal lamina on the dorsal surface of the neural tube is incomplete. It is possible that discontinuities in this extracellular matrix structure then permit cells of the neuroepithelium to emigrate. The cessation of migration coincides with the completion of basement membrane over the neural tube.

While all of these processes may have a role in initiation of crest migration, the disruption of the basal lamina appears to be the event of greatest consequence since: (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 initiation and containment of neural crest migration are correlated with discontinuous and complete basal laminae respectively. It remains to be investigated if a breakdown in the basal lamina allows the cells to escape, or if its discontinuity is symptomatic of other more basic disruptions in the neuroepithelium, such as loss of cell adhesions.

Several possibilities could account for the establishment of a complete basal lamina over the dorsal surface of the neural tube. As the crest cells leave, the tube may manufacture or deposit new extracellular matrix elements. Alternatively, as the crest migration depletes the number of cells within the dorsal neural tube, the lateral borders of the neural tube will be drawn together so that the edges of the basal lamina which are intact at all stages over the lateral and ventral surfaces of the neural tube will eventually meet. This latter idea is supported by the observation that the top of the tube broadens as crest cells first migrate, 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 limited to spaces bounded by basal lamina. Crest cells move initially only into interstitial spaces lateral to the NT. When the space between the ectoderm and somite is obstructed by the apposition of ectoderm and somite, crest cell migration is blocked (see Fig. 3, and Newgreen & Gibbins, 1982; Derby, 1978). Moreover, crest cells are found in the somite mesenchyme only after the epithelial somite breaks 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 as in the mouse mutant Patch, the crest cells can apparently move into these regions by migrating along the borders of the somites and the ectoderm. While we do not know yet the amount or composition of extracellular matrix in these blebs, it seems to be unlikely that this material differentially attracts crest cells since neural crest cells appear to migrate everywhere in the chick embryo if they are experimentally placed there (e.g. Erickson et al. 1980; Bronner-Fraser & Cohen, 1980). In addition, neural crest cells appear to be able to use many different combinations of macromolecules to support their migration in vitro (Maxwell, 1976; Greenberg, Seppä, Seppä & Hewitt, 1981; Newgreen & Thiery, 1980; Newgreen et al. 1982; Erickson & Turley, unpublished data). Thus it appears that the availability of space is the more important parameter for crest migration, and that they are unable to cross the basement membrane of their associated epithelia 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 the manuscript. This research was supported by NIH postdoctoral fellowship DE05080, NSF Grant 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. of O.

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