Absence of Neural Crest Cell Regeneration from the Postotic Neural Tube

DEVELOPMENTAL BIOLOGY 184, 222 –233 (1997) ARTICLE NO. DB978529

Absence of Neural Crest Cell Regeneration from the Postotic Neural Tube Hiroaki R. Suzuki and Margaret L. Kirby Developmental Biology Program, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia 30912-2640

The preotic neural tube has a variable ability for regeneration of neural crest depending on the neuraxial level. There is robust regeneration of neural crest in the caudal midbrain/rostral hindbrain. In contrast, removal of the cardiac neural crest results in cardiovascular abnormalities suggesting the lack of regeneration in this area, although the regenerative capacity of the cardiac crest region has never been tested directly. Premigratory cardiac neural crest was ablated bilaterally using laser irradiation or extirpation by tungsten needle, and the remaining ventral neural tube was labeled with DiI to examine any neural crest regeneration from the neural tube. The results indicate that there is very little regeneration of crest cells from the cardiac region of the neural tube if the ablation is done prior to the 5-somite stage and no regeneration after the 6-somite stage with either ablation procedure. Furthermore no compensatory response occurs from the adjacent regions of the neural crest. By contrast, we were able to confirm that regeneration of neural crest occurs in the preotic rhombencephalic neural tube even after laser irradiation. An analysis in the trunk region suggests that the trunk neural tube is similar to the cardiac region in that it does not regenerate crest cells in the ventral migratory pathway after ablation. However, melanocytes generated cranial and caudal to the ablated region migrate radially and fill in the ablated region so that there is no interruption of the normal pigment pattern. This study indicates that even though there is a variable capacity for crest regeneration in the preotic neural tube, the postotic neural tube does not have such regenerative ability. q 1997 Academic Press

INTRODUCTION Epithelial– mesenchymal transformation is an important step in the development of neural crest cells (Newgreen, 1985). The regulatory mechanisms that control the selection of cells that will make this transition from stationary neural tube cells is unknown. Since neural crest cells arise as a part of the neuroepithelium, these cells carry positional information about their origin within the neuroaxis (Hunt et al., 1991). In this regard, the regulation and restriction of differentiation of crest cells originating from the different axial levels have been of intense interest. Two major divisions of the neural crest are identified based on the ability to generate ectomesenchyme: cranial (mid-diencephalon to somite 5) and trunk (somites 6– 28) (Le Douarin, 1982). Cranial neural crest cells, when heterotopically grafted to the trunk crest region, can adapt themselves to their new environment and differentiate into cell types characteristic of the trunk crest. In contrast, trunk crest cells show restricted ability of adaptation when transplanted to the cranial regions and do not generate a significant ectomesenchymal population (Le Douarin, 1982).

The distinct migration patterns of neural crest cells arising from the hindbrain and rhombomere-specific expression of Hox genes suggest that neural crest cells inherit different segmental values before leaving the neural tube (Hunt and Krumlauf, 1992; Hunt et al., 1991; Lumsden, 1990). Even so, there are a number of differences in the behavior of neural crest cells originating from the cranial rhombencephalon compared with the caudal rhombencephalon. When neural crest cells originating from the cranial rhombencephalon which normally form connective tissues of the first pharyngeal arch were transplanted slightly caudally, these cells populated the second pharyngeal arch region but formed the first pharyngeal arch derivatives instead of the second (Noden, 1983). In contrast, an explant of the cranial rhombencephalon when transplanted to the postotic hindbrain showed plasticity of phenotypic modifications (Grapin-Botton et al., 1995). The cardiac neural crest has been proposed as a subdivision of the cranial neural crest (Kirby and Bockman, 1984). It is derived from rhombomeres 6, 7, and 8 and is known by its unique contribution to the heart. When the cardiac neural crest is ablated prior to migration, the cardiac out0012-1606/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

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flow septation does not occur due to the lack of ectomesenchymal tissue (Kirby and Waldo, 1995). This result indicates that there is neither regeneration of neural crest cells from the remaining more ventral neural tube in the caudal rhombencephalon nor substitution from neighboring crest cells rostral or caudal to the ablation. On the other hand, removal of more rostral cranial neural crest results in a normal embryo. This process was studied in the cranial rhombencephalon by Scherson et al. (1993) and McKee and Ferguson (1984), who showed that cranial neural tube cells in the midbrain and hindbrain can reconstitute neural crest cells following ablation. More recently, Sechrist et al. (1995) have shown that the caudal forebrain/rostral midbrain has a less robust capacity for crest cell regeneration. The present study tested the regulative ability of the neural tube in the caudal rhombencephalon in varied circumstances. We used the method employed by Scherson et al. (1993) of focal injections of a tracer dye, DiI, to label the tissue immediately ventral to or abutting the ablated region. The result indicated that the neural tube cells of the cardiac region do not regenerate neural crest cells after either laser or extirpation surgery. Furthermore, we confirmed earlier studies by Yntema and Hammond (1945), showing that trunk neural crest does not regenerate. This suggests that in the cardiac and trunk neural crest regions (all postotic neural tube), the demarcation between cells of the neural tube and neural crest occurs early and irreversibly in contrast to preotic neural tube, where a rigid boundary of cells capable of epithelial –mesenchymal transformation does not occur until later in development.

MATERIALS AND METHODS Embryos. Fertile Arbor Acre chicken eggs (Seaboard Hatchery, Athens, GA) were incubated at 377C for 32–37 hr (stages 80 to 9/, Hamburger and Hamilton, 1951). A window was made by sanding the shell and the embryo was lightly stained with neutral red. The cardiac neural crest has been established as mid-otic placode to the caudal limit of somite 3. On the neuraxis this corresponds to rhombomeres 6, 7, and 8. The cranial boundary of rhombomere 6 and the otic placode are indistinct at this age and are estimated to be 1.5 somite lengths cranial to the first visible somite. Ablation of premigratory neural crest. Ablation of premigratory neural crest was performed by cautery using laser irradiation or extirpation by an electrolytically sharpened tungsten needle. The laser ablation of the cardiac neural crest has been described previously (Kirby et al., 1993) and produces an absence of cardiac outflow septation in 90% of embryos operated at the 9-somite stage. Embryos used for preotic or cardiac neural crest ablations in the present study had developed between 3 and 9 somites (Table 2). The neural folds were ablated bilaterally leaving 70– 80% of the ventral portion of the neural tube intact, as shown previously (Kirby et al., 1985). For ablation by tungsten needle, the vitelline membrane was torn over the area of the hindbrain, and the neural folds were detached from the neural tube by bilateral incisions. An identical lesion equivalent to 4.5 somites in length was placed using either laser irradiation or tungsten needle extirpation in the preotic neural folds. The cranial limit of the lesion was

placed 1.5 somites above somite 1. The region affected included the preotic hindbrain and the caudal part of the midbrain. For ablation of trunk neural crest, embryos were in a range of 15– 24 somites (stage 12–15). The dorsal portion of the neural tube was laser-ablated bilaterally at the level of the 6 caudal somites or along the most rostral segmental plate over an equivalent length. Injection of vital dye. Tissue labeling by a focal injection of DiI C 18 (Molecular Probes, D-282) has been described previously (Lumsden et al., 1991; Sechrist et al., 1993). DiI (3 mg/ml) was dissolved in dimethyl sulfoxide. Injection micropipets were pulled to about a 5-mm tip diameter from glass capillaries (1.65 mm o.d., 1.15 mm i.d., 7052 glass, without filament, Garner Glass Co., Claremont CA). These pipets were filled with the DiI solution, attached to a pneumatic picopump (PV830, World Precision Instruments), and mounted to a micromanipulator (Narishige). The tip of the micropipet was inserted into the neural tube, and the dye was ejected with several 5-msec pulses at a pressure of 1.4 – 4.2 kg/cm2 (138– 414 kPa). A single injection or multiple unilateral or bilateral injections were made. In some embryos, intact neural folds abutting just cranial or caudal to the ablation were labeled with DiI to examine compensatory migration into the ablated area. The position(s) of the injection(s) was noted and the egg was sealed with cellophane tape and returned to the incubator for an additional 1 –3 days. During all procedures including ablation, the embryos were observed under subdued light and kept moist by adding Tyrode’s solution. Analysis of labeled cells. Embryos reaching stages 14– 24 were fixed overnight in 10% buffered Formalin at 47C, rinsed in PBS, and stored in PBS containing 2.5% DABCO anti-fade agent (Lumsden et al., 1991). All embryos were observed and the DiI-labeled cells were scored using an epifluorescence microscope with a rhodamine filter set (Zeiss Axioskop). The whole embryo or a half embryo, bissected sagittally, was mounted on a concave slide without a coverslip using a solution of 2.5% DABCO and 45– 90% glycerol in PBS. The embryos were further viewed using a laser confocal microscope (Bio-Rad MRC600 attached to a Zeiss Axioskop). Raw data files were stored in the optical disk and processed with Bio-Rad COMOS 6.03 software on an IBM/PC. The fluorescence images of multiple optical depths and the bright-field image were superimposed and printed with Adobe Photoshop 3.0 software in which pseudopurple color represented fluorescence. The position of the DiI-labeled cells was noted along the developing pharyngeal arches. Somites were also used as landmarks but were less reliable because the first somite begins to break down into mesenchyme at the 18-somite stage (stage 13-) and disappears before the 30-somite stage (stage 18-) (Romanoff, 1960). In addition, unlike undisturbed embryos, the experimental embryos often developed slight abnormalities in somites, i.e., they were often smaller and stuck together. The relative distance between the first somite and the otic placode increases slowly between stages 10 and 18 due to the growth in rhombomeres 6 and 7 (Vaage, 1969). In the normal embryo at stage 18, the center of the otic vesicle lies within the caudal part of pharyngeal arch 2, the center of somite 1 (if visible) corresponds to the cleft between arches 3 and 4, and the junction between somites 2 and 3 corresponds to the cleft between arches 4 and 6. Before stage 18, somite 1 refers to the true first somite, whereas it is omitted in the somite counts in embryos older than 3 days incubation (Hamburger and Hamilton, 1951; Suzuki and Ide, 1987). In order to confirm the position of the DiI-labeled cells, the embryos were transferred to 5% sucrose/PBS for 2– 3 hr and then to 20% sucrose/PBS at 47C overnight, infiltrated with 7.5% gelatin (Sigma) in 15% sucrose/PBS for 2 –3 hr (Stern and Holland, 1993).

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The embryos were placed in embedding molds with the infiltration solution and allowed to solidify. The gelatin blocks were frozen and sectioned on a cryostat at approximately 25 mm. The sections were viewed and photographed on an Olympus BX40 microscope using epifluorescence and Kodak Ektachrome EL400. Immunohistochemistry. A cocktail of antibodies against the 68- and 160-kDa neurofilaments was used (Sigma, N5139, N5264), which recognize nerve processes in the peripheral nervous system and the spinal cord. A 1/200 dilution was used for each antibody. As a secondary antibody, fluorescein-conjugated goat anti-mouse IgG was used at a 1/200 dilution. For whole-mount staining, embryos were fixed overnight in methanol at 0207C and transfered to PBS at room temperature. Cell membranes were permeabilized for 2– 3 hr at room temperature with PBS containing 0.1% Triton X100, and the embryos were treated overnight with a blocking solution containing 0.05% Triton X-100 and 4% goat serum in PBS at 47C. The embryos were reacted with 1.5 ml of the primary antibody diluted in the blocking solution (47C, 24– 36 hr), rinsed several times with the blocking solution (12 hr), reacted with the secondary antibody (24 hr), and finally rinsed with the blocking solution (12 hr). For paraffin sections, embryos were fixed overnight with Bouin’s fixative, dehydrated through graded alcohols, and embedded in paraffin. Serial 8-mm sections were mounted on slides subbed with 2% TESPA (3-aminopropyltriethoxysilane). The sections were deparaffinized and reacted with anti-neurofilament antibodies as described above. The reaction and rinsing steps for primary and secondary antibodies were done for a total of 30 min each at room temperature. Finally, the sections were mounted with Gel/Mount (Biomeda). Similarly, 4% paraformaldehyde-fixed, paraffin-sectioned embryos were reacted with HNK-1 antibody (a straight culture supernatant of HNK-1-producing hybridoma), followed by a fluorescein-tagged secondary antibody. The whole-mount and sectioned specimens were viewed using an epifluorescent microscope (Olympus BX40 or Zeiss Axioskop) and recorded photographically with Kodak Ektachrome EL400. Analysis of melanocyte reconstitution after trunk neural crest ablation. Using quail embryos at 18- to 28-somite stages, trunk neural crest was ablated bilaterally as described above to create a wide gap of 7 –8 segments. These embryos along with sham-operated embryos were allowed to develop to late incubation stages or hatching. The pigment and feather patterns were recorded photographically to note any differences.

RESULTS Normal migration of cardiac neural crest cells marked with DiI. Previous studies of the cardiac neural crest have used quail/chick chimeras and immunohistochemistry or analysis of phenotypes after ablation of the premigratory cardiac crest cells (Kirby et al., 1983; Kuratani and Kirby, 1991, 1992; Nishibatake et al., 1987). We examined the normal patterns of migration of cardiac neural crest cells originating from rhombomeres 6, 7, and 8 using DiI labeling. One focal injection was made in the neural fold at the 3to 8-somite stages. This is prior to the onset of neural crest emigration at this level. A single injection frequently resulted in bilateral labeling, with each side showing similar labeling patterns qualitatively, but not necessarily quantitatively. The patterns of migration in whole-mounts were

observed by confocal microscopy and the consensus migration pathways (confirmed in histological sections) were based on a total of 69 embryos. The numbers of embryos and migration destinations are shown in Table 1. Injections at rhombomeres (r) 4 and 5 resulted in labeled cells in arch 2 (Fig. 1). Such injections rarely labeled arch 1. Injections at the r5/r6 junction mainly labeled arches 2 and 3, with crest cells often crossing over the otic vesicle. Injections at the level of rhombomeres 6 – 8, i.e., the cardiac crest region, resulted in labeled cells in arches 3– 6, with a gradual shift rostrocaudally in the predominance of labeled cells corresponding to the original rostrocaudal level of the injection (Fig. 1). There was a broad overlap in the course of migration, in that each pharyngeal arch received contributions from extended axial levels. Injections at the level of somites 4/5 resulted in a few scattered cells appearing in arches 3– 6, with most of the labeled cells migrating via the ventromedial pathway caudally to populate the enteric plexus (Fig. 1 and data not shown). These cells have been shown previously to contribute to the enteric nervous system (Peters-Van der Sanden et al., 1993; Kuratani and Kirby, 1992). Labeled cells originating from the cardiac crest region migrated via the dorsolateral pathway into the circumpharyngeal region and pharyngeal arches (Fig. 2). The cells in the pharyngeal arches formed a sheath around the aortic arch arteries and a subpopulation migrated into the outflow tract (Fig. 2 and data not shown; Waldo et al., 1996). In summary (Fig. 3), the posterior hindbrain generates a cranial-type crest population that migrates via the dorsolateral pathway and populates the caudal pharyngeal arches with some overlap, that is, there is not necessarily a oneto-one correspondence of rhombomere origin with the pharyngeal arch destination, as occurs in the cranial rhombencephalon with arches 1 and 2. Ablation of the cardiac neural crest. When presumptive cardiac crest cells were ablated bilaterally and the remaining ventral neural tube was labeled by multiple DiI injections, the embryos did not show signs of crest cell regeneration, and the labeled cells were restricted to the neural tube 1– 2 days after the surgery and labeling proce-

TABLE 1 Summary of DiI Injections Rhombomere

Pharyngeal arch 2 3 4 6

4 –5

6

7

8

4

5

10/10 2/10 2/10 1/10

4/6 6/6 5/6 3/6

1/10 10/10 7/10 3/10

0/18 9/18 18/18 11/18

0/13 0/13 1/13 6/13

0/12 0/12 0/12 1/12

Note. Number of embryos with label/total number of injections.

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dure (Fig. 4). Using progressively younger embryos, it could be shown that very young embryos at the 3- to 5-somite stages showed a limited amount of regeneration (10 of 18 embryos with some regeneration) which, however, did not reconstitute the entirety of the ablated crest population (Table 2). The neural folds in the cardiac crest region were still open at the 5-somite stage. At the 6-somite stage the neural folds approximated in about half of the embryos and were closed in most of the 7-somite embryos. Thus, it appears that closure of the neural folds in the cardiac crest region demarcates an earlier period of limited regeneration of neural crest from a later period, when regeneration is no longer possible (24 of 26 embryos with no regeneration). This does not appear to be identical to the regenerative capacity in the preotic region, where regeneration occurs most robustly prior to neural tube closure but continues well after the period of closure (Sechrist et al., 1995; and unpublished observation). We used two different procedures for removing neural folds: extirpation by tungsten needle or cauterization by

laser ablation. The method used did not affect the outcome of the results. After either procedure, the ablated neural tube did not regenerate crest cells at the 6-somite stage or later. This may indicate that the boundary between dorsal cells that will undergo the epithelial– mesenchymal transition and become neural crest cells versus those that will remain in the neural tube is still somewhat flexible prior to neural tube closure. Next, we examined whether the regions cranial or caudal to the ablation showed any compensatory behavior. After ablation of the cardiac neural crest by either laser or tungsten needle, the intact neural folds just cranial and/or caudal to the ablation site were labeled by DiI. Injections anterior to the ablation resulted in labeled cells in arches 1 or 2 (3 by laser, 6 by tungsten), with no cells migrating to arches 3, 4, and 6. While a few cells from the caudal injection did migrate into the caudal arches, there was not a reconstitution of cardiac crest cells from these regions (7 by laser, 4 by tungsten; Fig. 4). This cannot be considered reconstitution of ablated cells since it is part of the broad overlap in

FIG. 1. DiI injection into normal embryos at 3- to 8-somite stages. Rostral is to the left. DiI labeling is in red. The cells are in the dorsolateral pathway with some deep labeling shown in Fig. 3. (A) Injection at the rhombomere 4/5 junction. DiI-labeled cells are seen primarily in arch 2. (B) Injection at the rhombomere 5/6 junction. Labeled cells are seen in arch 2, with a few labeled cells in arch 3. (C) Injection at rhombomere 7. Labeled cells are seen in arch 3, with a few cells in arch 4. (D) Injection at the somite 1/2 junction. Labeled cells are seen evenly in arches 3 and 4, with a few cells in arch 6. (E) Injection at the somite 2 level. Labeled cells are confined to arches 4 and 6. (F) Injection at the somite 3/4 junction. Scattered cells are seen in the arch 6 region and also posteriorly along somites 5 and 6. The crest cells in the arch region are reduced in number compared to more cranial levels. (G) Injection at somite 5. Labeled cells can be seen migrating ventrally and posteriorly toward the wing bud. Abbreviations: otic vesicle (ov), pharyngeal arches (a1, a2, . . .), pouch of arch 4 (p4), somites (s3, s4, . . .). Scale bar in E, 0.25 mm for A, B, and E. Scale bar in G, 0.25 for C, D, F, and G.

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FIG. 2. Histological sections and whole-mounts showing neural crest migration to arches 3 and 4. (A) A stage 15 embryo with DiI injected into rhombomere 7. Labeled cells migrated to pharyngeal arch 3, with some forming the tract of the glossopharyngeal nerve (IX). (B) A cross section of a stage 14 embryo similar to the one shown in A. Fluorescent cells can be seen in the dorsolateral pathway migrating toward pharyngeal arch 3. The cells form a cohesive mass, with its tip (*) located lateral to the pharynx. (C) A stage 14/ embryo with an injection at the level of somite 2. The cells are migrating toward pharyngeal arch 4. In a cross section (D), the leading edge of migrating cells is located more ventrally than in (B). (E and F) Cross section through arch 3, showing HNK-1 immunostaining of a stage 15 embryo. The layered image in (E) is enlarged in (F). The HNK-1 staining shows an identical pattern to that seen with DiI labeling. (*) Indicates the leading edge of migrating cells. (G) A cross section of a stage 17– 18 embryo showing that DiI-labeled neural crest cells have reached the ventral midline and form a layer lining the floor of the pharynx (p). Fuorescent cells have migrated to the junction of the outflow tract (ot) with the aortic sac (as). A few labeled cells (arrows) are seen in the outflow tract. Abbreviations: p, pharynx; sm, somatic mesoderm; m, myocardium; e, endocardium; c, pericardium. Scale bar in C, 100 mm for A, C. Scale bar in G, 100 mm for B, D, E, G.

migration which occurs in the normal embryo as described above (Fig. 3). Ablation of the preotic or trunk neural crest. Since our results with postotic crest were dissimilar to those of Scherson et al. (1993), we tested whether our ablation and labeling techniques repeated their observations for the adjacent preotic crest. The preotic neural crest was ablated bilaterally using laser, and the remaining neural tube labeled with DiI. Neural crest regeneration was consistent quantitatively and qualitatively with the results of Scherson et al. (9 of 10 at the 4- to 7-somite stages, Fig. 4). In two embryos, DiI was injected into the intact neural folds cranial and caudal to the ablation site. There was no significant deviation in the

course of migration from these regions (not shown) compared to the published pathways for preotic crest cells (Lumsden et al., 1991; Sechrist et al., 1993). Since ablation studies by Yntema and Hammond (1945) showed no reconstitution of neural crest from the spinal level of the neural axis, we reexamined the regulative ability of the neural tube caudal to rhombomere 8. Trunk neural folds at the level of the 6 most caudally developed somites were ablated bilaterally using laser irradiation and the remaining ventral neural tube was labeled by multiple DiI injections. No fluorescent cells arose from the labeled neural tube 1– 2 days after the ablation (15 of 15, at 15- to 24somite stages; Fig. 5). To determine whether younger neural

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limited contribution from the adjacent intact regions is consistent with filling in from the nonablated regions at the ends of the ablation (Teillet et al., 1987). Melanocytes are the last group of neural crest cells to leave the neural tube. They migrate dorsolaterally and radially, disregarding segmental boundaries (Serbedzija et al., 1989; Erickson and Goins, 1995). We examined embryos at stage 23 –24 that had been labeled with DiI. These cells migrated radially from the unablated regions cranial and caudal to the ablation and populated the epidermal fields lateral to the ablated and unablated regions of the neural tube (Fig. 7). From viewing this pattern of migration, we predicted that the pigmentation pattern in the ablated region would be normal or only slightly affected. To test this, trunk neural crest was ablated bilaterally to create a wide gap of 7 –8 segments in quail embryos of 18– 27 somites. The operated embryos developed to a stage with feathers but most died a few days before hatching. The feather patterns and pigmentation were normal (n Å 15). One operated quail embryo survived to hatching and its feather pattern and pigmentation were normal (Fig. 7). Dissection of the vertebral column from these birds showed that the dorsal root ganglia were missing in the region of the ablation (n Å 2). These results indicate that pigment cells are reconstituted from intact neural tube cranial and caudal to a region of ablation and develop a distinct feather pattern matching that of the local environment.

FIG. 3. Summary of DiI injection into normal embryos. The bar marks the injection site. Rostral is to the left. Abbreviations: rhombomere (r2, r3, . . .), somite (s1, s2, . . .), pharyngeal arch (a1, a2, . . .), otic vesicle (ov).

tube behaved differently, the neural folds were ablated along the most rostral segmental plate. Again no fluorescent cells were found outside of the neural tube, indicating that there is no crest cell regeneration from the trunk neural tube (8 of 8, at 18- to 22-somite stages, Fig. 5). Cells migrating via the ventral pathway from the intact neural tube, cranial or caudal to the ablation, did not deviate from their normal migratory pathway and populated the dorsal root and sympathetic ganglia of their own segmental level and one segmental level within the region of the ablation (Fig. 5). The absence of reconstitution of the dorsal root ganglia was confirmed in stage 24 whole-mount embryos processed immunohistochemically to visualize neurofilament. The antibodies stained the segmental dorsal root ganglia and ventral roots (Fig. 6). The dorsal root ganglia were missing in the region of the ablation, while the development of the ventral roots was unaffected (Fig. 6). Careful examination of serial cross sections showed that the dorsal root ganglia did not form at all throughout the middle of the ablated region; however, small dorsal root ganglia were seen at the cranial and caudal ends of the ablated region. This

DISCUSSION Ablation of premigratory neural crest cells in the caudal midbrain and rostral hindbrain has been shown previously to be followed by complete reconstitution of the neural crest cells from the neural tube (Scherson et al., 1993; Hunt et al., 1995; Sechrist et al., 1995; McKee and Ferguson, 1984); however, ablation of the region of the neural tube located immediately caudal to the otic placode results in a severely abnormal phenotype involving derivatives of the caudal pharynx and cardiovascular system. We have applied focal injections of a cell tracer in the cardiac crest region to determine by direct observation whether the potential for regeneration of the crest cell population exists. Consistent with the phenotype associated with ablation, our results demonstrate that the ability for reconstitution of the crest cell population does not extend to the postotic neural tube. This differential ability of reconstitution of the neural crest cells in the preotic region compared with the cardiac region of neural tube explains why no phenotype is associated with ablation of premigratory preotic crest (McKee and Ferguson, 1984), while a severely abnormal phenotype is associated with ablation of premigratory cardiac crest (Kirby et al., 1983). Interestingly, the caudal forebrain and rostral midbrain also have a reduced capacity for reconstitution of neural crest (Sechrist et al., 1995). Thus it appears that only the caudal midbrain and rostral hindbrain have the ability for almost complete reconstitution of neural crest cells. The

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FIG. 4. Ablation of preotic and cardiac neural crest. (A) A 6-somite stage embryo immediately after bilateral laser ablation of the neural crest. Somite 1 level (s1). (B) The same embryo as in A showing somite 4 (s4) level with the neural folds intact. (C) Confocal image of a stage 18 embryo in which the cardiac neural crest was ablated (bracket) at the 8-somite stage. Two focal injections of DiI were made into the intact neural fold immediately cranial and caudal to the ablation. Labeled preotic cells migrated to arch 2, while a few cells from below the lesion migrate to arches 4– 6 but do not reconstitute the cardiac crest. (D) Epifluorescence image of a right lateral view of a stage 16 embryo in which the cardiac neural crest was ablated (bracket) at the 8-somite stage. A single DiI injection was made within the ablation at the somite 1 level. No labeled cells could be seen in arches 3– 6. Intense labeling was present in the roof of the neural tube and in cellular debris nearby. (E) Cross section through the caudal pharyngeal region of the embryo shown in D. No labeled cells can be seen outside of the neural tube. (F) Left lateral image of a stage 16 embryo with preotic neural crest ablated at the 5-somite stage (bracket). A single DiI injection was made into the rhombomere 3/rhombomere 4 level. Neural crest cells can be seen leaving the neural tube (arrow). (G) Cross section of the cranial pharyngeal region of the embryo shown in F. Neural crest cells can be seen migrating dorsolaterally toward the pharynx (arrow). Abbreviations: s1, s4, somite level; a1, etc., pharyngeal arch; nt, neural tube; e, ectoderm; n, notochord; ov, otic vesicle; cv, cardinal vein; da, dorsal aorta; va, aortic sac; p, pharynx.

boundary in the hindbrain between the neural tube capable of regeneration of crest versus that incapable of regeneration appears to be rather sharply placed at the junction of r5/6, which is the level of the otic placode. Scherson et al. (1993) initially reported that after unilateral ablation, regeneration of crest cells in the mid- and hindbrain neuroepithelium occurred at the 4- to 7-somite stages and was progressively diminished at the 8- to 12-somite stages. They estimated that the capacity for regulation in the midand hindbrain ended 4.5– 6 hr after the onset of crest emigration. Crest cells start to emigrate from the midbrain and preotic hindbrain at the 6- to 7-somite stage and the 8- 9-somite stages, respectively (Lumsden et al., 1991; Sechrist et al., 1993). Sechrist et al. (1995) recently reexamined the process using bilateral deep ablation and concluded that a robust regulative response in the hindbrain occurs maximally at the 3to 5-somite stage, which is the period when we observed

minimal regulative capacity in the postotic hindbrain. Since postotic cardiac crest cells begin emigration at the 10-somite stage, the cardiac neural tube should be fully capable of regenerating crest cells at the 6- to 9-somite stage if there were a similar potential for regulation. The cardiac neural tube, however, exhibited only a limited capacity for regeneration at the 3- 5-somite stage. The trunk region did not show even this minimal capacity for regeneration, even along the rostral segmental plate, indicating that the ability of regeneration is completely lost. We used two different methods for making the ablation. Comparison of the results from both techniques led to the conclusion that the two techniques yielded identical results. This suggests that laser ablation does not inflict deleterious heat- or photo-induced damage on neighboring tissue with respect to regenerative potential of the neural tube. Since there were no differences in the reconstitutive ability

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TABLE 2 Ablation of Presumptive Cardiac Neural Crest Cells Laser Somite stage 3 4 5 6 7 8 9 Totals

Tungsten

Re

P-re

No

Re

P-re

1 1

3

1 4

1

1 2 1

1

4

2

2

3 4 6 3

5

21

No 2 1 2 3 1 2 11

Note. Presumptive cardiac neural crest cells were ablated by laser or tungsten and the remaining neural tube was labeled by DiI. Re, regeneration of the crest cells; P-re, partial regeneration; No, no regeneration.

of the neural tube with either laser or extirpation, we used only laser to examine the reconstitutive ability of the trunk neural crest. Our results indicate that the inability for regeneration of cardiac crest extends into the trunk region of the neural tube/crest, as was originally reported by Yntema and Hammond (1945). The cardiac crest extends caudally from the level of the midotic placode, and trunk neural crest

is traditionally thought to begin at the level of somite 5. It appears that there is continuity in the inability of cardiac and trunk neural tube to reconstitute neural crest. In contrast, the preotic neural tube is clearly capable of regenerating neural crest. Cardiac neural crest is a subdivision of cranial crest because of its ability to generate ectomesenchyme, a characteristic not shared by trunk neural crest (Leblanc et al., 1995). Thus the current results support the idea that the cardiac region of neural tube and neural crest represents a transition zone with some characteristics shared with the preotic region and others shared with the trunk region (Kuratani and Kirby, 1992). What is the mechanism regulating the difference in regenerative ability? There are many molecular differences between preotic and postotic neural tube, such as expression of different sets of homeobox-containing genes (Hunt et al., 1995). Distinct expression patterns of rhombomere-specific genes demarcate pairs of rhombomeres or single rhombomeres (Hox, Krox-20, HNK-1) in the preotic hindbrain, while expression of hox genes is limited to single rhombomere patterns in the postotic rhombomeres (Hunt et al., 1995; Kuratani, 1991). In addition, there are significant morphological differences between preotic and postotic neural tube. The neural tube of preotic hindbrain is two to three times thicker than the postotic neural tube. Thus preotic neural tube has a distinct advantage in cell numbers. In fact preotic neural tube begins regenerating crest cells as little as 5 hr after ablation (Scherson et al., 1993; Hunt et al.,

FIG. 5. Ablation of trunk neural crest cells. (A) Confocal image of a stage 17– 18 embryo in which the trunk neural crest was ablated (bracket) along the caudalmost 6 somites (s12–17) at the 17-somite stage. Multiple DiI injections were made within the neural tube in the ablated region. No labeled cells can be seen leaving the neural tube, indicating that there is no reconstitution of crest cells. (B) Confocal image of a stage 17 –18 embryo in which the neural crest was ablated (bracket) along the cranialmost segmental plate (presumptive s21– 26) at the 20-somite stage. Multiple DiI injections were made in the site of the ablation with no sign of crest regeneration. Wing bud (w). Bar, 1 mm. (C and D) The same stage 17 embryo at different focal planes. The neural crest was ablated (bracket) at the 23-somite stage along the caudalmost 6 somites (s18–23). Two focal injections of DiI were made, one at the level of somite 17 and the other in the middle of the ablated region (s21). The focus in (C) is on the cells migrating from the region cranial to the ablation. Cells can be seen migrating (arrow) but they do not deviate to fill in the ablated region. The focus in (D) is on the label in the region of the ablation. No cells can be seen migrating from the neural tube. Bar, 100 mm. (E) Cross section of the embryo in D cranial to the ablated region (indicated as e in D). Labeled cells can be seen in the ventral pathway medial to the dermamyotome (dm) and in the region of the primary sympathetic trunk (arrow). (F) Cross section of the embryo in D in the ablated region (level f in D). No labeled cells can be seen in the normal neural crest migratory pathways or terminal locations. Neural tube (nt), notochord (n), descending aorta (da), cardinal vein (cv). Bar, 100 mm for E and F.

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FIG. 6. Lateral views of control (A) and experimental embryos (B) with an ablation (bracket) just caudal to the brachial plexus (on the right) at stage 24. The embryos are stained in whole-mount with antibodies against neurofilament. The segmental organization of the peripheral nervous system can be seen. Fasciculated spinal nerves (s) and the dorsal root ganglia (drg) are apparent in the control embryo but cannot be seen in the ablated region of the experimental embryo. The ventral roots of the experimental embryo are not affected by the ablation and maintain a segmental appearance. (C and D) Cross sections of the control and experimental embryos shown in A and B, respectively. In the control embryo a well-developed dorsal root ganglion (drg) can be seen, while the dorsal root ganglia are absent bilaterally in the experimental embryo, confirming the observation from the whole-mount preparation. Neural tube (nt), notochord (n), dermamyotome (dm). Bar, 100 mm for C and D.

1995), suggesting that cell rearrangement rather than cell proliferation is the initial step in regulation. After deep bilateral ablation, Sechrist et al. (1995) concluded that preotic neural tube did not reproduce all of the loss, resulting in abnormalities such as reduced size of arch 1 and the trigeminal ganglia. This indicates that the regulative capacity does depend on the size of the cellular reservoir. The neural tube in the cardiac region is notably smaller than preotic neural tube but still somewhat larger than trunk neural tube. Finally, the process of regenerating crest cells from the ventral neural tube involves respecifying the ventral identity to a more dorsal identity. Size of the neural tube in various regions would affect the distance that any ventralizing signals may influence. Dorsalization of the remaining ventral neural tube after neural crest ablation may be initiated because the ventral neural tissue is released from suppression by the dorsal neural tube or the contact of damaged edges between the neural tube and surface ectoderm triggers induction of the remaining neural tube (Selleck and Bronner-Fraser, 1995; Dickinson et al., 1995). Several studies are now available that provide a complete visualization of the neural crest migratory pathways using DiI as a tracer (Serbedzija et al., 1990; 1991; Fukiishi and Morriss-Kay, 1992; Lumsden et al., 1991; Sechrist et al.,

1993). Fukiishi and Morriss-Kay (1992) used DiI labeling to examine the migration of rat cardiac neural crest cells into the pharyngeal arches and outflow tract and we have shown this migration with DiI in the chick. Generally these studies confirm the data from quail –chick chimeras and studies with HNK-1 and E/C8. It appears to be a rule that neural crest cells derived from a single preotic rhombomere migrate to a single pharyngeal arch. Thus, cells from rhombomere 2 migrate to arch 1 and cells from rhombomere 4 migrate to arch 2 (although still controversial, it appears that an insignificant number of neural crest cells is derived from rhombomeres 3 and 5 (Sechrist et al., 1993; Birgbauer et al., 1995)). This is in contrast with the three postotic rhombomeres that provide the cardiac neural crest. Cardiac neural crest cells originating from more rostral axial levels (i.e., rhombomere 6) populate the more rostral pharyngeal arches, maintaining the original rostrocaudal order. However, a broad, overlapping migration can be seen with DiI labeling (this paper) and with quail– chick chimeras into all of the caudal arches and cardiac outflow tract (MiyagawaTomita et al., 1991). In addition, the regions cranial and caudal to rhombomeres 6– 8 also contribute a few scattered cells to arches 3– 6. By observing living embryos after a smaller focal injection of DiI, Birgbauer et al. (1995) con-

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FIG. 7. (A) Trunk neural crest was ablated (bracket) along the caudalmost 6 somites (s18 – 23) at the 23-somite stage. Two focal injections were made, each into the intact neural fold just above and below the ablation (s17 and s24 levels). At stage 24, melanocytes can be seen migrating radially from the injection sites and are beginning to fill in the ablated region. Confocal image, right lateral view. Bar, 1 mm. (B) Stage 24 embryo in which the trunk neural crest was ablated (bracket) at the 20-somite stage along the caudalmost 6 somites (s15– 20). A single DiI injection in the region just above the ablation was made (s14 level). Melanocytes migrate radially from the injection site (i), with some moving into the field of the ablation. Two streaks of cells in the ventral pathway (arrows) can be seen out of the focal plane. Bar, 100 mm. (C and D) Newly hatched normal quail showing the typical feather pattern from dorsal and ventral views, respectively. (E and F) Newly hatched quail that had the trunk neural crest ablated along s19– s25 at the 24-somite stage. The feather distribution and pigmentation is normal.

cluded that the cells in rhombomere 5 contribute to both arches 2 and 3. It is interesting that the preotic hindbrain neural crest and trunk crest are similar in that the first wave of migrating cells follows segmental restrictions rather strictly, while the transitional cardiac region allows migration that seems not to be bound by segmental boundaries except very generally. Pigment cells originating in the trunk follow the dorsolateral pathway and are also not bound by segments. Two distinct populations of cells exist in the early neuroepithelium: neural crest cells and neural tube cells. By definition neural crest cells are the cells that migrate away from the neural tube (Horstadius, 1950). Thus the cells in the neural tube that have the potential for epithelial –mesenchymal transformation are the cells that comprise the neural crest morphogenetic field. Regeneration of neural crest depends on the potential of cells remaining in the neural tube to undergo epithelial –mesenchymal transformation. Where the potential exists, it is not unlimited but constrained, as are all developmental potentials, by time. Why there should be widely varying potential for such transformation in the preotic neural tube with almost no potential for reconstitution of the migratory population

postotically remains an intriguing mystery. The factors that may be involved in this potential may include basic architecture, molecular patterning instructions, and normal migration pathways. Now that these differences are completely known, it is possible to resolve the basis for the variability in potential for epithelial– mesenchymal transformation.

ACKNOWLEDGMENTS We thank Simon Conway, Karen Waldo, Brian Condie, and Nancy Manley for critical reading of the manuscript, Karen Waldo and Sachiko Miyagawa-Tomita for information about pharyngeal arch anatomy, Donna Kumiski for preparing HNK-1 stained slides, Yasuyo Shigetani and Shigeru Kuratani for sharing unpublished results, Patrick Tam and Marianne Bronner-Fraser for valuable comments, and Karen Waldo for help with the final figures. This study was supported by PHS Grants HL36059, HL51533, and HD17063.

REFERENCES Birgbauer, E., Sechrist, J., Bronner-Fraser, M., and Fraser, S. (1995). Rhombomeric origin and rostrocaudal reassortment of neural

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crest cells revealed by intravital microscopy. Development 121, 935–945. Bronner-Fraser, M., and Stern, C. (1991). Effects of mesodermal tissues on avian neural crest cell migration. Dev. Biol. 143, 213– 217. Dickinson, M. E., Selleck, M. A. J., McMahon, A. P., and BronnerFraser, M. (1995). Dorsalization of the neural tube by the nonneural ectoderm. Development 121, 2099 – 2106. Erickson, C. A., and Goins, T. L. (1995). Avian neural crest cells can migrate in the dorsolateral path only if they are specified as melanocytes. Development 121, 915–924. Fukiishi, Y., and Morriss-Kay, G. M. (1992). Migration of cranial neural crest cells to the pharyngeal arches and heart in rat embryos. Cell Tissue Res. 268, 1 –8. Grapin-Botton, A., Bonnin, M.-A., McNaughton, L. A., Krumlauf, R., and Le Douarin, N. M. (1995). Plasticity of transposed rhombomeres: hox gene induction is correlated with phenotypic modifications. Development 121, 2707 –2721. Hamburger, V., and Hamilton, H. L. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49–92. Ho¨rstadius, S. (1950). ‘‘The Neural Crest: Its Properties and Derivatives in the Light of Experimental Research.’’ Oxford Univ. Press, London. Hunt, P., Whiting, J., Muchamore, I., Marshall, H., and Krumlauf, R. (1991). Homeobox genes and models for patterning the hindbrain and branchial arches. Development 112(Suppl. 1), 187 –196. Hunt, P., and Krumlauf, R. (1992). Hox codes and positional specification in vertebrate embryonic axes. Annu. Rev. Cell Biol. 8, 227–256. Hunt, P., Ferretti, P., Krumlauf, R., and Thorogood, P. (1995). Restoration of normal Hox code and branchial arch morphogenesis after extensive deletion of hindbrain neural crest. Dev. Biol. 168, 584–597. Kalcheim, C., and Teillet, M.-A. (1989). Consequences of somite manipulation on the pattern of dorsal root ganglion development. Development 106, 85– 93. Kirby, M. L., Gale, T. F., and Stewart, D. E. (1983). Neural crest cells contribute to aorticopulmonary septation. Science 220, 1059 –1061. Kirby, M. L., Aronstam, R. S., and Buccafusco, J. J. (1985). Changes in cholinergic parameters associated with failure of conotruncal septation in embryonic chick hearts after neural crest ablation. Circ. Res. 56, 392– 401. Kirby, M. L., and Waldo, K. L. (1995). Neural crest and cardiovascular patterning. Circ. Res. 77, 211–215. Kirby, M. L., Kumiski, D. H., Myers, T., Cerjan, C., and Mishima, N. (1993). Backtransplantation of chick cardiac neural crest cells cultured in LIF rescues heart development. Dev. Dyn. 198, 296– 311. Kirby, M. L., and Bockman, D. E. (1984). Neural crest and normal development: A new perspective. Anat. Rec. 209, 1 –6. Kuratani, S. C. (1991). Alternate expression of the HNK-1 epitope in rhombomeres of the chick embryo. Dev. Biol. 144, 215 –219. Kuratani, S. C., and Kirby, M. L. (1991). Initial migration and distribution of the cardiac neural crest in the avian embryo: an introduction to the concept of the circumpharyngeal crest. Am. J. Anat. 191, 215– 227. Kuratani, S. C., and Kirby, M. L. (1992). Migration and distribution of circumpharyngeal crest cells in the chick embryo. Formation of the circumpharyngeal ridge and E/C8/ crest cells in the vertebrate head region. Anat. Rec. 234, 263– 280.

Leblanc, G. G., Holbert, T. E., and Darland, T. (1995). Role of the transforming growth factor-b family in the expression of cranial neural crest-specific phenotypes. J. Neurobiol. 26, 497– 510. Le Douarin, N. (1982). ‘‘The Neural Crest.’’ Cambridge Univ. Press, Cambridge. Lumsden, A. (1990). The cellular basis of segmentation in the developing hindbrain. Trends Neurosci. 13, 329–335. Lumsden, A., Sprawson, N., and Graham, A. (1991). Segmental origin and migration of neural crest cells in the hindbrain region of the chick embryo. Development 113, 1281 – 1291. McKee, G. J., and Ferguson, M. W. J. (1984). The effects of mesencephalic neural crest cell extirpation on the development of chicken embryos. J. Anat. 3, 491–512. Miyagawa-Tomita, S., Waldo, K., Tomita, H., and Kirby, M. L. (1991). Temporospatial study of the migration and distribution of cardiac neural crest in quail-chick chimeras. Am. J. Anat. 192, 79– 88. Newgreen, D. F. (1985). Control of the timing of commencement of migration of embryonic neural crest cells. Exp. Biol. Med. 10, 209–221. Nishibatake, M., Kirby, M. L., and van Mierop, L. H. (1987). Pathogenesis of persistent truncus arteriosus and dextroposed aorta in the chick embryo after neural crest ablation. Circulation 75, 255– 264. Noden, D. (1983). The role of the neural crest in patterning of avian cranial skeletal, connective, and muscle tissues. Dev. Biol. 96, 144–165. Peters-Van der Sanden, M. J. H., Kirby, M. L., Gittenberger-de Groot, A. C., Tibboel, D., Mulder, M. P., and Meijers, C. (1993). Ablation of various regions within the avian vagal neural crest has differential effects on ganglion formation in the fore-, midand hindgut. Dev. Dyn. 196, 183–194. Rickmann, M., Fawcett, J. W., and Keynes, R. J. (1985). The migration of neural crest cells and the growth of motor axons through the rostral half of the chick somite. J. Embryol. Exp. Morphol. 90, 437– 455. Romanoff, A. L. (1960). ‘‘The Avian Embryo: Structural and Functional Development,’’ p. 928. Macmillan Co., New York. Scherson, T., Serbedzija, G., Fraser, S., and Bronner-Fraser, M. (1993). Regulative capacity of the cranial neural tube to form neural crest. Development 118, 1049 –1062. Sechrist, J., Serbedzija, G. N., Scherson, T., Fraser, S. E., and Bronner-Fraser, M. (1993). Segmental migration of the hindbrain neural crest does not arise from its segmental generation. Development 118, 691 –703. Sechrist, J., Nieto, M. A., Zamanian, R. T., and Bronner-Fraser, M. (1995). Regulative response of the cranial neural tube after neural fold ablation: spatiotemporal nature of neural crest regeneration and up-regulation of Slug. Development 121, 4103 –4115. Serbedzija, G. N., Bronner-Fraser, M., and Fraser, S. E. (1989). A vital dye analysis of the timing and pathways of avian trunk neural crest cell migration. Development 106, 809–816. Serbedzija, G. N., Fraser, S. E., and Bronner-Fraser, M. (1990). Pathways of trunk neural crest cell migration in the mouse embryo as revealed by vital dye labelling. Development 108, 605–612. Serbedzija, G. N., Burgan, S., Fraser, S. E., and Bronner-Fraser, M. (1991). Vital dye labelling demonstrates a sacral neural crest contribution to the enteric nervous system of chick and mouse embryos. Development 111, 857– 866. Selleck, M. A. J., and Bronner-Fraser, M. (1995). Origins of the avian neural crest: the role of neural plate-epidermal interactions. Development 121, 525– 538.

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Stern, C. D., and Holland, P. W. H. eds. (1993). ‘‘Essential Developmental Biology: A Practical Approach.’’ IRS Press at Oxford Univ. Press, Oxford. Suzuki, H. R., and Ide, H. (1987). Positional heterogeneity of interaction between fragments of avian limb bud in organ culture. Dev. Biol. 124, 41–49. Teillet, M. A., Kalcheim, C., and LeDouarin, N. M. (1987). Formation of the dorsal root ganglia in the avian embryo: segmental origin and migratory behaviour of the neural crest progenitor cells. Dev. Biol. 120, 329– 347. Vaage, S. (1969). The segmentation of the primitive neural tube in

chick embryos (Gallus domesticus): a morphological, histochemical and autoradiographical investigation. Adv. Anat. Embryol. Cell Biol. 41, 1 –88. Waldo, K. L., Kumiski, D., and Kirby, M. L. (1996). Cardiac neural crest is essential for the persistence rather than the formation of an arch artery. Dev. Dyn. 205, 281– 292. Yntema, C. L., and Hammond, W. S. (1945). Depletions and abnormalities in the cervical sympathetic system of the chick following extirpation of neural crest. J. Exp. Zool. 100, 237–262. Received for publication November 25, 1996 Accepted February 3, 1997

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