Colonization of the post-umbilical bowel by cells

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Development 111, 647-655 (1991) Printed in Great Britain © The Company of Biologists Limited 1991

Colonization of the post-umbilical bowel by cells derived from the sacral neural crest: direct tracing of cell migration using an intercalating probe and a replication-deficient retrovirus

HOWARD D. POMERANZ*, TAUBE P. ROTHMAN and MICHAEL D. GERSHON Department of Anatomy and Cell Biology, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, New York, NY 10032, USA

* Author for correspondence

Summary

Experiments were done to test the hypothesis that the avian gut is colonized by cells derived from both vagal and sacral regions of the neural crest. Afluorescentdye, dil (l,l-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), and a replication-deficient retrovirus (LZ10; Galileo et al. 1990) were employed as tracers. Since LZ10 was constructed with lacZ of E. coli as a reporter gene, infected cells were identified by demonstrating /J-galactosidase immunoreactivity. Dil and LZ10 were injected between the neural tube and surface ectoderm (before the migration of crest cells away from the injection sites) at vagal, truncal (dil only), or sacral axial levels. The bowel was examined 4 days later in order to allow crest-derived cells sufficient time to migrate to the gut. Following injections of either tracer into the vagal crest, labelled cells were found in the gizzard and duodenum/When dil or LZ10 was injected into the sacral crest, labelled cells were seen in the postumbilical bowel and ganglion of Remak. In the hindgut,

marked cells were concentrated in the mesenchyme, just internal to the serosa, and were never observed rostral to the umbilicus. Nofluorescentcells were ever found in the bowel following truncal injections of dil, although such cells were observed in sympathetic ganglia. Labelled cells were always found in dorsal root ganglia, no matter which tracer or level of the crest was injected. In embryos injected with LZ10, infected cells in the gut and dorsal root ganglia displayed a neural crest marker (NC1 immunoreactivity). These observations confirm that the gut is colonized by cells from the sacral as well as the vagal region of the neural crest and that the emigres from the sacral crest are confined to the post-umbilical bowel.

Introduction

the grafts were located either at vagal or sacral axial levels of the host embryos. Vagal crest-derived cells were found to colonize the entire gut, while sacral crestderived cells were limited to the post-umbilical bowel. Since the publication of the pioneering studies of Le Douarin and Teillet, the conclusion that sacral crestderived cells normally migrate to the gut has been challenged by some investigators and supported by others. For example, Allan and Newgreen (1980), using morphological criteria and the development of neurons in chorio-allantoic membrane (CAM) grafts to identify 'neuroblasts', reported that these cells appear in a continuous proximodistal sequence throughout the bowel. An ascent of 'neuroblasts' developing from sacral crest-derived cells, which would be expected to colonize first the most distal portion of the gut, was not observed. Allan and Newgreen therefore concluded that the post-umbilical bowel contains no neural

Yntema and Hammond (1954, 1955) first established that the neurons and glia that constitute the enteric nervous system (ENS) are derived from the neural crest. These investigators observed that enteric ganglia failed to develop when the post-otic and anterior spinal levels of the crest were deleted in chick embryos. The origin of enteric neural precursors from the vagal region of the neural crest (corresponding to somites 1-7) has been confirmed by Le Douarin and Teillet (1973, 1974), who also concluded that the gut receives a second contribution of crest-derived cells that emigrate from sacral levels (caudal to somite 28) to the post-umbilical bowel. These investigators replaced the premigratory crest of chicks with that of quails (or the premigratory crest of quails with that of chicks) and observed that cells from the grafts would migrate to the bowel when

Key words: sacral neural crest, development, enteric nervous system, hindgut, dil, retrovirus, post-umbilical bowel.

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H. D. Pomeranz and others

precursors until after it becomes colonized by cells from the vagal crest and that there is probably no migration of sacral crest-derived cells to the gut. Allan and Newgreen suggested that crest-derived cells from the sacral level of the neuraxis might have reached the postumbilical bowel of the chimeric embryos studied by Le Douarin and Teillet (1973,1974) because crest cells of a different species are more invasive in host embryos than crest cells of the host species and thus reach sites that would not have been colonized by the host's own crestderived cells. More recently, it has been reported (Meijers et al. 19896; van Dongen et al. 1989) that the development of enteric neurons in the hindgut of chick embryos in situ can be prevented by severing the bowel proximal to the hindgut at day E4. When this was done, only bundles of axons and Schwann cells were observed in the region of the hindgut where the myenteric plexus is normally located. It was concluded that cells from the sacral neural crest do not give rise to enteric neurons in the avian hindgut. Support for the idea that the post-umbilical bowel of chicks is colonized by sacral crest-derived cells has been obtained through the use of an immunocytochemical marker for cells of neural crest origin (Pomeranz and Gershon, 1990). These studies utilized the monoclonal antibody NC-1 (equivalent to HNK-1), which recognizes migrating crest-derived cells, including those committed to neuronal or glial phenotypes (Vincent et al. 1983; Vincent and Thiery, 1984; Tucker et al. 1984, 1986, 1988). Separate rostral and caudal populations of NC-1-immunoreactive cells were detected in chick embryos at day E3.5 in continuous streams extending between the sacral crest and the hindgut. The rostral group, coexpressed neural markers, while the caudal population did not. The rostral, dually labelled cells appeared to become embedded in the mesenchyme of the dorsal bowel and later to enter the mesentery to give rise to the ganglion of Remak. The caudal NC-1immunoreactive group, which did not express neural markers, ascended within the colorectum and, in contrast to the rostral cells, fully encircled the gut. NC1-immunoreactive neurons and glia were found to develop in organotypic tissue cultures and chorioallantoic membrane grafts of post-umbilical bowel explanted at days E4 and E5, ages known to precede the colonization of the hindgut by cells from the vagal crest (Le Douarin, 1982). These observations are consistent with the view that NC-1 immunoreactive cells, which do not express neural markers, migrate from the sacral crest to the hindgut; however, the evidence is not conclusive.- Many of the NC-1 immunoreactive cells in the explants did not acquire neurofilament immunoreactivity; moreover, the NC-1 antibody recognizes a carbohydrate epitope that is not specifically expressed only by crest-derived cells (Vincent and Thiery, 1984; Newgreen et al. 1990). In fact, other investigators (Meijers et al. 1989a), who have also observed the presence of HNK-1 (equivalent to NC-l)-immunoreactive cells in the hindgut that do not express neurofilament immunoreactivity, have concluded that these cells are not crest-derived at all, but mesenchymal

cells (presumably of mesodermal origin) that attract crest-derived cells to the hindgut. It is clearly necessary to resolve the question as to whether or not sacral crest-derived cells contribute to the formation of the post-umbilical bowel. This part of the gut is frequently the site of congenital abnormalities of the ENS, the understanding of which depends on knowing the lineage of neural and glial precursor cells (Garrett and Howard, 1981). Because of the difference in the methods previously used to study the migration of crest-derived cells to the bowel, it would be desirable to test the hypothesis that the post-umbilical gut is colonized by cells from the sacral crest by means that involve neither the construction of interspecies chimeras nor the morphological detection of 'neuroblasts' in conventionally stained specimens. Conceivably, the apparent discrepancy between data derived from studies of chick-quail chimeras (Le Douarin and Teillet, 1973, 1974) and those that focus on the formation of neurons (Allan and Newgreen, 1980; van Dongen et al. 1989) could be the result of a failure of sacral crest-derived cells that migrate to the hindgut to give rise to neurons. If sacral crest-derived cells do not normally form neurons or do so only after they interact within the enteric mesenchyme with vagal crest-derived cells (a possibility first suggested by Allan and Newgreen, 1980), the presence in the bowel of sacral crest-derived cells would go undetected in experiments that use only neurogenesis as a crest marker and which prevent vagal crest-derived, cells from entering the hindgut. The cranial neural crest gives rise to ectomesenchymal cells (Le Lievre and Le Douarin, 1975; Johnston et al. 1979; Le Douarin, 1982; Noden, 1983, 1984, 1988); the sacral crest might do so as well in the bowel. The current study was thus undertaken to trace the migration of sacral crest cells directly by using two cell markers: an intercalating carbocyanine dye and a replication-deficient retro virus. The intercalating fluorescent carbocyanine dye, (1, l-dioctadecyl-3,3,3' ,3' -tetramethylindocarbodil cyanine perchlorate), has been extensively used as a marker in previous studies (Honig and Hume, 1986; Godement et al. 1987; Bovolenta and Dodd, 1990) and has been successfully employed to analyze the pathways of truncal neural crest migration (Serbedzija et al. 1989). Dil does not spread from labelled to unlabelled . cells (Honig and Hume, 1986,1989), does not adversely affect the survival of cells that it labels (Honig and Hume, 1986), and can be injected directly into the vicinity of the neural crest in order to label crest cells in large numbers without introducing a population of cells from a different species. Dil has the disadvantage that it becomes diluted by continued cell division and crestderived cells are known to divide as they migrate. Another marker that can be used to follow the migration and lineage of cells is a replication-deficient retrovirus (Sanes et al. 1986; Price et al. 1987; Gray et al. 1988; Galileo et al. 1990). When the recombinant retrovirus infects a dividing cell, its genome integrates into the DNA of the infected cell and is inherited by that cell's progeny. Markers encoded by the virus thus

Sacral crest colonizes the hindgut are not diluted or lost as a result of cell division. A disadvantage of the virus is that it infects relatively small numbers of cells; therefore, the current experiments were performed with both dil and a retrovirus, two methods that complement one another, to trace directly the migration of cells from the neural crest to the bowel. Our studies demonstrate that NC-1 immunoreactive cells do migrate from sites of origin in the sacral neural crest to the post-umbilical bowel. Preliminary reports of these observations have been presented (Pomeranz et al. 1989, 1990). Materials and methods

Preparation of eggs Fertilized eggs from White Leghorn chickens (Gallus gallus) were obtained from Truslow Farms (Chestertown, MD) and were incubated at 37°C in a humidified, forced draft incubator. When injections of dil or LZ10 were to be made into the vagal region of the neuraxis, embryos were allowed to develop until they had acquired 7 to 12 somites. Embryos that had reached the age of 17-19 somites were used for injections into the truncal crest. For sacral injections embryos were incubated until they had acquired 26 or more somites. Since rostral somites are no longer clearly visible in embryos of 26 somites, the vitelline veins, which form an apex at the approximate level of the 24th somite, were used as a landmark. Before opening the eggs, albumin was removed with a sterile needle. The eggs were washed in 70 % ethanol, a window was cut in the shell over the embryo and India ink (Pelikan™, Hannover, W. Germany) was injected under the blastodisc so that the embryos could be visualized. The vitelline membrane was severed with a tungsten needle to allow access to the embryo. After the injection, the egg was sealed with adhesive tape and returned to the incubator.

Microinjection of dil Dil was prepared according to the method of Honig and Hume (1986). Briefly, injections were made with a 2.5% solution (w/v) of dil (Molecular Probes, Eugene, OR) in 100% ethanol. Prior to each use the dye solution was sonicated and then centrifuged to remove any crystals that might clog the micropipette tip. Injections of the dil were performed as described by Serbedzija et al. (1989). A micropipette with a tip diameter of approximately 10 /an made from thin-walled glass capillary tubes (World Precision Instruments, New Haven, CT) was backfilled with the dil solution and centered over the neural tube with a Narashige micromanipulator. The pipette was then lowered to insert the tip between the dorsal surface of the neural tube and the overlying ectoderm. Dil was injected with pressure delivered by applying up to 3 bursts of N2 (100ms; 5.6kPa) from a picrospritzer™ (General Valve, Fairfield, NJ). Embryos were incubated for 1-4 days following injection.

Microinjection of the LZ10 virus The LZ10 retrovirus, generously supplied by Dr Joshua Sanes of Washington University, St Louis, was derived from the Rous sarcoma virus (Galileo et al. 1990). The retrovirus was constructed such that a reporter gene, lacZ of Escherichia coli, was fused in frame to a large fragment of the RSV gag gene (Galileo et al. 1990). The pol and env genes of the virion were also deleted. As a result, infectious particles are not released in embryos by cells carrying the viral construct. The

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viral marker protein is restricted to the cytoplasm within the progeny of cells originally infected with virions obtained from a quail fibroblast packaging cell line (QT6-62). The QT6-62 packaging cells were incubated overnight in a minimal amount of medium (Medium 199 (Cat. No. 320-1150AG, GIBCO BRL, Grand Island, NY), tryptose phosphate broth (10%; GIBCO), fetal calf serum (5%; GIBCO), DMSO (1%), penicillin-streptomycin (1%; GIBCO) and gentamicin (400/igml"1; Sigma Chemical Co., St Louis, MO)) in order to generate a high titer of virus. The following day the growth medium was collected and centrifuged at 1600 revs mill"1. Hexadimethrine (polybrene (SjUgmT1); Sigma Chemical Co.) was added to the viral concentrate to facilitate infection of cells in the embryos. The viral suspension was maintained in a water bath at 37 °C until it was loaded into a micropipette by backfilling and injected into embryos as described above for injections of dil. Embryos were incubated for 3-4 days following injection in order to allow crest-derived cells sufficient time to migrate to the gut. Infected cells were detected immunocytochemically with antibodies to bacterial /S-galactosidase (/S-gal), the lacZ gene product (see below).

Immunocytochemistry All embryos were fixed at room temperature for 1-4 h in 4 % formaldehyde (freshly prepared from paraformaldehyde) in O.lMperiodate-lysine buffer at pH 7.4 (McLean and Nakane, 1974). After fixation, all tissues were briefly rinsed in 0.1M phosphate-buffered saline (PBS) at pH7.4. Embryos in which dil was injected into the vagal crest were then embedded in 7% agar and sectioned (50-100 ^m) with a vibratome. Several days after injection of dil into the sacral crest, the foregut and hindgut were dissected from the embryos and embedded next to one another in the same block of agar for sectioning with a vibratome. Sections were mounted on slides with Gel/mount (Biomeda Corp., Foster City, CA). Embryos that had been injected with the LZ10 retrovirus were cryoprotected by infiltration overnight with 30 % sucrose in PBS at 4°C. The tissues were then placed in molds containing an embedding compound (OCT; Miles Laboratories, Naperville, IL), rapidly frozen with liquid N 2 , and stored at — 80°C until used. Sections were cut at 10-15/an using a cryostat-microtome and thaw-mounted onto glass slides that had been coated 3 times with chromium alum-gelatin. The mounted specimens were washed twice with PBS to remove embedding compound and then fixed to the slides with 2% formaldehyde in PBS at pH7.4. The tissues were permeabilized for 30min with 0.1 % Triton X-100 (Sigma Chemical Co.) in PBS and incubated for 30min at room temperature in a solution containing 0.1M Tris-HCl, 1.5% NaCl, 0.1% Triton X-100, and 10% horse serum (GIBCO) to reduce background staining. Tissues were then exposed overnight in a humidified chamber at 4°C to primary antisera (diluted with 4% horse serum in the above buffer). Primary immune reagents included: (i) monoclonal antibody NC-1 (diluted 1:10; provided by Dr Jean-Paul Thiery, Ecole Normale Supe'rieure, Paris, France), and (ii) a polyclonal rabbit antiserum to bacterial /S-gal (1:100, Organon TeknikaCappel, Malvern, PA) After washing several times, the tissues were exposed for three hours at room temperature to appropriate affinity-purified, species-specific, secondary antibodies (1:400, goat anti-mouse IgM, Organon TeknikaCappel; goat anti-rabbit IgG, Kirkegaard and Perry Labs, Gaithersburg, MD) coupled to tetramethylrhodamine isothiocyanate (TRITC), fluorescein isothiocyanate (FITC) or biotin in the above buffer. Those preparations that were exposed to a biotinylated secondary antibody were subsequently treated for one h with avidin-FITC (1:200, Vector

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Fig. 1. Residual fluorescence at the site of injection of dil. Bright-field (A) and fluorescence (B) photomicrographs are shown of the dorsal surface of a chick embryo that was injected at the truncal level two days previously with dil. The site where dil was injected (—>) remains fluorescent. Scale bar=1000/im. (C) A 50-100/an thick section cut through the vagal region of a chick embryo two days after injection of dil. Note that dil fluorescence is localized on the dorsal surface of the embryo. The dye has stained tissue (including the dorsal neural tube, overlying epidermis {-^), and intervening mesenchyme) in the region from which the vagal crest emigrated. (nt=neural tube, a=aorta) Scale bar=100^m. Laboratories, Burlingame, CA) to visualize sites of bound antibody. As an immunocytochemical control, non-immune serum (1:100) was substituted for primary immune reagents. Specific immunostaining was defined as that not present in the controls. Specimens were mounted in 9.0 mM p-phenylenediamine in 9 parts glycerol and 1 part PBS (final pH=8.0) in order to retard bleaching of FTTC (Johnson and Nogueira Araujo, 1981). The tissue sections were then viewed with vertical illumination in a Leitz Ortholux fluorescence microscope. TRTTC and dil fluorescence were visualized using an 'N2' dichroic mirror/filter cube (exciting filter BP 530-560; dichroic mirror RKP 580; barrier filter LP 580) that passed no FTTC fluorescence. FITC fluorescence was visualized using an 'L2' dichroic mirror/filter cube (exciting filter BP 450-490; dichroic mirror RKP 510; barrier filter LP 525/20) that passed no TRITC fluorescence. Tissues were photographed with Kodak EL 135 or TMAX film and processed at ASA 800. Results

Tracing the migration of cells following injection of dil into the vagal or sacral regions of the neuraxis In order to determine sites to which cells migrate from the vagal or sacral regions of the neural crest, dil was injected between the neural tube and the surface ectoderm at these axial levels. 28 embryos were injected, 5 at the vagal level and 23 at the sacral level. Because of the difference in ages of the embryos used for vagal (7 to 12 somites) or sacral injections (26 or more somites), results could not be confounded by spread of the dye in the anterior-posterior direction. At the time vagal injections were made, the sacral crest had not yet formed and, more importantly, at the time sacral injections were made, the vagal crest had long since departed from the neuraxis (the. migration of vagal crest cell is completed before embryos reach a length of 15 somites; Le Douarin, 1982). The injected

dye could be seen to spread laterally to cover the entire dorsolateral aspect of the neural tube so as to include the neural crest (Fig. 1A,B). The dye also was seen to spread proximodistally for a distance of 1-2 somites. Embryos were fixed and examined 1-4 days after injection. The site where dil was injected could easily be recognized in whole chick embryos or in sections because it was marked by the intense fluorescence of residual dil in the injected region (Fig. 1). Injection sites were examined to confirm that the injections had been placed in the correct locations. Embryos were selected for analysis and considered 'vagal injections' (Fig. 1C) if the residual dil was found adjacent to the rhombencephalon and had not spread beyond the dorsal surface of the neural tube. Similarly, 'sacral injections' were considered those in which residual dil was found between the developing spinal cord and surface ectoderm and remained caudal to the level of somite 28. Two to four days after the injection of dil into the region of the vagal crest, the foregut was found to contain many dil-fluorescent cells (Fig. 2). These cells appeared in the gizzard and presumptive duodenum, but were not observed in the esophagus. Dil-fluorescent cells were most numerous in the gizzard, within which they were located in the enteric mesenchyme. The fluorescent cells were concentrated in the outer mesenchyme of the gizzard under the serosa. Some cells approached the mucosa, but did not appear to come into contact with the epithelium. The dil-labelled cells were mainly round or stellate in appearance; however, some of the cells had extended varicose neuritic processes, which were also fluorescent. The intensity of the dil fluorescence varied considerably. Since the vibratome sections were quite thick, it is likely that at least some of this variation is due to the location of the

Sacral crest colonizes the hindgut

Fig. 2. Numerous dil-labelled cells can be found in the gizzard 4 days after injection of dil into the vagal crest of a 12 somite embryo. Note that dil-labelled cells (arrowheads) extend from the serosal surface (s) of the bowel to just external to the mucosal epithelium (m). Most of these cells are round or mesenchymal in appearance and display considerable variation in the intensity of their fluorescence. Some of the dil-labelled cells can be seen to have extended varicose neurites (—»). Scale bar=100,um.

cells within the section. Fluorescence appeared to be most intense when the fluorescent cell was situated near the upper surface of the section where it was directly exposed to the exciting beam of light. Cells that were located more deeply in the section tended to nuoresce less brightly. The variation was especially visible in photomicrographs in which the most clearly visible cells were those that were included in the plane of focus. The locations in which dil-fluorescent cells were found corresponded to those in which NC-1-immunoreactive cells were observed in comparable material, although it was not possible to use double label immunocytochemistry (antibodies do not penetrate into the thick sections used for viewing dil fluorescence) to make certain that cells labeled with dil were also NC-1-immunoreactive. Two to four days after the injection of dil into the region of the sacral crest, the post-umbilical bowel was found to contain many dil-fluorescent cells, but no dillabelled cells were observed in the gut rostral to the umbilicus (Fig. 3). In addition, dil-labelled cells were also seen in the ganglion of Remak (not illustrated), which contained no dil-stained cells following vagal injections of the dye. Most of the dil-!abe!!ed cells in the post-umbilical bowel were located within the enteric mesenchyme, just internal to the serosa (Fig. 3). This intra-enteric distribution is similar to that observed for dil-labelled cells in the foregut following vagal injections of dil. Control experiments were performed in which the crest was injected with dil at the level of the trunk. Nine embryos that had developed to the age of 15-20 somites were injected at the level of the most caudal somite. Following these injections, dil-labelled cells were observed in dorsal root and sympathetic ganglia; however, in no instance were dil-fluorescent cells found

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Fig. 3. Four days after the injection of dil into the sacral crest fluorescent cells are found in the bowel caudal, but not rostral, to the umbilicus. Note that many cells (—*) are labelled with dil in the post-umbilical gut (hg); however, no labelled cells are observed in the preumbilical bowel (fg). The dil-labelled cells are observed in the enteric mesenchyme internal to the serosal epithelium. Scale bar=100Jum.

in the gut. Similar results were previously obtained for truncal injections of dil by Serbedzija et al. (1989). Tracing the migration of cells following injection of the LZ10 retrovirus into the vagal or sacral regions of the neuraxis The LZ10 replication-deficient retrovirus was injected into the vagal or sacral regions of the neuraxis in order to confirm the results obtained with dil. 51 embryos were injected, 27 at the vagal level and 24 at the sacral level. LZlO-infected cells were detected immunocytochemically with antibodies to /3-gal. NC-1 immunoreactivity was demonstrated simultaneously in order to correlate viral infection with the localization of another crest marker in the same tissue sections. When the LZ10 retrovirus was injected into the vagal region of the neuraxis, ^-gal-labelled cells later were found in the pre-umbilical gut (Fig. 4). These cells were confined to regions where NC-1-immunoreactive cells were concentrated and all of the /3-gal-labelled cells in the bowel coexpressed NC-1 immunoreactivity. Following vagal injections, LZlO-infected cells were also observed in dorsal root ganglia (DRG) at anterior cervical levels. When the LZ10 retrovirus was injected into the sacral region of the neuraxis, ^-gal-labelled cells were observed in DRG (Fig. 5A-D) and the mesenchyme of the post-umbilical bowel (Fig. 5E-G). No /3-gallabelled cells were found in the pre-umbilical gut. In the DRG and in the bowel some ^-gal-labelled cells were found to co-express NC-1 immunoreactivity. The ^-gallabelled cells were often found in clusters or coherent chains of cells (Figs 4F, 5B, 5D and 5G). These chains reached lengths of 8-10 cells. These arrays appeared also to be several cells thick, because they appeared in many adjacent sections.

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Fig. 4. Four days after the injection of LZIO into the vagal crest /3-galactosidase-immunoreactive cells are found in developing ganglia of the myenteric plexus of the presumptive gizzard; these cells co-express NC-1 immunoreactivity. (A,B,C) NC-1 immunoreactivity visualized with light appropriate to demonstrate TRITC fluorescence; (D,E,F) pgalactosidase immunoreactivity visualized with light appropriate to demonstrate FITC fluorescence. The same field is shown in A and D, B and E, C and F. Note that there are many more NC-1-immunoreactive cells than /3-galactosidase-labelled cells in the enteric mesenchyme. When NC-1 immunoreactivity is visualized at low (A) and high powers of magnification (B), developing myenteric ganglia can be discerned in the outer gut mesenchyme, just internal to the serosa (s). ^-Galactosidase immunoreactive cells are found in these ganglia (D,E). The NC-1- and /3-galactosidase-immunoreactive cells were often found in coherent chains of cells (C,F). These chains reached lengths of 8-10 cells. The /3-galactosidaselabelled cells in (F) were not strongly immunoreactive with the NC-1 monoclonal antibody (C). The —> indicates the same cells viewed under each type of illumination. (m=mucosa, s=serosa) Scale bar for A and D=50/im. Scale bar for B and F.=30fjm. Scale bar for C and F=30//m. Discussion Experiments were done to test the hypothesis that sacral crest-derived cells colonize the avian postumbilical bowel. Two relatively non-invasive techniques were used to follow the migration of crestderived cells. The intercalating dye, dil, and the

replication-deficient retrovirus, LZIO, were injected into the neuraxis of chick embryos. Embryos were not subjected to surgical transplantation, the embryo's own cells were marked, and crest-derived cells could be identified in the bowel even though the labelled cells could not yet be recognized as 'neuroblasts'. The interpretation of the current data, therefore, is not

Sacral crest colonizes the hindgut

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Fig. 5. Four days after the injection of LZ10 into the sacral crest /3galactosidase-immunoreactive cells are found in DRG and the post-umbilical bowel; many of these cells co-express NC-1 immunoreactivity. Panels A, C, E and F show NC-1 immunoreactivity visualized with light appropriate to demonstrate TRITC fluorescence; B, D and G show /3-galactosidase immunoreactivity visualized with light appropriate to demonstrate FITC fluorescence. Panels A and B, C and D, F and G are pairs of pictures showing NC-1 and ^3-galactosidase immunoreactivities in the same respective fields. A dorsal root ganglion (g) is illustrated in A and B. A region of the same ganglion is shown at higher magnification in C and D. The arrows point to the same line of /Sgalactosidase-labeled cells in panels B and D. Note (in D) that /S-galactosidase immunoreactivity is cytoplasmic and outlines the non-fluorescent nuclei of labelled cells. The /J-galactosidaseimmunoreactive cells form coherent chains within the ganglion. Note that most of the NC-1-immunoreactive cells of the DRG do not demonstrate figalactosidase-immunoreactivity. The post-umbilical bowel is shown in E. The region of the same section of the bowel in E illustrated at higher magnification in F and G is indicated by the white box. The arrowheads indicate the same cells viewed under each type of illumination. The ^3-galactosidaseimmunoreactive cells are located just internal to the serosal epithelium (s). Note that the /S-galactosidaseimmunoreactive cells contact one another so as to form a coherent chain. The /J-galactosidase-labelled cells are also NC-1immunoreactive. (m=mucosa; r=ganglion of Remak) Scale bars for A and B = 100jan. Scale bars for C and D=20/im. Scale bar for E=100^m. Scale bars for F and G=20/im.

confounded by the possibility (Allan and Newgreen, 1980) that crest-derived cells of a heterologous species might reach sites in chimeric embryos to which they would not have migrated had they been left undisturbed in situ. Moreover, the identification of crest-derived cells is not dependent on their developing along a neuronal lineage; thus, crest-derived cells would be recognized even if they do not give rise to neurons in the hindgut and develop exclusively in this region of the bowel as glia or mesectoderm. Finally, the identification of cells as crest-derived is not dependent on the presumed specificity of the carbohydrate epitope that reacts with the NC-1 and HNK-1 monoclonal antibodies (Meijers et al. 1989a,b; Pomeranz and Gershon, 1990). Since there are some potential disadvantages inherent in the use either of dil or the LZ10 retrovirus, we employed both of them. Theflawsinvolved in the use of one of these tracers tend not to apply to the other. Experiments with each tracer confirmed that cells

migrate to the bowel from vagal and sacral levels of the neural crest and do not migrate to the gut from the neural crest of the trunk. Serbedzija and BronnerFraser (personal communication) have also observed that cells labelled with dil migrate to the hindgut from the sacral region of the crest. The experiments in which /J-gal immunoreactivity was demonstrated simultaneously with that of the NC-1 epitope confirmed that both vagal and sacral emigre's in the bowel are NC-1immunoreactive. These data fit well with the previous observations that a continuous stream of NC-1immunoreactive cells extends to the post-umbilical bowel from the sacral crest and that the hindgut contains many NC-1-immunoreactive cells before it is colonized by cells from the vagal crest (Pomeranz and Gershon, 1990). Our observations establish that at least some of the early NC-1-immunoreactive cells in the post-umbilical bowel must be derived from the sacral crest, which explains why neurons can develop in vitro

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in segments of hindgut explanted before the arrival of vagal crest-derived cells (Pomeranz and Gershon, 1990). It cannot be concluded, however, that, because some of the many NC-1 immunoreactive cells that encircle the post-umbilical bowel are crest-derived, all of them are. The possibility that a subset of the NC-1 immunoreactive population is of mesodermal origin is thus not excluded by these data. Also remaining to be demonstrated is whether or not the sacral CTest-derived cells that colonize the post-umbilical gut actually give rise to neurons in vivo. The fact that such cells can develop into neurons in organotypic tissue cultures or in CAM grafts (Pomeranz and Gershon, 1990) does not establish that these cells are neurogenic in the microenvironment in which they develop in situ. It is of interest that many of the retrovirally infected cells found in both the DRG and gut contacted one another in such a way as to form long coherent chains or clusters. This cellular grouping could be explained if each of the cells of such a chain was derived from a common ancestor. Since the injected material was prepared so as to contain as many virions as possible, it seems highly likely that more than a single crest cell would become infected following the injection of LZ10. Moreover, crest-derived cells appear to disperse and to divide as they migrate (Le Douarin, 1982). The linear aggregates of infected cells in DRG or bowel, therefore, cannot be presumed to be clones of a single virally infected premigratory crest cell. They could, however, be clones of a single infected post-migratory crest-derived progenitor, the progeny of which remain coherent after that cell reaches its site of terminal differentiation in the DRG or gut. Some regions of the gut that were expected to become colonized by vagal crest-derived cells were not found to contain labelled cells following injection of either dil of LZ10 into the vagal crest. For example, no labelled cells were observed in the esophagus, or in the bowel distal to the duodenum. The data of Le Douarin and Teillet (1973,1974) indicate that vagal crest-derived cells colonize the entire gut. In the current study, 4 days were allotted for crest cell migration following injection of the tracers. This time should have been sufficient for cells to have migrated beyond the duodenum (Le Douarin, 1982; Payette et al. 1984). It is possible that crest-derived cells that migrate over greater distances divide more than those that arrive sooner at their sites of terminal differentiation. If so, continued cell division might dilute the dil marker below its threshold for detectability. Neurofilament-immunoreactive (Payette et al. 1984) and NC-1-immunoreactive cells (Tucker et al. 1986) have each been found to appear in the gut for the first time in the region of the proventriculus where the vagus nerves reach the bowel. These observations suggest that vagal crest-derived cells enter the gut near the presumptive stomach and migrate from there to colonize the esophagus and distal gut. The concentration of dil-fluorescent cells in the gizzard and duodenum may thus reflect the earlier arrival and development of neurons in these regions (with an associated smaller number of mitoses) than elsewhere

in the bowel. The relatively small number of infected cells in embryos following the injection of LZ10 may make these cells difficult to find if they migrate long distances and become isolated. These considerations serve to emphasize that the dil and LZ10 tracers are useful when positive results are obtained. In contrast, failure to find labelled cells in a given location after the injection of either of these labels into the neural crest does not establish that crest-derived cells do not enter that site. Many published studies of lineage and migration of crest-derived cells have drawn conclusions based on the assumption that the chick hindgut remains aneuronal until day E6-7 (see Le Douarin, 1982). The observations that sacral crest-derived cells migrate to the post-umbilical gut and evidently colonize it by day E4 (Pomeranz and Gershon, 1990) mean that the hindgut, prior to day E6, should no longer be considered a crestfree tissue. Moreover, whether or not sacral crestderived cells give rise to neurons in the bowel in situ, they clearly have the capacity to do so in vitro. As a result, neurons arise in cultures or even CAM grafts of explants of bowel if crest-derived cells are present in them at the time of explantation. Nevertheless, the experimental use of the entire post-umbilical gut as a source of aneuronal enteric tissue is not necessarily invalidated. Since the sacral crest-derived emigre's appear to ascend within the post-umbilical bowel (Pomeranz and Gershon, 1990), the zone adjacent to the junction of the cecal appendages is the last region of the gut to receive crest-derived emigre's. If bowel is obtained from the region of the cecal appendages of an early embryo, therefore, the explanted gut will probably be devoid of crest-derived cells. Fortunately, this region of the bowel appears to have been the one most frequently explanted in previous studies and treated as aneuronal gut (see for example, Ciment and Weston, 1983; Smith et al. 1977; Rothman et al. 1990). Moreover, as noted above, it remains possible that the post-umbilical bowel might contain crest-derived cells that do not give rise to neurons. A distinction should thus be made between tissue that is crest-free and that which is aneuronal. In summary, we have demonstrated the use of the intercalating carbocyanine dye, dil, and a replicationdeficient retrovirus to trace crest-derived cells from their origins in the vagal and sacral regions of the neuraxis to the gut. We conclude that the post-umbilical bowel is colonized by cells from the sacral region of the neural crest. The derivatives formed by these cells remain to be identified. The abundance and location of the sacral emigres makes it necessary to determine in future experiments, whether the enteric sacral crestderived cells give rise to mesectoderm, to neurons, to glia, or to some combination of these derivatives. The authors would like to thank Dr Janet Jacobs-Cohen for her assistance in preparing the retrovirus suspension for injection, and Ms Manami Eiki and Ms Edith Abreu for excellent technical assistance. The authors also would like to thank Dr Joshua Sanes for the generous donation of the

Sacral crest colonizes the hindgut replication-deficient retrovirus, LZ10, and Dr Jean-Paul Thiery for providing the NC-1 antibody. This work was supported by NIH grants NS15547, HD17736, HD20470, GM07367, and a predoctoral fellowship from Lilly Research Laboratories. References ALLAN, I. J. AND NEWGREEN, D. F. (1980). The origin and

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