- Email: [email protected]
Analysis of the effects of endothelin-3 on the development of neural crest cells in the embryonic mouse gut
Analysis of the Effects of Endothelin-3 on the Development of Neural Crest Cells in the Embryonic Mouse Gut By Mark N. Woodward, Emma L. Sidebotham, M. Gwen Connell, Simon E. Kenny, Camille R. Vaillant, David A. Lloyd, and David H. Edgar Liverpool, England
Background/Purpose: Mutations in the endothelin-3 (ET-3) and endothelin-B receptor (EDNR-B) genes cause terminal colonic aganglionosis in mice and are linked to Hirschsprung’s disease. These experiments are designed to determine if the development of terminal enteric ganglia depends on changes in proliferation, apoptosis, or differentiation of enteric neural crest (NC) cells in response to ET-3. Methods: Gut from embryonic lethal-spotted mice (lacking ET-3) and controls were investigated in vivo. NC-derived cells were identified immunohistochemically and their proliferation, apoptosis and differentiation monitored by bromodeoxyuridine incorporation, the terminal deoxytransferase poly dU nick end labelling (TUNEL) reaction, and appearance of neuronal nitric oxide synthase (NOS), respectively.
T
HE ENTERIC nervous system (ENS) in the colon is derived mainly from vagal neural crest (NC) cells that enter the mesenchyme of the foregut and then migrate toward the rectum.1 Although many mechanisms regulating the differentiation of NC-derived cells remain unknown, several of the factors that influence development of the ENS have been elucidated recently.2,3 In particular, endothelin-3 (ET-3) and the endothelin B receptor (EDNR-B) have been shown to be necessary for complete colonization of the colon because mice with mutations in the genes coding for these proteins display aganglionosis of the terminal colon and rectum.4,5 This restricted aganglionosis is similar to that seen in human patients with short-segment Hirschsprung disease (HD), which has been linked also with mutations in the genes
From the Departments of Child Health, Veterinary Preclinical Sciences, and Human Anatomy, the University of Liverpool, Liverpool, England. Supported by Research Fellowships from the Digestive Disorders Foundation (MNW), the Royal College of Surgeons of England (ELS, MNW) and the Pilkington Charitable Trust (ELS). Project Grant from Action Research (CRV and DHE). Address reprint requests to Emma L. Sidebotham, Institute of Child Health, Alder Hey Children’s Hospital, Eaton Rd, Liverpool L12 2AP, England. © 2003 Elsevier Inc. All rights reserved. 0022-3468/03/3809-0009$30.00/0 doi:10.1016/S0022-3468(03)00389-0 1322
Results: No differences in apoptosis or proliferation of NC cells were apparent between lethal-spotted embryos and controls. Although no temporal differences in the differentiation of NOS neurones were evident, these cells appeared more cranially in the gut in the absence of ET-3 than in controls. Conclusions: ET-3 has no detectable influence on proliferation, apoptosis, or timing of differentiation of NC-derived cells in the gut. However, the more proximal location of differentiated neurones in the absence of ET-3 is consistent with a restricted role in migration of NC-derived cells. J Pediatr Surg 38:1322-1328. © 2003 Elsevier Inc. All rights reserved. INDEX WORDS: Hirschsprungis disease, enteric nervous system, neural crest, endothelin-3.
coding for ET-3 and EDNR-B6,7 or alternatively is associated with decreased levels of ET-3 expression.8 Although ET-3 is necessary for colonization of the terminal colon by NC-derived cells, high levels of ET-3 mRNA expression have been localized to the mesenchyme of the embryonic mouse cecum and proximal colon at the time EDNR-B– expressing NC cells are migrating through this region.9 Furthermore, it has been shown that the requirement for ET-3/EDNR-B signalling is restricted to the relatively short period of development between embryonic days (E) 10 and 12.5 when the NC cells have arrived in the vicinity of the cecum.10,11 The time and place at which ET-3 acts are consistent with earlier observations showing that abnormalities in the behavior of NC cells first become apparent at the cecum in ET-3 -deficient lethal-spotted (ls/ls) mice, although aganglionosis is restricted to the terminal colon in postnatal mice.12,13 Much work has been undertaken to elucidate the effect of ET-3 on NC cells in tissue culture.14-18 However, these reports have produced apparently conflicting results in that some show ET-3 influences the proliferation or differentiation of cultured NC cells, whereas other reports show no effect on these parameters. Taken together, however, these reports indicate that the responses of NC cells to ET-3 are likely to depend on the source from which they were isolated, their developmental stage, and the conditions under which they were main-
Journal of Pediatric Surgery, Vol 38, No 9 (September), 2003: pp 1322-1328
ENDOTHELIN-3 AND ENTERIC NEURAL CREST CELLS
tained in culture.14-18 Thus, to determine how ET-3 influences NC cells and is thereby necessary for complete colonization of the colon, the responses of enteric NC-derived cells to ET-3 need to be examined at the appropriate time and place within the environment of the embryonic gut wall. We therefore have compared NC-derived cell proliferation and differentiation in situ in the bowel of ls/ls (ET-3 deficient) embryonic mice and in control ls/⫹ siblings at the time ET-3 is known to be necessary for gut colonization.10,11 In addition, we have examined NC cell apoptosis because activation of the EDNR-B has been reported also to prevent the apoptotic death of some cell types.19 MATERIALS AND METHODS
Animals and Genotyping Six- to 8-week-old male lethal-spotted (ls/ls) mice were mated with female C57 mice to produce litters of heterozygote (ls/⫹) offspring. The female ls/⫹ progeny subsequently were time mated with male homozygote ls/ls mice and the date of the vaginal plug counted as embryonic day (E) 0.5. Pregnant mice were killed at E11.5, E12.5, and E13.5 by cervical dislocation. The age of embryos was confirmed by staging based on examination of the external features. E11.5 uteri were kept in Dulbecco’s modified Eagle medium (DMEM; Gibco Life Technologies) at 37°C until all embryos were dissected. The gut was dissected under aseptic conditions and oriented using tungsten needles on Millipore-CM culture plate inserts (Millipore, Watford, Herts, UK) in 6-well Nunclon culture dishes (Gibco Life Technologies, Paisly, Strathclyde, UK). The gut explants then were either fixed immediately over 4% paraformaldehyde or after culture. Genotyping was undertaken to identify ls/ls embryos from their heterozygote control littermates by extraction of total cellular RNA from samples of esophagus and stomach using the acid phenol-chloroform method.20 RNA rather than DNA genotyping was used to simplify the identification of restriction digestion fragments uninterrupted by intronic sequences. First strand cDNA synthesis was performed using Superscript II reverse transcriptase (GIBCO-BRL) as previously described.8 cDNA samples were amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using ET-3 primers that had previously been designed in this laboratory.9 The ET-3 primers amplified a product that spanned the missense 758 C 3 T change of the ls allele mutation.4 RT-PCR conditions established were 35 cycles including denaturation at 94°C for 1 minute, annealing at 51°C for 1 minute, and extension at 72°C for 1 minute followed by a final extension step of 10 minutes. After RT-PCR, an Xmn1 (Boehringer Mannheim, Lewer, East Sussex, UK) restriction digest was performed on the PCR product. The Xmn1 restriction enzyme cuts the sequence GAANN2NNTTC, which is lost in the ls mutation.4 Electrophoresis on ethidium bromide stained 2% (wt/vol) agarose gels showed a single 244 base-pair band in homozygote ls/ls embryos, whereas 244, 161 and 83-bp bands were seen in heterozygote ls/⫹ embryos.
Immunohistochemistry Tissues were fixed in 4% paraformaldehyde for 2 hour, rinsed in PBS for 1 hour, immersed in 15% (wt/vol) sucrose in phosphate-buffered saline (PBS) for 24 hours at 4°C, then embedded in 7.5% (wt/vol) gelatin/15% (wt/vol) sucrose in PBS before freezing with isopentane/ liquid nitrogen. Serial longitudinal 10-m sections were cut with a cryostat.
1323
Neural crest-derived cells were identified by staining for protein gene product 9.5 (PGP 9.5) using a rabbit antiserum (Biogenesis, Poole, Dorset, UK) at 1:4000 dilution or the p75 low-affinity nerve growth factor receptor (p75) using a rat antiserum (Chemicon, Chandlersford, Hampshire, UK) at 1:100. Differentiation of neural crest cells was determined by the appearance of nitric oxide synthase (NOS)positive neurons in the gut from E11.5 through E13.5 using a goat antiserum (kindly donated by Dr Piers Emson, Babraham Institute, Cambridge, UK) diluted 1:1000. NOS was selected as a marker of neuronal differentiation, because this is one of the first specific neuronal phenotypes to differentiate and has been shown to be present in the region of cecum and colon during embryogenesis of the gut.21,22 Sections were incubated overnight at 4°C in primary antisera diluted in PBS containing 1% (wt/vol) bovine serum albumin. Sections were washed in PBS and incubated for 1 hour in appropriate secondary antisera; Cy3-conjugated donkey antirabbit immunoglobulin (1:500 dilution; Jackson Laboratories, West Grove, PA), Texas red-conjugated goat antirabbit immunoglobulin (1:200 dilution; Jackson Laboratories), FITC-conjugated sheep antirat immunoglobulin (1:32 dilution; Sigma Gillingham, Dorset, UK), and biotinylated donkey antisheep immunoglobulin (1:100 dilution, Sigma) followed by FITC-conjugated streptavidin (1:50 dilution, Amersham, Chalfont, St Giles, Bucks, UK). Omission of primary antisera resulted in the total absence of fluorescent signal. For double staining, omission of either one of the primary antisera resulted in loss of the appropriate fluorochrome signal.
Cell Proliferation Assay E11.5 uteri were kept in Dulbecco’s modified Eagle medium (DMEM; Gibco Life Technologies) at 37°C until all embryos were dissected. The gut was dissected under aseptic conditions and oriented using tungsten needles on Millipore-CM culture plate inserts (Millipore) in 6-well Nunclon culture dishes (Gibco Life Technologies). The gut explants were placed over 1 mL DMEM supplemented with 10% (vol/vol) fetal calf serum (Gibco Life Technologies), 1% (wt/vol) L-glutamine, and 50 g/mL penicillin/streptomycin and the cultures maintained in a 5% (vol/vol) CO2 incubator at 37°C. The culture medium was supplemented with 10 mol/L bromodeoxyuridine (BrdU) for 1 hour before fixing the tissue with 4% paraformaldehyde.23 Sections were processed initially for immunolabelling for PGP 9.5 as above, washed in PBS, treated with 4 mol/L HCl for 15 minutes and then washed again in PBS until the pH reached 6.5 or above. Sections then were incubated with mouse anti-BrdU (DAKO) diluted 1:25 in PBS for 1 hour at 38°C, washed in PBS for 3 ⫻ 10 minutes, incubated with biotinylated rabbit antimouse immunoglobulin (DAKO, UK) 1:100 in PBS for 30 minutes, washed again, and finally incubated with streptavidin/FITC (Amersham) 1:50 in PBS for 1 hour.
Detection of Apoptosis E11.5 gut from lethal-spotted crosses, fixed immediately on dissection, was examined using a modified TUNEL reaction for apoptotic cells.24 Sections were processed initially for immunolabelling for PGP 9.5 as above, washed in distilled H2O for 2 ⫻ 10 minutes treated with 1.5% (vol/vol) TRITON X100 in PBS for 30 minutes and washed again in H2O for 4 ⫻ 2 minutes. Positive controls were incubated with DN buffer (30 mmol/L Tris HCl; pH 7.2; 140 mmol/L potassium cacodylate; 4 mmol/L MgCl2; 0.1 mmol/L dithiothreitol) for 2 to 3 minutes followed by 5 g/mL type II DNAse (Sigma) in DN buffer for 10 minutes at room temperature and then washed in H2O, for 4 ⫻ 2 minutes. Sections then were incubated with TdT buffer (30 mmol/L Tris HCl, pH 7.2; 140 mmol/L sodium cacodylate; 1 mmol/L cobalt chloride) for 2 to 3 minutes followed by TUNEL reaction mix for 2 hours at 38°C. TUNEL reaction mix comprised 15 U terminal deoxynucleotidyl transferase (TdT, Pharmacia) and 10 mol/L biotinylated
1324
WOODWARD ET AL
deoxyuridine triphosphate (dUTP; Boehringer Mannheim) in 100 L TdT buffer. TdT enzyme was omitted from reaction mix for negative controls. Sections were allowed to cool, washed in H2O for 3 ⫻ 10 minutes and then in PBS for 10 minutes before incubation with streptavidin/FITC (Amersham) diluted 1:50 in PBS for 1 hour.
Determination of Cell Numbers Counts of neural crest cells in each experiment were carried out on a minimum of 10 sections from between 4 and 10 samples of gut and matching numbers of control samples. The area in which cells were counted extended from the cecum to the most caudal PGP 9.5-positive cells, which were restricted to the proximal colon at the times at which counts were performed.11,13 Statistical analysis to compare counts of experimental groups with controls was performed using a 2-tailed student t test with a significance level of .05.
RESULTS
Genotyping of Embryos RT-PCR was performed using ET-3 primers on total RNA, and subsequently an Xmn1 restriction digest was undertaken on the PCR product. The expected single 244-bp band was amplified from all embryos. After restriction digestion, this single band remained in samples from homozygote ls/ls embryos, whereas additional 161 and 83-bp bands corresponding to digestion fragments of the wild-type allele appeared in heterozygote ls/⫹ embryos (Fig 1). A total of 29 ls/ls and 25 ls/⫹ embryos were genotyped at the time-points studied, which approximates to the expected 50:50 Mendelian distribution. Differentiation of Neural Crest Cells No NOS-positive cells were apparent in the gut of either ls/ls or ls/⫹ control mice at E11.5 (Fig 2A,B). By
Fig 2. Appearance of NOS-expressing neurons in embryonic ls/ls and control ls/ⴙ mouse gut. At E11.5, no expression of NOS is apparent in the ileocecal region of control (A) or ls/ls (B) gut. By E 12.5 a very few sporadic NOS-positive neurones have appeared in the cecum of control gut (E, arrow) but are absent in the terminal small intestine (C), whereas in ls/ls gut, NOS is evident in the terminal ileum (D, arrow), but no NOS-positive cells are seen in the cecum (F). By E13.5, many more NOS-positive cells are evident, but the pattern of expression in the ileocecal region persists. NOS-positive cells are evident in the cecum (I) of control gut, but the small intestine remain devoid of NOS neurons (G), whereas in ls/ls gut, NOS-positive cells are readily apparent in the small intestine (H), and NOS cells are seen only sporadically in the caecum (J, arrow). Diagram of the gut of control (K) and ls/ls (L) gut shows the distribution of NOS-positive cells (dotted areas) along the length of the gut at E13.5 (bar ⴝ 50 m) ce, cecum; co, colon; re, rectum; st, stomach; si, small intestine.
Fig 1. Genotyping of embryos produced from crosses of male ls/ls with female ls/ⴙ mice. Agarose gel electrophoresis of PCR products amplified from the cDNA of ls/ⴙ (1, 2) and ls/ls (3, 4) embryos. A single 244-bp band is amplified from both homozygous and heterozygous embryos (1, 3). After digestion with Xmn1, 161 bp and 31 bp bands are produced from the wild-type allele of heterozygotes (2), but the is allele remains uncleaved (4).
E12.5, small numbers of NOS-positive neurons were present in the stomach (not shown) in both ls/ls embryos and controls. In controls, no NOS labelling was detected in the small intestine (Fig 2C), but occassional NOSpositive cells were seen in the cecum (Fig 2E, arrow). In
ENDOTHELIN-3 AND ENTERIC NEURAL CREST CELLS
contrast, NOS neurons were present in the ls/ls distal small intestine (Fig 2D, arrow), but no NOS labelling was detected in ls/ls cecum (Fig 2F). By E13.5 many more NOS-positive cells were apparent in both control and ls/ls mice. However, the differences in location of positive cells in the ileocecal region persisted with many NOS-positive cells in control cecum (Fig 2I) but no NOS labelling in the small intestine (Fig 2G), whereas in ls/ls gut there were many NOS-positive cells in the terminal ileum (Fig 2H) but only occasional cells in the cecum (Fig 2J, arrow). NOS-positive cells were present along the length of the colon in control mice but were restricted to the proximal colon in ls/ls mice at this stage reflecting the limited colonization of the colon by NC-derived cells (Fig 2K,L).11 Proliferation of Neural Crest Cells The low affinity neurotrophin receptor p75 is displayed by most if not all NC cells in the embryonic mouse gut,25 but being an integral membrane protein it is lost during permeabilization procedures to assay cell proliferation and apoptosis. However, double labelling immunohistochemistry has shown that essentially all the p75-positive cells found at the leading edge of migration in the proximal colon at E11.5 also stain for PGP9.5, thus confirming PGP9.5 as a suitable marker to identify NC cells in these studies.26 Gut was taken from ls/ls and ls/⫹ embryos at E11.5 and pulsed in organ culture for 1 hour with BrdU. The BrdU was incorporated into large numbers of epithelial and mesenchymal cells throughout the gut specimens (Fig 3A and C). NC-derived cells, which labelled with PGP9.5, were also readily identifiable (Fig 3B and D, arrows). Small numbers of NC-derived cells also labelled for BrdU. Counts of BrdU-labelled PGP-positive cells in the cecum and proximal colon showed that there was no significant difference in the proportions of NC cells that had incorporated BrdU in the ls/ls and ls/⫹ control guts (Fig 3E). These determinations were powered to be able to detect a minimal difference of 14% in populations at the significance level of 85%. Apoptosis of Neural Crest Cells The TUNEL reaction to detect nuclear DNA fragmentation characteristic of apoptosis24 was performed on E11.5 gut from ls/ls and controls (ls/⫹). No epithelial or mesenchymal labelling was observed in negative controls for the TUNEL reaction, whereas all cells were labelled after pretreatment of sections with DNAse (not shown). Although small numbers of apoptotic epithelial and mesenchymal cells were seen throughout the specimens (Fig 4A and C), there was no colocalization of PGP9.5 immunoreactivity (Fig 4B and D) with any of the
1325
TUNEL-labelled cells. Thus, TUNEL labelling did not provide any evidence that NC cells undergo apoptosis at this stage of embryonic development either in the presence or absence of ET-3. DISCUSSION
Several studies have described the potential ways in which ET-3 can influence the behavior of NC-derived cells in vitro. However, findings from these studies have suggested widely differing consequences of the ET-3/ EDNR-B interaction, dependent on the time of isolation of cells, the cell population studied, and the culture conditions used.14,15,17,18,27 It has therefore been difficult to draw any firm conclusions regarding the mechanisms by which ET-3 prevents terminal aganglionosis of the colon. Comparison of NC-derived cell proliferation, apoptosis, and differentiation in situ in the gut of ls/ls embryos and age-matched ls/⫹ sibling controls permitted us to study the effects of ET-3 under the necessary controlled conditions to define its action during ENS development. The current in vivo study did not show any significant ET-3 effects on enteric NC-derived cell proliferation or apoptosis. No temporal differences in the differentiation of NC-derived cells were apparent, judged by the appearance of NOS immunoreactivity, which defines the differentiation of one of the first neuronal phenotypes in the gut. However, the position at which NC cells differentiated into NOS-positive neurons was shifted rostrally from the cecum and proximal colon to the distal small intestine in ls/ls gut. These observations indicate that rather than regulating the timing of neuronal differentiation, ET-3 is necessary for the correct migration of NC-derived cells in the gut wall, their differentiation being independent of ET-3. Previous studies have found that more PGP9.5-positive cells were found in cultures of immunoselected enteric NC-derived cells after 7 days culture in the presence of the EDNB-R antagonist, BQ788 vitro.18 Because this marker is expressed by most if not all migrating NC-derived cells at E11.5,26 then this observation is consistent with the hypothesis that ET-3 exerts a survival-promoting or mitotic effect of ET-3 on the cells at some point during this period of culture.18 However, our observations in vivo indicate that NC-derived cells do not undergo programmed cell death in the absence or in the presence of ET-3, ruling out the possibility that ET-3 exerts its effect of NC-derived cell survival. Furthermore, we were unable to detect an effect of ET-3 on the mitotic index of NC-derived cells in the embryonic gut wall, and so these experiments do not provide any support for the hypothesis that ET-3 regulates cell proliferation to achieve full colonization of the gut.
1326
WOODWARD ET AL
Fig 3. Proliferation of neural crest cells in the embryonic gut. Longitudinal sections of ileocecal region of E11.5 control (A, B) and ls/ls (C, D) gut were dual labelled for BrdU (A, C) and PGP-9.5 (B, D) immunoreactivity. Numerous proliferating epithelial and mesenchymal cells were identified throughout the specimens (A, C). A number of PGP-immunoreactive cells that had incorporated BrdU also were identified (B, D). The proportion of NC cells that were dual labelled was equivalent in specimens and controls (Bar ⴝ 20 m). (E) Histogram shows the percentage of PGP9.5-positive cells at the leading edge of migration in cecum and proximal colon that had incorporated BrdU (ⴞSEM; n ⴝ 9). There was no significant difference (t test; P ⴝ .56) between the numbers of NC cells incorporating BrdU in control (ls/ⴙ) and ls/ls gut.
ENDOTHELIN-3 AND ENTERIC NEURAL CREST CELLS
1327
Fig 4. Apoptosis of neural crest cells in the embryonic gut. Sections were stained for TUNEL labelling (A, C), and PGP-9.5 immunoreactivity (B, D). Small numbers of apoptotic epithelial and mesenchymal cells were identified throughout the specimens (A, C, arrowheads). Numerous PGP-immunoreactive cells were identified at the level of the presumptive myenteric plexus (B, D) but there was no evidence of colocalization of immunoreactivities in any of the specimens. (bar ⴝ 50 m) e, epithelium; m, mesenchyme.
Although our results do not show any significant effects of ET-3 on the proliferation or differentiation of the bulk population of NC-derived cells in the embryonic gut, we cannot rule out the possibility that ET-3 affects these aspects of the behavior of a small subpopulation of cells. It has been shown recently that a small number of cells at the leading edge of NC cell migration differ from more rostral cells as they do not yet display all the differentiation markers characteristic of NC-derived cells of the ENS.25 Previous studies have not used ubiquitous markers of NC cells and so differences in the behavior of cells at the leading edge of migration are particularly liable to be missed.13,28,29 This omission is potentially of
significance in the light of the observation that a very few NC-derived cells in this position are sufficient to colonize the entire colon.30 Any effect of ET-3 on such a small subpopulation of stem cells would be masked within the larger population of unresponsive NC-derived cells but, nevertheless, may be responsible for the migratory defect seen in the absence of ET-3.
ACKNOWLEDGMENT Additional support The authors thank Dr Piers Emson of the ARC, Babraham, Cambridge, England for the generous donation of NOS antibodies.
REFERENCES 1. Le Douarin NM, Teillet MA: The migration of neural crest cells to the wall of the digestive tract in avian embryo. J Embryol Exp Morphol 30:31-48, 1973 2. Gershon MD: Genes and lineages in the formation of the enteric nervous system. Curr Opin Neurobiol 7:101-109, 1997 3. Taraviras S, Pachnis V: Development of the mammalian enteric nervous system. Curr Opin Genet Dev 9:321-327, 1999 4. Baynash AG, Hosoda K, Giaid A, et al: Interaction of endothelin-3 with endothelin-B receptor is essential for development of
epidermal melanocytes and enteric neurons. Cell 79:1277-1285, 1994 5. Hosoda K, Hammer RE, Richardson JA, et al: Targeted and natural (piebald-lethal) mutations of endothelin-B receptor gene produce megacolon associated with spotted coat color in mice. Cell 79:1267-1276, 1994 6. Puffenberger EG, Hosoda K, Washington SS, et al: A missense mutation of the endothelin-B receptor gene in multigenic Hirschsprung’s disease. Cell 79:1257-1266, 1994
1328
7. Kusafuka T, Puri P: Genetic aspects of Hirschsprung’s disease. Semin Pediatr Surg 7:148-155, 1998 8. Kenny SE, Hofstra RMW, Buys CHCM, et al: Reduced endothelin-3 expression in sporadic Hirschsprung’s disease. Br J Surg 87:580585, 2000 9. Leibl MA, Ota T, Woodward MN, et al: Expression of endothelin 3 by mesenchymal cells of embryonic mouse caecum. Gut 44:246-252, 1999 10. Shin MK, Levorse JM, Ingram RS, et al: The temporal requirement for endothelin receptor-B signalling during neural crest development. Nature 402:496-501, 1999 11. Woodward MN, Kenny SE, Vaillant C, et al: Time-dependent effects of endothelin-3 on enteric nervous system development in an organ culture model of Hirschsprung’s disease. J Pediatr Surg 35:2529, 2000 12. Kapur RP, Yost C, Palmiter RD: A transgenic model for studying development of the enteric nervous system in normal and aganglionic mice. Development 116:167-175, 1992 13. Coventry S, Yost C, Palmiter RD, et al: Migration of ganglion cell precursors in the ileoceca of normal and lethal spotted embryos, a murine model for Hirschsprung disease. Lab Invest 71:82-93, 1994 14. Hearn CJ, Murphy M, Newgreen D: GDNF and ET-3 differentially modulate the numbers of avian enteric neural crest cells and enteric neurons in vitro. Dev Biol 197:93-105, 1998 15. Lahav R, Ziller C, Dupin E, et al: Endothelin-3 promoters neural crest cell-proliferation and mediates a vast increase in melanocyte number in culture. Proc Nat Acad Sci U S A 93:3892-3897, 1996 16. Opdecamp K, Kos L, Amheiter H, et al: Endothelin signalling in the development of neural crest-derived melanocytes. Biochem Cell Biol 76:1093-1099, 1998 17. Stone J, Spirling L, Richardson M: The neural crest population responding to endothelin-3 in vitro includes multipotent cells. J Cell Sci 110:1673-1682, 1997 18. Wu JJ, Chen JX, Rothman TP, et al: Inhibition of in vitro enteric neuronal development by endothelin-3: Mediation by endothelin B receptors. Development 126:1161-1173, 1999 19. Shichiri M, Marumo F, Hirata Y: Endothelin-B receptor-mediated suppression of endothelial apoptosis. J Cardiovasc Pharmacol 31:S138-141, 1998
WOODWARD ET AL
20. Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156-159, 1987 21. Branchek TA, Gershon MD: Time course of expression of neuropeptide Y, calcitonin gene-related peptide, and NADPH diaphorase activity in neurons of the developing murine bowel and the appearance of 5-hydroxytryptamine in mucosal enterochromaffin cells. J Comp Neurol 285:262-273, 1989 22. McHugh KM: Molecular analysis of smooth muscle development in the mouse. Dev Dyn 204:278-290, 1995 23. Kono T, Takada M, Wu JY, et al: Double immunohistochemical detection of transmitter phenotype of proliferating cells using bromodeoxyuridine. Neurosci Lett 132:113-116, 1991 24. Gavrieli Y, Sherman Y, Ben-Sasson SA: Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119:493-501, 1992 25. Young HM, Ciampoli D, Hsuan J, et al: Expression of ret-, p75(NTR)-, Phox2a-, Phox2b-, and tyrosine hydroxylase-immunoreactivity by undifferentiated neural crest-derived cells and different classes of enteric neurons in the embryonic mouse gut. Dev Dyn 216:137-152, 1999 26. Sidebotham EL, Woodward MN, Kenny SE, et al: Assessment of protein gene product 9.5 as a marker of neural crest-derived precursor cells in the developing enteric nervous system. Pediatr Surg Int 17:304-307, 2001 27. Reid K, Turnley AM, Maxwell GD, et al: Multiple roles for endothelin in melanocyte development: Regulation of progenitor number and stimulation of differentiation. Development 122:3911-3919, 1996 28. Natarajan D, Grigoriou M, Marcos-Gutierrez CV, et al: Multipotential progenitors of the mammalian enteric nervous system capable of colonising aganglionic bowel in organ culture. Development 126: 157-168, 1999 29. Webster W: Embryogenesis of the enteric ganglia in normal mice and in mice that develop congenital aganglionic megacolon. J Embryol Exp Morphol 30:573-585, 1973 30. Sidebotham EL, Woodward MN, Kenny SE, et al: Localisation and endothelin-3 dependence of stem cells of the enteric nervous system. J Pediatr Surg 37:145-150, 2002
Background/Purpose: Mutations in the endothelin-3 (ET-3) and endothelin-B receptor (EDNR-B) genes cause terminal colonic aganglionosis in mice and are linked to Hirschsprung’s disease. These experiments are designed to determine if the development of terminal enteric ganglia depends on changes in proliferation, apoptosis, or differentiation of enteric neural crest (NC) cells in response to ET-3. Methods: Gut from embryonic lethal-spotted mice (lacking ET-3) and controls were investigated in vivo. NC-derived cells were identified immunohistochemically and their proliferation, apoptosis and differentiation monitored by bromodeoxyuridine incorporation, the terminal deoxytransferase poly dU nick end labelling (TUNEL) reaction, and appearance of neuronal nitric oxide synthase (NOS), respectively.
T
HE ENTERIC nervous system (ENS) in the colon is derived mainly from vagal neural crest (NC) cells that enter the mesenchyme of the foregut and then migrate toward the rectum.1 Although many mechanisms regulating the differentiation of NC-derived cells remain unknown, several of the factors that influence development of the ENS have been elucidated recently.2,3 In particular, endothelin-3 (ET-3) and the endothelin B receptor (EDNR-B) have been shown to be necessary for complete colonization of the colon because mice with mutations in the genes coding for these proteins display aganglionosis of the terminal colon and rectum.4,5 This restricted aganglionosis is similar to that seen in human patients with short-segment Hirschsprung disease (HD), which has been linked also with mutations in the genes
From the Departments of Child Health, Veterinary Preclinical Sciences, and Human Anatomy, the University of Liverpool, Liverpool, England. Supported by Research Fellowships from the Digestive Disorders Foundation (MNW), the Royal College of Surgeons of England (ELS, MNW) and the Pilkington Charitable Trust (ELS). Project Grant from Action Research (CRV and DHE). Address reprint requests to Emma L. Sidebotham, Institute of Child Health, Alder Hey Children’s Hospital, Eaton Rd, Liverpool L12 2AP, England. © 2003 Elsevier Inc. All rights reserved. 0022-3468/03/3809-0009$30.00/0 doi:10.1016/S0022-3468(03)00389-0 1322
Results: No differences in apoptosis or proliferation of NC cells were apparent between lethal-spotted embryos and controls. Although no temporal differences in the differentiation of NOS neurones were evident, these cells appeared more cranially in the gut in the absence of ET-3 than in controls. Conclusions: ET-3 has no detectable influence on proliferation, apoptosis, or timing of differentiation of NC-derived cells in the gut. However, the more proximal location of differentiated neurones in the absence of ET-3 is consistent with a restricted role in migration of NC-derived cells. J Pediatr Surg 38:1322-1328. © 2003 Elsevier Inc. All rights reserved. INDEX WORDS: Hirschsprungis disease, enteric nervous system, neural crest, endothelin-3.
coding for ET-3 and EDNR-B6,7 or alternatively is associated with decreased levels of ET-3 expression.8 Although ET-3 is necessary for colonization of the terminal colon by NC-derived cells, high levels of ET-3 mRNA expression have been localized to the mesenchyme of the embryonic mouse cecum and proximal colon at the time EDNR-B– expressing NC cells are migrating through this region.9 Furthermore, it has been shown that the requirement for ET-3/EDNR-B signalling is restricted to the relatively short period of development between embryonic days (E) 10 and 12.5 when the NC cells have arrived in the vicinity of the cecum.10,11 The time and place at which ET-3 acts are consistent with earlier observations showing that abnormalities in the behavior of NC cells first become apparent at the cecum in ET-3 -deficient lethal-spotted (ls/ls) mice, although aganglionosis is restricted to the terminal colon in postnatal mice.12,13 Much work has been undertaken to elucidate the effect of ET-3 on NC cells in tissue culture.14-18 However, these reports have produced apparently conflicting results in that some show ET-3 influences the proliferation or differentiation of cultured NC cells, whereas other reports show no effect on these parameters. Taken together, however, these reports indicate that the responses of NC cells to ET-3 are likely to depend on the source from which they were isolated, their developmental stage, and the conditions under which they were main-
Journal of Pediatric Surgery, Vol 38, No 9 (September), 2003: pp 1322-1328
ENDOTHELIN-3 AND ENTERIC NEURAL CREST CELLS
tained in culture.14-18 Thus, to determine how ET-3 influences NC cells and is thereby necessary for complete colonization of the colon, the responses of enteric NC-derived cells to ET-3 need to be examined at the appropriate time and place within the environment of the embryonic gut wall. We therefore have compared NC-derived cell proliferation and differentiation in situ in the bowel of ls/ls (ET-3 deficient) embryonic mice and in control ls/⫹ siblings at the time ET-3 is known to be necessary for gut colonization.10,11 In addition, we have examined NC cell apoptosis because activation of the EDNR-B has been reported also to prevent the apoptotic death of some cell types.19 MATERIALS AND METHODS
Animals and Genotyping Six- to 8-week-old male lethal-spotted (ls/ls) mice were mated with female C57 mice to produce litters of heterozygote (ls/⫹) offspring. The female ls/⫹ progeny subsequently were time mated with male homozygote ls/ls mice and the date of the vaginal plug counted as embryonic day (E) 0.5. Pregnant mice were killed at E11.5, E12.5, and E13.5 by cervical dislocation. The age of embryos was confirmed by staging based on examination of the external features. E11.5 uteri were kept in Dulbecco’s modified Eagle medium (DMEM; Gibco Life Technologies) at 37°C until all embryos were dissected. The gut was dissected under aseptic conditions and oriented using tungsten needles on Millipore-CM culture plate inserts (Millipore, Watford, Herts, UK) in 6-well Nunclon culture dishes (Gibco Life Technologies, Paisly, Strathclyde, UK). The gut explants then were either fixed immediately over 4% paraformaldehyde or after culture. Genotyping was undertaken to identify ls/ls embryos from their heterozygote control littermates by extraction of total cellular RNA from samples of esophagus and stomach using the acid phenol-chloroform method.20 RNA rather than DNA genotyping was used to simplify the identification of restriction digestion fragments uninterrupted by intronic sequences. First strand cDNA synthesis was performed using Superscript II reverse transcriptase (GIBCO-BRL) as previously described.8 cDNA samples were amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using ET-3 primers that had previously been designed in this laboratory.9 The ET-3 primers amplified a product that spanned the missense 758 C 3 T change of the ls allele mutation.4 RT-PCR conditions established were 35 cycles including denaturation at 94°C for 1 minute, annealing at 51°C for 1 minute, and extension at 72°C for 1 minute followed by a final extension step of 10 minutes. After RT-PCR, an Xmn1 (Boehringer Mannheim, Lewer, East Sussex, UK) restriction digest was performed on the PCR product. The Xmn1 restriction enzyme cuts the sequence GAANN2NNTTC, which is lost in the ls mutation.4 Electrophoresis on ethidium bromide stained 2% (wt/vol) agarose gels showed a single 244 base-pair band in homozygote ls/ls embryos, whereas 244, 161 and 83-bp bands were seen in heterozygote ls/⫹ embryos.
Immunohistochemistry Tissues were fixed in 4% paraformaldehyde for 2 hour, rinsed in PBS for 1 hour, immersed in 15% (wt/vol) sucrose in phosphate-buffered saline (PBS) for 24 hours at 4°C, then embedded in 7.5% (wt/vol) gelatin/15% (wt/vol) sucrose in PBS before freezing with isopentane/ liquid nitrogen. Serial longitudinal 10-m sections were cut with a cryostat.
1323
Neural crest-derived cells were identified by staining for protein gene product 9.5 (PGP 9.5) using a rabbit antiserum (Biogenesis, Poole, Dorset, UK) at 1:4000 dilution or the p75 low-affinity nerve growth factor receptor (p75) using a rat antiserum (Chemicon, Chandlersford, Hampshire, UK) at 1:100. Differentiation of neural crest cells was determined by the appearance of nitric oxide synthase (NOS)positive neurons in the gut from E11.5 through E13.5 using a goat antiserum (kindly donated by Dr Piers Emson, Babraham Institute, Cambridge, UK) diluted 1:1000. NOS was selected as a marker of neuronal differentiation, because this is one of the first specific neuronal phenotypes to differentiate and has been shown to be present in the region of cecum and colon during embryogenesis of the gut.21,22 Sections were incubated overnight at 4°C in primary antisera diluted in PBS containing 1% (wt/vol) bovine serum albumin. Sections were washed in PBS and incubated for 1 hour in appropriate secondary antisera; Cy3-conjugated donkey antirabbit immunoglobulin (1:500 dilution; Jackson Laboratories, West Grove, PA), Texas red-conjugated goat antirabbit immunoglobulin (1:200 dilution; Jackson Laboratories), FITC-conjugated sheep antirat immunoglobulin (1:32 dilution; Sigma Gillingham, Dorset, UK), and biotinylated donkey antisheep immunoglobulin (1:100 dilution, Sigma) followed by FITC-conjugated streptavidin (1:50 dilution, Amersham, Chalfont, St Giles, Bucks, UK). Omission of primary antisera resulted in the total absence of fluorescent signal. For double staining, omission of either one of the primary antisera resulted in loss of the appropriate fluorochrome signal.
Cell Proliferation Assay E11.5 uteri were kept in Dulbecco’s modified Eagle medium (DMEM; Gibco Life Technologies) at 37°C until all embryos were dissected. The gut was dissected under aseptic conditions and oriented using tungsten needles on Millipore-CM culture plate inserts (Millipore) in 6-well Nunclon culture dishes (Gibco Life Technologies). The gut explants were placed over 1 mL DMEM supplemented with 10% (vol/vol) fetal calf serum (Gibco Life Technologies), 1% (wt/vol) L-glutamine, and 50 g/mL penicillin/streptomycin and the cultures maintained in a 5% (vol/vol) CO2 incubator at 37°C. The culture medium was supplemented with 10 mol/L bromodeoxyuridine (BrdU) for 1 hour before fixing the tissue with 4% paraformaldehyde.23 Sections were processed initially for immunolabelling for PGP 9.5 as above, washed in PBS, treated with 4 mol/L HCl for 15 minutes and then washed again in PBS until the pH reached 6.5 or above. Sections then were incubated with mouse anti-BrdU (DAKO) diluted 1:25 in PBS for 1 hour at 38°C, washed in PBS for 3 ⫻ 10 minutes, incubated with biotinylated rabbit antimouse immunoglobulin (DAKO, UK) 1:100 in PBS for 30 minutes, washed again, and finally incubated with streptavidin/FITC (Amersham) 1:50 in PBS for 1 hour.
Detection of Apoptosis E11.5 gut from lethal-spotted crosses, fixed immediately on dissection, was examined using a modified TUNEL reaction for apoptotic cells.24 Sections were processed initially for immunolabelling for PGP 9.5 as above, washed in distilled H2O for 2 ⫻ 10 minutes treated with 1.5% (vol/vol) TRITON X100 in PBS for 30 minutes and washed again in H2O for 4 ⫻ 2 minutes. Positive controls were incubated with DN buffer (30 mmol/L Tris HCl; pH 7.2; 140 mmol/L potassium cacodylate; 4 mmol/L MgCl2; 0.1 mmol/L dithiothreitol) for 2 to 3 minutes followed by 5 g/mL type II DNAse (Sigma) in DN buffer for 10 minutes at room temperature and then washed in H2O, for 4 ⫻ 2 minutes. Sections then were incubated with TdT buffer (30 mmol/L Tris HCl, pH 7.2; 140 mmol/L sodium cacodylate; 1 mmol/L cobalt chloride) for 2 to 3 minutes followed by TUNEL reaction mix for 2 hours at 38°C. TUNEL reaction mix comprised 15 U terminal deoxynucleotidyl transferase (TdT, Pharmacia) and 10 mol/L biotinylated
1324
WOODWARD ET AL
deoxyuridine triphosphate (dUTP; Boehringer Mannheim) in 100 L TdT buffer. TdT enzyme was omitted from reaction mix for negative controls. Sections were allowed to cool, washed in H2O for 3 ⫻ 10 minutes and then in PBS for 10 minutes before incubation with streptavidin/FITC (Amersham) diluted 1:50 in PBS for 1 hour.
Determination of Cell Numbers Counts of neural crest cells in each experiment were carried out on a minimum of 10 sections from between 4 and 10 samples of gut and matching numbers of control samples. The area in which cells were counted extended from the cecum to the most caudal PGP 9.5-positive cells, which were restricted to the proximal colon at the times at which counts were performed.11,13 Statistical analysis to compare counts of experimental groups with controls was performed using a 2-tailed student t test with a significance level of .05.
RESULTS
Genotyping of Embryos RT-PCR was performed using ET-3 primers on total RNA, and subsequently an Xmn1 restriction digest was undertaken on the PCR product. The expected single 244-bp band was amplified from all embryos. After restriction digestion, this single band remained in samples from homozygote ls/ls embryos, whereas additional 161 and 83-bp bands corresponding to digestion fragments of the wild-type allele appeared in heterozygote ls/⫹ embryos (Fig 1). A total of 29 ls/ls and 25 ls/⫹ embryos were genotyped at the time-points studied, which approximates to the expected 50:50 Mendelian distribution. Differentiation of Neural Crest Cells No NOS-positive cells were apparent in the gut of either ls/ls or ls/⫹ control mice at E11.5 (Fig 2A,B). By
Fig 2. Appearance of NOS-expressing neurons in embryonic ls/ls and control ls/ⴙ mouse gut. At E11.5, no expression of NOS is apparent in the ileocecal region of control (A) or ls/ls (B) gut. By E 12.5 a very few sporadic NOS-positive neurones have appeared in the cecum of control gut (E, arrow) but are absent in the terminal small intestine (C), whereas in ls/ls gut, NOS is evident in the terminal ileum (D, arrow), but no NOS-positive cells are seen in the cecum (F). By E13.5, many more NOS-positive cells are evident, but the pattern of expression in the ileocecal region persists. NOS-positive cells are evident in the cecum (I) of control gut, but the small intestine remain devoid of NOS neurons (G), whereas in ls/ls gut, NOS-positive cells are readily apparent in the small intestine (H), and NOS cells are seen only sporadically in the caecum (J, arrow). Diagram of the gut of control (K) and ls/ls (L) gut shows the distribution of NOS-positive cells (dotted areas) along the length of the gut at E13.5 (bar ⴝ 50 m) ce, cecum; co, colon; re, rectum; st, stomach; si, small intestine.
Fig 1. Genotyping of embryos produced from crosses of male ls/ls with female ls/ⴙ mice. Agarose gel electrophoresis of PCR products amplified from the cDNA of ls/ⴙ (1, 2) and ls/ls (3, 4) embryos. A single 244-bp band is amplified from both homozygous and heterozygous embryos (1, 3). After digestion with Xmn1, 161 bp and 31 bp bands are produced from the wild-type allele of heterozygotes (2), but the is allele remains uncleaved (4).
E12.5, small numbers of NOS-positive neurons were present in the stomach (not shown) in both ls/ls embryos and controls. In controls, no NOS labelling was detected in the small intestine (Fig 2C), but occassional NOSpositive cells were seen in the cecum (Fig 2E, arrow). In
ENDOTHELIN-3 AND ENTERIC NEURAL CREST CELLS
contrast, NOS neurons were present in the ls/ls distal small intestine (Fig 2D, arrow), but no NOS labelling was detected in ls/ls cecum (Fig 2F). By E13.5 many more NOS-positive cells were apparent in both control and ls/ls mice. However, the differences in location of positive cells in the ileocecal region persisted with many NOS-positive cells in control cecum (Fig 2I) but no NOS labelling in the small intestine (Fig 2G), whereas in ls/ls gut there were many NOS-positive cells in the terminal ileum (Fig 2H) but only occasional cells in the cecum (Fig 2J, arrow). NOS-positive cells were present along the length of the colon in control mice but were restricted to the proximal colon in ls/ls mice at this stage reflecting the limited colonization of the colon by NC-derived cells (Fig 2K,L).11 Proliferation of Neural Crest Cells The low affinity neurotrophin receptor p75 is displayed by most if not all NC cells in the embryonic mouse gut,25 but being an integral membrane protein it is lost during permeabilization procedures to assay cell proliferation and apoptosis. However, double labelling immunohistochemistry has shown that essentially all the p75-positive cells found at the leading edge of migration in the proximal colon at E11.5 also stain for PGP9.5, thus confirming PGP9.5 as a suitable marker to identify NC cells in these studies.26 Gut was taken from ls/ls and ls/⫹ embryos at E11.5 and pulsed in organ culture for 1 hour with BrdU. The BrdU was incorporated into large numbers of epithelial and mesenchymal cells throughout the gut specimens (Fig 3A and C). NC-derived cells, which labelled with PGP9.5, were also readily identifiable (Fig 3B and D, arrows). Small numbers of NC-derived cells also labelled for BrdU. Counts of BrdU-labelled PGP-positive cells in the cecum and proximal colon showed that there was no significant difference in the proportions of NC cells that had incorporated BrdU in the ls/ls and ls/⫹ control guts (Fig 3E). These determinations were powered to be able to detect a minimal difference of 14% in populations at the significance level of 85%. Apoptosis of Neural Crest Cells The TUNEL reaction to detect nuclear DNA fragmentation characteristic of apoptosis24 was performed on E11.5 gut from ls/ls and controls (ls/⫹). No epithelial or mesenchymal labelling was observed in negative controls for the TUNEL reaction, whereas all cells were labelled after pretreatment of sections with DNAse (not shown). Although small numbers of apoptotic epithelial and mesenchymal cells were seen throughout the specimens (Fig 4A and C), there was no colocalization of PGP9.5 immunoreactivity (Fig 4B and D) with any of the
1325
TUNEL-labelled cells. Thus, TUNEL labelling did not provide any evidence that NC cells undergo apoptosis at this stage of embryonic development either in the presence or absence of ET-3. DISCUSSION
Several studies have described the potential ways in which ET-3 can influence the behavior of NC-derived cells in vitro. However, findings from these studies have suggested widely differing consequences of the ET-3/ EDNR-B interaction, dependent on the time of isolation of cells, the cell population studied, and the culture conditions used.14,15,17,18,27 It has therefore been difficult to draw any firm conclusions regarding the mechanisms by which ET-3 prevents terminal aganglionosis of the colon. Comparison of NC-derived cell proliferation, apoptosis, and differentiation in situ in the gut of ls/ls embryos and age-matched ls/⫹ sibling controls permitted us to study the effects of ET-3 under the necessary controlled conditions to define its action during ENS development. The current in vivo study did not show any significant ET-3 effects on enteric NC-derived cell proliferation or apoptosis. No temporal differences in the differentiation of NC-derived cells were apparent, judged by the appearance of NOS immunoreactivity, which defines the differentiation of one of the first neuronal phenotypes in the gut. However, the position at which NC cells differentiated into NOS-positive neurons was shifted rostrally from the cecum and proximal colon to the distal small intestine in ls/ls gut. These observations indicate that rather than regulating the timing of neuronal differentiation, ET-3 is necessary for the correct migration of NC-derived cells in the gut wall, their differentiation being independent of ET-3. Previous studies have found that more PGP9.5-positive cells were found in cultures of immunoselected enteric NC-derived cells after 7 days culture in the presence of the EDNB-R antagonist, BQ788 vitro.18 Because this marker is expressed by most if not all migrating NC-derived cells at E11.5,26 then this observation is consistent with the hypothesis that ET-3 exerts a survival-promoting or mitotic effect of ET-3 on the cells at some point during this period of culture.18 However, our observations in vivo indicate that NC-derived cells do not undergo programmed cell death in the absence or in the presence of ET-3, ruling out the possibility that ET-3 exerts its effect of NC-derived cell survival. Furthermore, we were unable to detect an effect of ET-3 on the mitotic index of NC-derived cells in the embryonic gut wall, and so these experiments do not provide any support for the hypothesis that ET-3 regulates cell proliferation to achieve full colonization of the gut.
1326
WOODWARD ET AL
Fig 3. Proliferation of neural crest cells in the embryonic gut. Longitudinal sections of ileocecal region of E11.5 control (A, B) and ls/ls (C, D) gut were dual labelled for BrdU (A, C) and PGP-9.5 (B, D) immunoreactivity. Numerous proliferating epithelial and mesenchymal cells were identified throughout the specimens (A, C). A number of PGP-immunoreactive cells that had incorporated BrdU also were identified (B, D). The proportion of NC cells that were dual labelled was equivalent in specimens and controls (Bar ⴝ 20 m). (E) Histogram shows the percentage of PGP9.5-positive cells at the leading edge of migration in cecum and proximal colon that had incorporated BrdU (ⴞSEM; n ⴝ 9). There was no significant difference (t test; P ⴝ .56) between the numbers of NC cells incorporating BrdU in control (ls/ⴙ) and ls/ls gut.
ENDOTHELIN-3 AND ENTERIC NEURAL CREST CELLS
1327
Fig 4. Apoptosis of neural crest cells in the embryonic gut. Sections were stained for TUNEL labelling (A, C), and PGP-9.5 immunoreactivity (B, D). Small numbers of apoptotic epithelial and mesenchymal cells were identified throughout the specimens (A, C, arrowheads). Numerous PGP-immunoreactive cells were identified at the level of the presumptive myenteric plexus (B, D) but there was no evidence of colocalization of immunoreactivities in any of the specimens. (bar ⴝ 50 m) e, epithelium; m, mesenchyme.
Although our results do not show any significant effects of ET-3 on the proliferation or differentiation of the bulk population of NC-derived cells in the embryonic gut, we cannot rule out the possibility that ET-3 affects these aspects of the behavior of a small subpopulation of cells. It has been shown recently that a small number of cells at the leading edge of NC cell migration differ from more rostral cells as they do not yet display all the differentiation markers characteristic of NC-derived cells of the ENS.25 Previous studies have not used ubiquitous markers of NC cells and so differences in the behavior of cells at the leading edge of migration are particularly liable to be missed.13,28,29 This omission is potentially of
significance in the light of the observation that a very few NC-derived cells in this position are sufficient to colonize the entire colon.30 Any effect of ET-3 on such a small subpopulation of stem cells would be masked within the larger population of unresponsive NC-derived cells but, nevertheless, may be responsible for the migratory defect seen in the absence of ET-3.
ACKNOWLEDGMENT Additional support The authors thank Dr Piers Emson of the ARC, Babraham, Cambridge, England for the generous donation of NOS antibodies.
REFERENCES 1. Le Douarin NM, Teillet MA: The migration of neural crest cells to the wall of the digestive tract in avian embryo. J Embryol Exp Morphol 30:31-48, 1973 2. Gershon MD: Genes and lineages in the formation of the enteric nervous system. Curr Opin Neurobiol 7:101-109, 1997 3. Taraviras S, Pachnis V: Development of the mammalian enteric nervous system. Curr Opin Genet Dev 9:321-327, 1999 4. Baynash AG, Hosoda K, Giaid A, et al: Interaction of endothelin-3 with endothelin-B receptor is essential for development of
epidermal melanocytes and enteric neurons. Cell 79:1277-1285, 1994 5. Hosoda K, Hammer RE, Richardson JA, et al: Targeted and natural (piebald-lethal) mutations of endothelin-B receptor gene produce megacolon associated with spotted coat color in mice. Cell 79:1267-1276, 1994 6. Puffenberger EG, Hosoda K, Washington SS, et al: A missense mutation of the endothelin-B receptor gene in multigenic Hirschsprung’s disease. Cell 79:1257-1266, 1994
1328
7. Kusafuka T, Puri P: Genetic aspects of Hirschsprung’s disease. Semin Pediatr Surg 7:148-155, 1998 8. Kenny SE, Hofstra RMW, Buys CHCM, et al: Reduced endothelin-3 expression in sporadic Hirschsprung’s disease. Br J Surg 87:580585, 2000 9. Leibl MA, Ota T, Woodward MN, et al: Expression of endothelin 3 by mesenchymal cells of embryonic mouse caecum. Gut 44:246-252, 1999 10. Shin MK, Levorse JM, Ingram RS, et al: The temporal requirement for endothelin receptor-B signalling during neural crest development. Nature 402:496-501, 1999 11. Woodward MN, Kenny SE, Vaillant C, et al: Time-dependent effects of endothelin-3 on enteric nervous system development in an organ culture model of Hirschsprung’s disease. J Pediatr Surg 35:2529, 2000 12. Kapur RP, Yost C, Palmiter RD: A transgenic model for studying development of the enteric nervous system in normal and aganglionic mice. Development 116:167-175, 1992 13. Coventry S, Yost C, Palmiter RD, et al: Migration of ganglion cell precursors in the ileoceca of normal and lethal spotted embryos, a murine model for Hirschsprung disease. Lab Invest 71:82-93, 1994 14. Hearn CJ, Murphy M, Newgreen D: GDNF and ET-3 differentially modulate the numbers of avian enteric neural crest cells and enteric neurons in vitro. Dev Biol 197:93-105, 1998 15. Lahav R, Ziller C, Dupin E, et al: Endothelin-3 promoters neural crest cell-proliferation and mediates a vast increase in melanocyte number in culture. Proc Nat Acad Sci U S A 93:3892-3897, 1996 16. Opdecamp K, Kos L, Amheiter H, et al: Endothelin signalling in the development of neural crest-derived melanocytes. Biochem Cell Biol 76:1093-1099, 1998 17. Stone J, Spirling L, Richardson M: The neural crest population responding to endothelin-3 in vitro includes multipotent cells. J Cell Sci 110:1673-1682, 1997 18. Wu JJ, Chen JX, Rothman TP, et al: Inhibition of in vitro enteric neuronal development by endothelin-3: Mediation by endothelin B receptors. Development 126:1161-1173, 1999 19. Shichiri M, Marumo F, Hirata Y: Endothelin-B receptor-mediated suppression of endothelial apoptosis. J Cardiovasc Pharmacol 31:S138-141, 1998
WOODWARD ET AL
20. Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156-159, 1987 21. Branchek TA, Gershon MD: Time course of expression of neuropeptide Y, calcitonin gene-related peptide, and NADPH diaphorase activity in neurons of the developing murine bowel and the appearance of 5-hydroxytryptamine in mucosal enterochromaffin cells. J Comp Neurol 285:262-273, 1989 22. McHugh KM: Molecular analysis of smooth muscle development in the mouse. Dev Dyn 204:278-290, 1995 23. Kono T, Takada M, Wu JY, et al: Double immunohistochemical detection of transmitter phenotype of proliferating cells using bromodeoxyuridine. Neurosci Lett 132:113-116, 1991 24. Gavrieli Y, Sherman Y, Ben-Sasson SA: Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119:493-501, 1992 25. Young HM, Ciampoli D, Hsuan J, et al: Expression of ret-, p75(NTR)-, Phox2a-, Phox2b-, and tyrosine hydroxylase-immunoreactivity by undifferentiated neural crest-derived cells and different classes of enteric neurons in the embryonic mouse gut. Dev Dyn 216:137-152, 1999 26. Sidebotham EL, Woodward MN, Kenny SE, et al: Assessment of protein gene product 9.5 as a marker of neural crest-derived precursor cells in the developing enteric nervous system. Pediatr Surg Int 17:304-307, 2001 27. Reid K, Turnley AM, Maxwell GD, et al: Multiple roles for endothelin in melanocyte development: Regulation of progenitor number and stimulation of differentiation. Development 122:3911-3919, 1996 28. Natarajan D, Grigoriou M, Marcos-Gutierrez CV, et al: Multipotential progenitors of the mammalian enteric nervous system capable of colonising aganglionic bowel in organ culture. Development 126: 157-168, 1999 29. Webster W: Embryogenesis of the enteric ganglia in normal mice and in mice that develop congenital aganglionic megacolon. J Embryol Exp Morphol 30:573-585, 1973 30. Sidebotham EL, Woodward MN, Kenny SE, et al: Localisation and endothelin-3 dependence of stem cells of the enteric nervous system. J Pediatr Surg 37:145-150, 2002