GDNF and ET-3 Differentially Modulate the Numbers of Avian

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DEVELOPMENTAL BIOLOGY 197, 93–105 (1998) ARTICLE NO. DB988876 GDNF and ET-3 Differentially Modulate the Numbers of Avian Enteric Neural Crest ViewCells metadata, citation and and Enteric similar papers at Neuronscore.ac.uk in Vitro brought to you by CORE provided by Elsevier - Publisher Connector

Catherine J. Hearn,* Mark Murphy,† and Don Newgreen* *The Murdoch Institute and †The Walter and Eliza Hall Institute, Parkville 3052, Victoria, Australia

Vagal (hindbrain) neural crest cells migrate rostrocaudally in the gut to establish the enteric nervous system. Glial-derived neurotrophic factor (GDNF) and its receptor(s), and endothelin-3 (ET-3) and its receptor, are crucial for enteric nervous system development. Mutations interrupting either of these signaling pathways cause aganglionosis in the gut, termed Hirschsprung’s disease in humans. However, the precise functions of GDNF and ET-3 in enteric neurogenesis are still unknown. We isolated precursor cells of the enteric nervous system from the vagal level neural crest of E1.7 quail embryos prior to entry into the gut and from the developing midgut at stages corresponding to migrating (E4.7) and longer resident differentiating cells (E7) using HNK-1 immunoaffinity and magnetic beads. These cells were tested for their response to GDNF and ET-3 in culture. ET-3 and GDNF had little effect in vitro on the growth, survival, migration, or neurogenesis of E1.7 vagal neural crest cells. In contrast, GDNF increased the proliferation rate and numbers of enteric neural precursors isolated from the E4.7 and E7 gut. Also, many more neurons and neurites developed in cultures treated with GDNF, disproportionately greater than the effect on cell numbers. At high cell density and in the presence of serum, ET-3, and GDNF had an additive effect on proliferation of neuron precursor cells. In defined medium, or low cell density, ET-3 reduced cell proliferation, overriding the proliferative effect of GDNF. Regardless of the culture condition, the stimulatory effect of GDNF on neuron numbers was strikingly diminished by the simultaneous presence of ET-3. We propose first that GDNF promotes the proliferation in the migratory enteric neural precursor cell population once the cells have entered the gut and is especially crucial for the differentiation of these cells into nonmigrating, nonproliferating enteric neurons. Second, we suggest that ET-3 modulates the action of GDNF, inhibiting neuronal differentiation to maintain the precursor cell pool, so ensuring sufficient population numbers to construct the entire enteric nervous system. Third, we suggest that generalized defects in enteric neural precursor cell numbers and differentiation due to mutations in the ET-3 and GDNF systems are converted to distal gut neural deficiencies by the rostrocaudal migration pattern of the precursors. Fourth, we suggest that additional factors such as those found in serum and produced by the enteric neural cells themselves are likely also to be involved in enteric nervous system development and consequently in Hirschsprung’s disease. ᭧ 1998 Academic Press Key Words: intestine; enteric nerves; cell migration; Hirschsprung’s disease; neural crest; ET-3; GDNF.

INTRODUCTION which transiently express sympathoadrenal markers including tyrosine hydroxylase (Durbec et al., 1996a; Blaugrund The enteric nervous system (ENS) in vertebrates derives et al., 1996). The sacral neural crest (caudal to somites 28 from neural crest cells that migrate into the developing gut in birds) contributes to the ENS of the hindgut and caudal (Yntema and Hammond, 1954). Vagal level neural crest cells part of the midgut (Pomeranz et al., 1991; Serbedzija et (originating from the hindbrain at the level of somites 1–7 al., 1991), although the population size and identity of the in birds) are a major and indispensable source of neurons derivatives are as yet unclear. A lengthy rostrocaudal migra- and glia throughout the gut (Le Douarin and Teillet, 1973; tion is necessary to populate the gut with cells from the Yntema and Hammond, 1954). Neural crest cells from the vagal neural crest, and this has been timetabled in birds postvagal level contribute most of the foregut enteric neu- (Allan and Newgreen, 1980; Le Douarin and Teillet, 1973; rons, giving rise to a lineage of early generated neurons Tucker et al., 1986) and rodents (Kapur et al., 1992; New-

0012-1606/98 $25.00 Copyright ᭧ 1998 by Academic Press All rights of reproduction in any form reserved. 93

AID DB 8876 / 6x3c$$$$41 04-18-98 14:37:17 dbas 94 Hearn, Murphy, and Newgreen green and Hartley, 1995; Webster, 1973). The appearance of In this study, we isolated quail neural crest cells from (i) the first differentiated neurons follows soon after coloniza- the vagal level prior to entry into the gut, (ii) the developing tion by neural crest cells (Allan and Newgreen, 1980; Fair- midgut while the neural crest cells were in a migratory man et al., 1995), with additional neurons continuing to phase, and (iii) at a later stage, during differentiation. To appear well into the postnatal period in both rodents (Ga- isolate and purify cells from the developing gut we utilized bella, 1967) and avians (Ali and McLelland, 1979). the HNK-1 epitope which is expressed by practically all Hirschsprung’s disease (HSCR) is a common congenital migratory and postmigratory neural crest cells in the avian condition in humans characterized by enteric aganglionosis embryo (Tucker et al., 1984). We then examined the in vitro of the caudal portion of the intestine. Partial ablation of the effects of ET-3 and GDNF on these ENS precursors. vagal neural crest cell population in chick embryos results in HSCR-like aganglionic segments of varying lengths al- ways including the most caudal intestine. The length of the MATERIALS AND METHODS aganglionic segment is proportional to the extent of loss of vagal neural crest cells (Yntema and Hammond, 1954). This Factors points to a simple reduction in the number of ENS precursor Human recombinant GDNF obtained from PeproTech (Rocky cells being able to produce the HSCR phenotype. Hill, NJ) was stored at 080ЊC and used at 125 nM in tissue culture Over a third of HSCR can be accounted for by mutations medium. Endothelin-3-synthesized peptide from Sigma (St. Louis, in the RET gene (Angrist et al., 1995; Attie´ et al., 1995). MO) was stored at 080ЊC and used at 100 nM. These concentrations RET is a transmembrane receptor tyrosine kinase whose had given maximal effect in previous experiments (Maxwell et al., ligand has recently been identified as glial-cell-line-derived 1996; Reid et al., 1996). neurotrophic factor (GDNF) (Durbec et al., 1996b; Trupp et al., 1996). GDNF is an atypical member of the TGF-b superfamily, a group of molecules which play important Neural Tube and Crest Explant Cultures roles in the regulation of cell survival, proliferation, and Quail (Coturnix coturnix japonica) embryos of 1.7 days incuba- differentiation (Moses and Serra, 1996). Although some tion (E1.7) were harvested and embryos of 10 to 14 somites (HH dominant mutations in GDNF have been found, other 10 to 11/ (Hamburger and Hamilton, 1951)) selected. A tissue block GDNF mutations have not resulted in a HSCR phenotype with the neural tube and somites 1 to 7 (i.e., the vagal level), was (Angrist et al., 1996; Ivanchuk et al., 1996; Salomon et al., microdissected free and incubated in 2 mg/ml dispase II (Boehringer 1996). Ret homozygous knockout mice suffer intestinal Mannheim) in Dulbecco’s modified Eagle medium (DME) for 20 aganglionosis of the entire gut, excluding the foregut, which min at room temperature, after which the neural tube was freed from surrounding tissues. These vagal neural tubes were separated has a substantial nonvagal contribution of ENS cells (Schu- into exact somite-number groups for all subsequent procedures. chardt et al., 1994). In contrast to humans, in mice the The isolated neural tubes were washed in DME containing 0.5% heterozygotes are normal. GDNF knockouts show an al- bovine serum albumen (BSA, fraction V, Boehringer-Mannheim) most identical phenotype (Moore et al., 1996; Pichel et al., and transferred to culture medium consisting of DME with 10% 1996; Sańchez et al., 1996). fetal calf serum (FCS, Trace Bioscientific, Australia), 20 mM Hepes, The endothelin signaling pathway has also been impli- 20 mM glutamine, 6 mg/ml penicillin, and 10 mg/ml streptomycin. cated in HSCR. A number of HSCR patients have mutations Alternatively, defined medium of N1 supplement plus 0.5% BSA in the endothelin-3 (ET-3) or the endothelin receptor B (Bottenstein et al., 1980) was used. (EDNRB) genes. These result in variable HSCR combined Bacteriological petri dishes (30 mm diameter, Disposable Prod- with other neural crest defects such as hypopigmentation ucts, Australia) were coated with human plasma fibronectin (100 ml, 5 mg/ml in F12, 30 min, 37ЊC; Boehringer-Mannheim). After of the skin, hair, and irises and cochlear deafness (Edery washing away nonbound fibronectin, parallel line pairs (400 mm et al., 1996; Puffenberger et al., 1994). In addition, mouse separation) were scored into the coated surface in a rosette pattern mutants with enteric aganglionosis, lethal spotted (ls/ls) using two scalpel blades glued together. l l and piebald lethal (s /s ), carry mutations in ET-3 and Neural tubes for each treatment were matched for somite num- EDNRB, respectively (Baynash et al., 1994; Hosoda et al., ber to ensure comparability. In each dish, 5–6 neural tubes were 1994). Mutations in the EDNRB gene are also responsible arranged on the rosette with the dorsal (i.e., neural crest) side facing for the spotting lethal (sl/sl) rat phenotype (Ceccherini et outward, and with the zone of peak enteric neural crest emigration al., 1995; Gariepy et al., 1996). (somite level 3–5) perpendicular to and within the scored lines. Clearly ET-3 and GDNF have important roles in ENS Cells cannot cross the scored lines, constraining cell migration to development, but their actions in this system are undefined. a defined corridor. The rosette formation was designed to equalize explant–explant interactions by, for example, explant-derived solu- These molecules could influence cells within the vagal neu- ble factors. Explants were allowed to attach in a 5% CO2 atmo- ral tube prior to migration, while migrating en route to sphere at 37ЊC in 500 ml of culture medium containing GDNF, ET- the gut or when enteric neuron precursors are migrating 3, or neither. After2hanadditional 200 ml of the same medium through or settling in the gut. It is not known whether they was added and incubation continued. At 20 h incubation, phase- affect proliferation, survival, migration, or differentiation contrast micrographs of cell migration were made on a Zeiss IM- of enteric neural precursors, or have a combination of roles. 35 microscope with a Nikon 601M camera. The number of mesen-

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chymal neural crest cells migrating away from the dorsal neural times in FCS/PBS before being suspended in culture medium and tube within the 400-mm-wide corridor defined by the scored lines stored at 4ЊC. was counted from photographic prints. Values are presented as The addition of 1 HNK-1-coated Dynabead per 16 gut cells was means { standard deviation. In addition, for each explant the dis- found to be optimal to extract as many HNK-1-positive cells as placement of the most distant neural crest cell from the origin in possible, while simultaneously minimizing contamination with the dorsal neural was measured. This was plotted against the num- HNK-1-negative cells, and without covering each cell with an ex- ber of cells in the same explants to evaluate the relationship be- cess of beads. This number gave an average of 1.25 beads per HNK- tween cell number and extent of cell migration. 1-positive cell based on initial observations that about 5% of cells in the midgut of an E4.7 embryo express HNK-1. The HNK-1- coated beads were diluted in 200 ml of 0.5% BSA in DME before Dissociated Neural Crest Cell Cultures being added to the total cell suspension (approximately 1 ml vol- ume for 30–40 pooled midguts). The cells and beads were then Vagal neural crest cells. Vagal level neural tubes were obtained incubated on ice with constant rotation for 60 min. Following this, from E1.7 quail embryos as described above. These were explanted the cells plus beads were placed on a magnet (MPC-E-1, Dynal) to onto uncoated 30-mm-diameter tissue culture dishes (Falcon) in allow the supernatant to be removed. The beads with attached cells were resuspended in culture medium and the magnetic extraction 500 ml of culture medium and cultured in 5% CO2 at 37ЊC. After 2 h an additional 400 ml of culture medium was added and incubation repeated before these cells were harvested and counted as above. continued for an additional 18 h when the neural tubes were de- Determining the yield and purity of enteric precursor cells. tached with a glass probe and removed. The cells that had migrated Magnetic extraction yielded 7000–10,000 cells for each E4.7 mid- from the dorsal neural tube (Newgreen et al., 1979) were then har- gut and 25,000–40,000 for each E7 midgut. This is 30–50% of vested by two brief washes with Ca2/- and Mg2/-free Hanks’ buff- the estimated number of HNK-1-positive cells present in these ered salt solution (HBSS) followed by 5 min at 37ЊC in HBSS with segments. 0.05% trypsin (Trace Biosciences, Aust). The culture medium, all The purity of the bead-separated population was tested by plating washes, and the enzyme solution were pooled and the cells were out total (unseparated) and separated cells and using immunocyto- pelleted by centrifugation for 10 min at 120g and then resuspended chemistry to detect the HNK-1 epitope. The cells were plated in in culture medium and counted. The yield was 1000–1500 neural fibronectin-coated (10 mg/ml, 45 min, room temperature) 60-well crest cells per vagal segment; the culturing of these cells is de- tissue culture grade Terasaki dishes (LUX, Nunc) at a density of scribed below. 5000 cells/12 ml well. These were incubated in 5% CO2 at 37ЊC for 2 or 20 h and then fixed with methanol (020ЊC). Immunocyto- chemistry involved a 1 h incubation with 1/10 HNK-1 antibody, followed by detection with alkaline phosphatase-conjugated goat HNK-1 Cell Separation of Migratory and anti-mouse IgG and IgM (1/150, DAKO) followed by NBT/BCIP Postmigratory Enteric Neural Precursors (Boehringer Mannheim) visualization. The total cells in each well were visualized by phase-contrast microscopy using a Zeiss IM-35 Dissection of tissues and cell dissociation. Segments of midgut and counts of HNK-1-positive and -negative cells in each popula- (duodenal flexure to ceca) were dissected from E4.7 and E7 quail tion were made. In the midgut segment of an E4.7 quail, less than embryos, corresponding to HH26-27 and HH32-34, respectively 5% of the total cells express the HNK-1 epitope. Following purifi- (Hamburger and Hamilton, 1951), and the mesentery including the cation with HNK-1-coated magnetic beads, the purity of HNK-1- nerve of Remak was removed. At HH26 neural crest cells have expressing cells was enriched to 90–95% of the final cell population colonized to the level of the ceca and differentiated neurons are (Figs. 1A and 1B). detected as far caudal as the umbilicus. At HH32, neural crest cells have fully colonized the gut almost to the level of the anus and differentiated neurons are found throughout the midgut (Allan and Culture and Analysis of Enteric Neural Crest Cells Newgreen, 1980; Fairman et al., 1995). The segments were incubated in 2 mg/ml dispase II in DME at 37ЊC for 15 min, followed Terasaki plates were coated with human plasma fibronectin (5 by 1 mM EDTA in Ca2/- and Mg2/-free PBS at 37ЊC for 15 min. ml, 50 mg/ml in DME, 45 min, room temperature). E1.7 neural crest This was replaced by 0.5% BSA in DME and a narrowed Pasteur cells and HNK-1-positive E4.7 and E7 cells were plated to a final pipet was used to triturate the tissue. This cell suspension was density of 2500 or 5000 cells (referred to as low and high density, allowed to settle for 5–10 min and the top 90% was taken. This respectively) in 12 ml of culture medium containing 10% FCS or ensured that the cell suspension contained at least 90% single cells. in N1 serum-free medium (as described above) and growth factors,

This was checked under a hemocytometer, and the cells were and allowed to settle on ice before being transferred to a 5% CO2 counted. An aliquot representing the total cell population in the atmosphere at 37ЊC. midgut at each stage was cultured at this point (see below). Neuron detection. Neurons were identified by neurofilament Isolation of enteric neural crest cells with HNK-1-coated mag- expression. This was detected by fixing cells in methanol (020ЊC) netic beads. Streptavidin-conjugated Dynabeads (M-280, Dynal) and then incubation with rabbit anti-neurofilament 150 kDa (1/ were washed three times in PBS plus 1% FCS and incubated with 200, Chemicon) for 2 h. Goat anti-rabbit IgG–alkaline phosphatase a 1/5 dilution of biotinylated donkey anti-mouse IgM (Jackson Im- (1/150, Sigma Immunochemicals) was applied for 1 h followed by munoresearch Laboratories) for 30 min at room temperature. The NBT/BCIP substrate (Boehringer-Mannheim). beads were washed three times in 1% FCS in PBS and then incu- Cell and neurite counting. To obtain time course observations, bated at room temperature for 30–60 min with HNK-1 monoclonal cells were plated in multiple Terasaki plates, each one being fixed antibody supernatant (Tucker et al., 1984) diluted 1/5 in 1% FCS at a particular time point for up to 5 days and stained for neurofil- in PBS. Following this, the beads were washed three additional ament. In other experiments, the same wells were followed over

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graph was taken of each, approx 5 ml of culture medium was pi- petted up and down three times and the same field was rephoto- graphed. Using the first micrograph of each pair, the positions of 60 cells in a contiguous area were plotted from the center of each well. This area was compared in the second micrograph and the previously plotted cells that had resisted being moved by the flow of medium were counted as attached cells, and expressed as a percentage of initial cell numbers. Each experiment was replicated in 8–10 wells. Live/dead assay. The percentage of dead cells was assayed at 2 h in 6–10 replicate wells using a live/dead viability/cytotoxicity kit (Molecular Probes, Oregon) reagents, as described in the kit instructions. Statistical analysis. For comparisons between treatment groups one-way analyses of variance (ANOVAs) were carried out to determine if there was a significant treatment effect. Once sig- nificance was established, multiple two tailed, type II t tests estab- FIG. 1. Immunolabeling cells with the HNK-1 antibody demon- lished P values for each pair of observations within a given experi- strates the purity of the enteric precursors isolated from the devel- ment. oping gut. (A) 5% of cells in the total population of cells in an E4.7 midgut segment are HNK-1-positive. (B) After the separation RESULTS process there is an 18-fold enrichment in HNK-1-positive cells and a 400-fold decrease in mesenchymal cells. Scale bar, 100 mm. Effects of ET-3 and GDNF on Cell Migration and Cell Number of Primary Neural Crest Cells The effects of ET-3 and GDNF were first examined for any effects on neural crest cells as they migrated from the the culture period with counts of total cell numbers being made neural tube. Vagal neural tube explants were grown in cul- by phase contrast microscopy each day. All experiments on the ture plates which had been scored with parallel lines to mid (E4.7) and later (E7) enteric neural precursor cells were repeated constrain neural crest cell migration to a 400-mm-wide cor- in the presence and absence of serum. To sample the number of ridor perpendicular to the dorsal side of the midvagal level total cells and neurons within each well, 2 fields under the 201 of the neural tube. Cell migration was analyzed over the phase objective of a Zeiss IM-35 microscope were counted and first 20 h in vitro by counting the number of cells migrating summed. Thus the area sampled in each Terasaki well was 0.56 1 from a 400-mm-long segment of the neural tube. Over this 0.75 mm2. Each experiment involved between 6 and 10 replicate wells. To give an indication of how extensive the axonal network time period, no difference in cell morphology was noted was in the presence of different molecules, the number of axons in control cultures versus cultures with GDNF or ET-3 in bisecting the midline of each well was counted. All values are defined or serum-containing medium. The average number represented as means { standard deviation. of mesenchymal neural crest cells, and the distance mi- Cell proliferation assays. E1.7 neural crest cells and E4.7 HNK- grated, varied widely even within treatment groups between 1-positive cells were plated as above in the presence of GDNF and/ exactly stage-matched explants, obscuring any potential or ET-3 for 20 h after which time cell proliferation labeling reagent factor-driven differences. The results for all treatment (bromodeoxyuridine and fluorodeoxyuridine, Amersham) was groups (total 74 explants), however, confirmed the strong added at a final dilution of 1/1000. Culturing was continued for 4 positive relationship between the number of neural crest h and the cells fixed in methanol (020ЊC). After washes in 1% cells and the distance which the furthest cell had migrated FCS in Tris-buffered saline (TBS) the cells were incubated with undiluted antibromodeoxyuridine (BrdU), mouse monoclonal anti- from its origin in the neural tube (Fig. 2). body (Amersham) for 1 h at room temperature, followed by an anti- To more accurately determine any effects of ET-3 or mouse IgG conjugated to alkaline phosphatase, also for 1 h. A short GDNF on neural crest cell number, the migrated neural NBT/BCIP (Boehringer-Mannheim) reaction visualized the cells crest cells were harvested and replated at defined cell densi- that had incorporated BrdU. Two fields of cells for each 6–10 repli- ties and cultured with these factors. Neither ET-3 nor cate wells were counted using a 201 objective. The number of GDNF had an observable effect on the total cell number in BrdU-positive cells was expressed as a percentage of the total cells these dissociated neural crest cultures (data not shown), and to give a proliferation index {SD for each well. BrdU incorporation studies also showed no observable effect Adhesion assay. Terasaki plates (non-tissue culture grade; Dis- of these factors, either alone or in combination (Table 1). posable Products, Australia) were coated with human plasma fibronectin (5 ml, 50 mg/ml in DMEM, 45 min, room temperature). Effects of ET-3 and GDNF on Cell Number and Cells were plated at 2500 cells/well with control, GDNF, ET-3, or Proliferation on Enteric Precursor Cells Isolated both as described above. After 10 min on ice to allow settling, cells from E4.7 Gut were transferred to 37ЊC for 45 min at which time the wells were photographed using an inverted phase-contrast microscope Zeiss In order to determine if ET-3 or GDNF influenced cell IM-35, 161 objective and a Nikon 601 camera. After the first photo- number in enteric precursor cells after they had begun to

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separation from other cells with the use of antibody-coated magnetic beads (as described under Materials and Methods). These purified cells were then cultured in the presence of ET-3 and GDNF. Under specific culture conditions, that is, in the presence of 10% FCS and at relatively high cell-plating densities (5000 cells/well), culturing with ET-3 and GDNF had clear effects on cell number. Under these conditions, there were more than twice the number of cells in cultures treated with either ET-3 or GDNF compared to controls (Fig. 3A). However, when these cells were cultured in the absence of FCS or at lower cell densities resulted in no effect of ET-3, while the stimulatory effect of GDNF persisted (data not shown). In the absence of FCS and at low cell density, neither factor showed any stimulatory effect (Fig. 4). In fact, under these conditions, there were less cells in cultures treated with ET-3 compared to controls (Fig. 3B). FIG. 2. The number of migrating vagal neural crest cells plotted In these cultures, changes in proliferation paralleled the against the distance of migration of the cell farthest from its origin changes in cell number observed with GDNF and ET-3 in the neural tube. Neural crest cell migration is confined to a 400- treatment. The rate of proliferation was measured over a 4- mm corridor perpendicular to the neural tube. There is a strong h period by BrdU incorporation at the end of the first day positive relationship between the number of cells and the distance in culture. The results of these experiments are summarized covered in their migration across the substrate (correlation Å 0.78). in Table 1. At both high cell density and in the presence of FCS, both ET-3 and GDNF stimulated proliferation. How- ever, for proliferation, there was some additivity of effect for the combination of ET-3 and GDNF, compared to either migrate into the gut, these cells were isolated from embry- factor alone (P õ 0.03). These proliferative effects, along onic gut dissected from quail embryos at embryonic day 4 with the basal proliferation rates of cultures, were depen- and 18 h (E4.7), which corresponds to stage 26–27 (Ham- dent on the culturing conditions. The presence of serum burger and Hamilton, 1951). The neural precursor cells were was associated with an increase of approximately 50% in purified by labeling with antibodies to HNK-1 followed by the basal proliferation rate compared to defined medium.

TABLE 1 The Rates of Proliferation in Culture of Enteric Neural Precursor Cells Isolated at Early (E1.7), Mid (E4.7), and Late (E7) Stages

Proliferation indexa

Embryonic Cell Control day Mediumb densityc (%) GDNF (%) ET-3 (%) ET-3 / GDNF (%)

1.7 FCS High 14.0 { 3.6 10.5 { 0.6 10.8 { 1.1 12.2 { 2.9 1.7 FCS Low 5.3 { 1.9 4.3 { 2.4 4.3 { 1.2 4.7 { 2.1 4.7 FCS High 8 { 4.3 12.5 { 2.7F** 11.5 { 2.7F** 16.8 { 4.3F** 4.7 FCS Low 4.8 { 1.3 9.8 { 1.1F** 6.1 { 1.2 5.7 { 2.7 4.7 N1 High 5.6 { 1.8 11.0 { 1.8F** 9.8 { 2.3F** 8.6 { 3.4F** 4.7 N1 Low 3.1 { 0.9 3.9 { 1.4 1.4 { 0.7f** 1.1 { 0.6f** 7 FCS High 11.7 { 1.7 13.6 { 3.7 11.7 { 4.1 18.2 { 4.2F* 7 FCS Low 9.9 { 3.5 14.5 { 3.8F* 9.7 { 2.7 12.4 { 2.7 7 N1 High 10.7 { 2.2 13.3 { 3.3 12.5 { 2.3 14.3 { 2.3F** 7 N1 Low 9.8 { 2.1 13.3 { 2.4F** 9.3 { 4.3 12.0 { 3.9

a The number of cells that incorporated BrdU in the period 20–24 h in vitro as a percentage of the total cells. b 10% FCS or N1 defined medium supplement in DME. c low, 2500 cells/well; high, 5000 cells/well. F, significantly increased with respect to controls; f, significantly decreased with respect to controls. * P õ 0.05. ** P õ 0.01.

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ment (E7), when neuronal differentiation begins. In the presence of serum and high cell density, E7 cells survived very well without added factors. If there were any factor-driven differences in cell number under these conditions, they were difficult to determine because final cell densities were very high under all treatment conditions. However, in defined medium, GDNF-treated cultures contained significantly more cells than control cultures irrespective of cell density (Fig. 4; P õ 0.01). ET-3 had no observable effect on cell number in cultures of E7 ENS precursors (Fig. 4). Additionally, the effects of the factors on proliferation were less pronounced in the E7 cultures compared to E4.7 cultures. Thus, ET-3 alone had no significant effect on proliferation on these cells, although it did show some additive effects in combination with GDNF (Table 1). Similarly, the stimulatory effects of GDNF on proliferation were less pronounced, and were only significant at low cell density or in combination with ET-3 (Table 1). It was possible that proliferative effects of these factors were masked by relatively higher basal proliferation rates in these cultures. The basal proliferation index of E7 cultures was practically unaf- fected by serum and increased only marginally by doubling cell density, in contrast to cells from younger donors. The difference in cell numbers between the GDNF-

FIG. 3. Cell numbers in cultures of E4.7 ENS precursor cells after 2 days. (A) In 10% FCS and at relatively high cell density (5000 cells/well), treatment with ET-3 or GDNF increase total cell numbers. (B) In N1 deﬁned medium and at low cell density (2500 cells/ well), there is no effect of GDNF on cell number, and fewer cells in the presence of ET-3.

Additionally, doubling of cell density was associated with a 70% increase in proliferation, which was additive to the increase seen with serum (Table 1). As with the effects observed on total cell number, there were less effects of the factors at lower cell density and in the absence of serum: at low cell density, ET-3 had little effect, and at both low cell density and in the absence of FCS, neither factor showed any stimulatory effect on proliferation. There was even a decrease in proliferation in the presence of ET-3 under these conditions, consistent with the decrease in cell number in the presence of ET-3 under the same conditions.

Effects of ET-3 and GDNF on Cell Number and Proliferation on Enteric Precursor Cells Isolated from E7 Gut FIG. 4. Cell numbers in cultures of E7 ENS precursors grown in N1 deﬁned medium for 2 days. GDNF induces an increases in cell The effects of ET-3 and GDNF were also examined on number at both (A) high (5000 cells/well) and (B) low cell density enteric precursor cells isolated at later stages of develop- (2500 cells/well).

AID DB 8876 / 6x3c$$$$43 04-18-98 14:37:17 dbas Effect of ET-3 and GDNF on Gut Neural Crest Cells 99 treated cultures and controls was seen as early as 2 h in vitro in serum-containing medium and/or high cell density. For example, in late (E7.0) enteric precursors cultured at low cell density and with serum, controls had 160 { 40 cells per counting field and GDNF treated cultures had 205 { 40 (P Å 0.034). There was, however, no difference in the proportions of dead cells per counting field (labeled in the live/dead assay) in the four treatment groups after the first 2 h in culture to account for this, with cell death ranging from 16 to 18% in cultures of late (E7.0) precursor cell cultures. To determine if differences in initial cell numbers in culture were due to GDNF-induced differential adhesion of mid and late enteric precursors to the culture dish, cell adhesion in the four treatment groups was compared at 45 min in vitro. From 55 to 65% of the cells were adherent, with no significant differences between the treatments.

Effects of GDNF and ET-3 on Neuron Number and Axon Outgrowth in Enteric Precursor Cultures To look for effects of ET-3 and GDNF on neuronal development, cultures of cells isolated at the different developmental stages were treated with the factors and incubated for up to 5 days. The cultures were then stained for neurofilament and neuron numbers were determined. In cultures of early (E1.7) neural crest cells, few cells (2.5%) had neu- FIG. 5. Neuron numbers in cultures of mid (E4.7) ENS precursors, plated at low cell density (2500 cells/well). (A) Cells cultured for ronal morphology or expressed neurofilament at the end of 4 days in serum-containing medium (10% FCS). (B) Cells cultured the culture. in N1 defined medium for 2.5 days. GDNF induces a significant In mid-stage enteric precursor cells cultured with serum, increase in the number of neurons in the presence or absence of GDNF stimulated a threefold increase in the number of serum. ET-3 inhibits neuronal generation when present with neurons after 4 days in culture (P õ 0.005, Figs. 5A and GDNF, or alone under serum-free conditions. 6C). ET-3 had no effect on the number of neurons present compared to controls and, in addition, antagonized the effect of GDNF in the stimulation of neuronal generation, since ET-3 plus GDNF treatment resulted in significantly with serum. GDNF induced an increase in the number of fewer neurons compared to GDNF treatment alone (Figs. neurons present, while ET-3 treatment decreased neuron 5A and 6C–6D, P õ 0.05). numbers compared to controls (Fig. 7B). In addition, when A similar pattern was observed when these cells were ET-3 and GDNF were applied simultaneously, ET-3 negated incubated under serum-free conditions, although the neu- the stimulatory effects of GDNF (Fig. 7B). rons appeared earlier than in serum treated cultures (2–3 As well as increasing the number of neurons present in days). There were greater numbers of neurons in cultures cultures, the presence of GDNF dramatically increased the treated with GDNF compared to controls (Fig. 5B, P õ 0.05), elaboration of neurite outgrowth within each well. For ex- but fewer neurons with ET-3 treatment (P õ 0.05). Cultures ample, in late-stage enteric precursor cultures after 4 days treated with ET-3 plus GDNF contained significantly fewer there were 3 times the number of neurons in the GDNF- neuron numbers than those treated with GDNF alone (Fig. treated group compared to controls, and yet 20 times the 5B, P õ 0.001). number of axons bisecting the midline of each well (Fig. 8). The effect of GDNF on neuron numbers was greater in cultures of late ENS precursors than in cultures of mid- stage precursor cells. In the presence of serum, the numbers DISCUSSION of neurons increased until 4–5 days in culture, by which time GDNF had induced an eightfold increase in neurons Cell Number Can Drive the Distribution of Neural (Fig. 7A). ET-3 alone had no measurable effect on neuron Crest Cells numbers in these cultures and, as with the mid-stage precursor cultures, antagonized the effect of GDNF in stimu- Vagal neural crest cells destined to form the ENS of the lating neuronal generation (Fig. 7A). With defined medium mid- and hindgut must migrate rostrocaudally through the (Fig. 7B), neurons appeared more rapidly (2–3 days) than developing gut which is rapidly elongating. The rates of

AID DB 8876 / 6x3c$$$$43 04-18-98 14:37:17 dbas 100 Hearn, Murphy, and Newgreen

FIG. 6. Cultures of mid (E4.7) enteric precursor cells, ﬁxed after 4 days and stained for neuroﬁlament. (A) controls, (B) GDNF, (C) ET- 3, and (D) ET-3 plus GDNF. Scale bar, 100 mm.

growth of the mid- and hindgut are low prior to the stage GDNF and ET-3 Have Little Effect on Cultured when vagal neural crest cells normally arrive, but increased Vagal Neural Crest Cells Prior to Entry to the Gut dramatically after arrival (Newgreen et al., 1996). Decreased GDNF increases the total cell number in cultures of trunk migratory drive of vagal neural crest cells at any point in neural crest cells, but this effect is only seen after at least the intestine could jeopardize the ability of the cells to fully 6 days of culturing and additionally requires the presence populate the remaining more caudal sections of the gut, due of chick embryo extract (Maxwell et al., 1996). GDNF did to the normal growth spurt of the intestine causing the not signiﬁcantly stimulate proliferation, increase in cell migration endpoint to recede faster than the rate of neural numbers, or alterations in cell morphology in dissociated or crest cell migration. As shown by our experiments on vagal explant cultures of E1.7 vagal neural crest cells. Messenger cells, and by Newgreen et al. (1979) and Rovasio et al. (1983) RNA for the GDNF receptor, ret, has been detected in the for trunk-level neural crest cells in culture, one source of vagal dorsal neural tube and early migrating neural crest migratory drive is cell population pressure. This is consis- cells in the chick embryo (Robertson and Mason, 1995). In tent with the reduction of the extent of migration in the mice, ret expression was not seen in vagal neural crest cells gut in vivo following partial ablation of the vagal neural until later, when they had migrated lateral to the dorsal crest cell population (Yntema and Hammond, 1954). It aorta (Lo and Anderson, 1995; Pachnis et al., 1993). More- would therefore be likely, based on the rostrocaudal migra- over, ret function is not required for the normal migration tion pattern of vagal enteric neural precursor cells, that the of mouse neural crest cells from the vagal and anterior trunk absence of those growth factors which normally aid in the neural crest to the dorsal aorta, since this is accomplished maintaintenance of steadily increasing cell numbers would in ret0/0 mice (Durbec et al., 1996a). Our experiments sug- result in a HSCR-like aganglionosis of the most distal gut. gest that avian embryos are functionally similar, since mi-

AID DB 8876 / 6x3c$$$$44 04-18-98 14:37:17 dbas Effect of ET-3 and GDNF on Gut Neural Crest Cells 101 grating quail vagal neural crest cells were unresponsive to GDNF until they had entered the gut. The proportion of enteric neural precursors expressing ret in the gut is unknown; however, given the complete intestinal aganglionosis observed in the fore- and hindgut of ret knockout mice (Schuchardt et al., 1994), it must be expressed by enough cells to massively affect the entire migrating (or differentiating) population of enteric neuronal precursors. Like GDNF, ET-3 did not stimulate the proliferation of dissociated or explant cultures of E1.7 vagal neural crest cells, nor were signiﬁcant effects on cell morphology seen. The gene for its receptor, EDNRB, is expressed by most avian and murine trunk neural crest cells prior to and fol- FIG. 8. The number of axons bisecting the midline of each culture well reﬂects if there are differences in the number and/or length lowing their emigration from the neural tube (Nataf et al., of axons under each treatment condition. In cultures of late (E7) 1996; Reid et al., 1996). Other studies have detected a prolif- enteric precursor cells, plated at 2500 cells/well in 10% FCS, there erative and morphological effect of ET-3 on trunk level neu- were three times more neurons with GDNF than in controls after ral crest cells after longer periods in culture, though this 4 days and yet 20 times the number of axons crossing the midline effect was greatly enhanced by the presence of Steel factor of each well.

for mouse cells (Reid et al., 1996) or chick embryo extract for chick cells (Lahav et al., 1996). Early migrating vagal neural crest cells may require the presence of additional factors which are not produced by the cells themselves and are not found in FCS which induce their responsiveness to or synergize with ET-3 or GDNF. Presumably the neural crest cells undergo a change, such that when they are migrating through the gut they become independent of exogenous sources of hypothetical ET-3 and GDNF cofactors. While early migrating neural crest cells appear not to be affected by GDNF or ET-3, other experiments have shown that other molecules are very important for the survival and proliferation of these cells, especially neurotrophin-3 (Kalcheim et al., 1992) and BDNF (Kalcheim et al., 1988).

Growth Factors Modulate Cell Number and Proliferation of Gut-Derived Neural Crest Cells Whereas neither ET-3 nor GDNF had any observable effect on early vagal neural crest cells prior to entering the gut, both factors influenced total cell number in cultures of precursors isolated from within the gut. In the presence of serum and at relatively high cell densities, both factors stimulated over a twofold increase in cell number in these cultures. Under these conditions, both factors increased the proliferation indices of enteric precursor cells; however, the stimulation of proliferation was relatively modest and it FIG. 7. Neuron numbers in cultures of E7 ENS precursors, plated was possible that the factors were acting as both survival at high density (5000 cells/well). (A) In serum-containing medium and proliferative factors. (10% FCS), neuron numbers peak at 4–5 days. (B) In N1-defined medium, neuron numbers peak at 2–3 days. Independent of the Under different culture conditions, in particular in the culture conditions, GDNF induces a large increase in the number absence of serum and at lower cell densities, the pattern of neurons present, an effect which is antagonized by ET-3. Note was somewhat different. ET-3 had a negative effect on the that in N1 medium there is a significant negative effect of ET-3 proliferation of mid-stage enteric precursors, and this effect alone on neuron number (P Å 0.02). was masked by the presence of serum or cell produced fac-

AID DB 8876 / 6x3c$$$$44 04-18-98 14:37:17 dbas 102 Hearn, Murphy, and Newgreen tors. Under such conditions, ET-3 mediated an increase in teric precursor cells into neurons and/or their resultant sur- proliferation and the effect of both molecules in combina- vival. We showed previously that GDNF could stimulate tion was additive. The effect of ET-3 and GDNF on prolifera- the generation of neurons in primary cultures of mouse tion was greatest in enteric precursor cells isolated from neural crest, but only in the presence of chick embryo ex- the E4.7 midgut, which include the cells at the wavefront tract (Maxwell et al., 1996). In addition, much of this activ- of neural crest cell colonization. Some of these effects may ity may have occurred in cells appearing after 6 days in have been due to the survival effects of these factors. culture. This finding suggests that GDNF was acting not GDNF-treated cultures had a greater total cell number, on neural crest stem cells but on committed neural crest- which by 1 day in culture could be explained by the differen- derived neuroblasts, which is consistent with the findings tial proliferation rates of GDNF-treated cultures; however, presented here. GDNF can also stimulate survival of other this effect was observed as early as 2 h in culture. One peripheral neurons including sympathetic and ciliary neu- possibility was that GDNF may aid the initial adherence rons (Buj-Bello et al., 1995; Ebendal et al., 1995). of enteric precursor cells to the fibronectin, keeping them While the RET mutations usually associated with HSCR within the counting fields in the centre of each well. How- are due to loss of function of the GDNF receptor RET, con- ever, this was not confirmed by specific adhesion assays. It stitutive activation of this receptor is associated with a sep- follows that these early effects of GDNF were on the sur- arate cluster of conditions (van Heyningan, 1994), typified vival of the cells. We attempted to demonstrate differential by familial medullary thyroid carcinoma and multiple endo- survival effects with a live/dead cell assay, but this did not crine neoplasia type 2A and 2B (Robertson and Mason, show any differences. While we thus cannot determine the 1997). A major component of multiple endocrine neoplasia precise reason for this early effect of GDNF, we still favor 2B is intestinal ganglioneuromatosis, where there is a prolif- the idea that it is on the survival of the cells, and the reason eration of nerves and nerve plexuses in the small and large that the live/dead cell assay showed no differences may intestine, leading to malfunction and intestinal obstruction have been due to the rapid detachment of the dead cells (Carney et al., 1976). Consistent with this, our results in from the substrate. vitro point to activation of Ret via exogenous GDNF, lead- The dependence of neural crest cell migration on popula- ing to increased neuron numbers and neurite growth. tion pressure sustained by increasing cell numbers, as men- In ret 0/0 mice and GDNF0/0 mice, no neurons exist tioned above, may explain why, in the functional absence distal to the stomach (Durbec et al., 1996a), which suggests of either GDNF or ET-3 or their receptors, neural crest cell that there is no differentiation or survival of enteric neurons migration is retarded and is not sustained along the entire derived from the vagal neural crest. This correlates with our length of the gut. in vitro findings that both survival and proliferation of the Since cell density and FCS modulated the proliferative precursors may be regulated by GDNF, and moreover that action of ET-3, it may be that ET-3 alone has little effect, GDNF treatment results in an at least eightfold increase in but functionally interacts with other exogenous and endoge- neuronal differentiation of these cells. However, in culture nous molecules to affect cell growth. These molecules may GDNF is not absolutely required for neurons to appear and be found in FCS and may also be derived from the ENS it may be that other factors may be present in these cultures, precursor cells themselves, as either released or cell-sur- which can act as survival/differentiation factors for the en- faced (contact mediated) forms. Moreover, cell number still teric neurons, but which are not present in vivo. decreased over time, albeit more slowly, with serum, high Both the stimulation of neuron numbers and axon growth cell density, GDNF, and ET-3; this implies that further by GDNF were partially inhibited by the additional pres- growth and survival factors operate in the development of ence of ET-3. In previous studies, as well as stimulating the ENS. There is also a possibility that GDNF and ET-3 proliferation in trunk neural crest cultures, ET-3 has been act on different populations of cells and some support for observed to temporarily inhibit the differentiation of these this comes from findings that GDNF and ET-3 null mutants cells into melanocytes (Lahav et al., 1996). Since melano- have different degrees of loss of enteric neurons. Future cytes divide slowly relative to their precursors (Maxwell, studies which involve determining receptor distribution in 1976), the net effect of this, paradoxically, is to increase the vivo and in vitro through ENS development should help to final numbers of melanocytes in vitro (Lahav et al., 1996). resolve this issue. The down-modulating effect that ET-3 has on a GDNF- induced increase in ENS neuron number is consistent with ET-3 having a similar effect on enteric precursor cells as on ET-3 and GDNF May Balance Neural Precursor melanocyte precursor cells, that is, delaying their differenti- Proliferation and Neuronal Differentiation ation into neurons and therefore prolonging their prolifera- GDNF increased the number of differentiated neurons in tion and migratory phases. cultures of neural crest cells isolated from the developing The inhibitory effect that ET-3 has on neuronal differenti- E4.7 and E7 midgut and also dramatically encouraged the ation in the presence of GDNF leads to the possibility that length and/or numbers of axons present. These findings aganglionosis due to functional loss of ET-3 may be, in part, show that GDNF stimulated the differentiation of the en- the result of inappropriately precocious and complete neu-

AID DB 8876 / 6x3c$$$$44 04-18-98 14:37:17 dbas Effect of ET-3 and GDNF on Gut Neural Crest Cells 103 ronal differentiation in the neural crest population, which EDNRB (or any other receptor) would conventionally be would be additive to the direct loss of proliferation poten- regarded as classically cell autonomous. It may be that the tial, both of which could impact on the distal distribution concept of cell autonomous/non-cell autonomous is inade- of the cells via a reduction in cell numbers. quate when the phenotypic outcome is decided at the cell In the ls/ls mouse (ET-3 mutant), aganglionosis accompa- population level (in this case the sum of ENS precursors; nies an increase in extracellular matrix molecules, espe- sl/sl mutant plus normal). cially laminin and chondroitin sulfate proteoglycan. Immu- nohistochemical studies suggest that aganglionosis is not sufficient to cause a secondary increase in ECM molecules (Newgreen et al., 1996; Rothman et al., 1996). 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