Epithelial Splicing Regulatory Protein 1 (ESRP1) is a new regulator of stomach smooth muscle development and plasticity.

In vertebrates, stomach smooth muscle development is a complex process that involves the tight transcriptional or post-transcriptional regulation of different signalling pathways. Here, we identified the RNA-binding protein Epithelial Splicing Regulatory Protein 1 (ESRP1) as an early marker of developing and undifferentiated stomach mesenchyme. Using a gain-of-function approach, we found that in chicken embryos, sustained expression of ESRP1 impairs stomach smooth muscle cell (SMC) differentiation and FGFR2 splicing profile. ESRP1 overexpression in primary differentiated stomach SMCs induced their dedifferentiation, promoted specific-FGFR2b splicing and decreased FGFR2c-dependent activity. Moreover, co-expression of ESRP1 and RBPMS2, another RNA-binding protein that regulates SMC plasticity and Bone Morphogenetic Protein (BMP) pathway inhibition, synergistically promoted SMC dedifferentiation. Finally, we also demonstrated that ESRP1 interacts with RBPMS2 and that RBPMS2-mediated SMC dedifferentiation requires ESRP1. Altogether, these results show that ESRP1 is expressed also in undifferentiated stomach mesenchyme and demonstrate its role in SMC development and plasticity.

Analysis of the expression of p53 during the morphogenesis of the gastroesophageal mucosa of Gallus gallus domesticus (Linnaeus, 1758).

Ontogenesis comprises a series of events including cell proliferation and apoptosis and resulting in the normal development of the embryo. Protein p53 has been described as being involved in the development of several animal species. The aim of this study was to analyze the expression of protein p53 during the morphogenesis of the gastroesophageal mucosa of Gallus gallus domesticus and to correlate it with the histogenesis of structures present in this tissue. We used 24 embryos (at 12-20 days of incubation) and the thymus of two chickens. Immunohistochemical analysis was performed with the ABC indirect method. The expression of p53 in the gastroesophageal mucosa increased during the formation of the organ, mainly at the stages during which tissue remodeling and cell differentiation began. In the esophagus at stages 42 and 45, we observed immunoreactive (IR) cells in the surface epithelium and in early esophageal glands. In the proventriculus at stages 39-45, IR cells were present in the epithelial mucosa and rarely in the proventricular glands. In the gizzard after stage 42, we found IR cells mainly in the medial and basal epithelial layers of the mucosa and especially within the intercellular spaces that appeared at this phase and formed the tubular gland ducts. Thus, protein p53 occurs at key stages of development: in the esophagus during the remodeling of esophageal glands, in the proventriculus during the differentiation of the epithelium of the mucosa and in the gizzard during the formation of tubular glands.

Expression pattern of the homeotic gene Bapx1 during early chick gastrointestinal tract development.

Regulation of the Bone Morphogenetic Protein (BMP) signaling pathway is essential for the normal development of vertebrate gastrointestinal (GI) tract, but also for the differentiation of the digestive mesenchymal layer into smooth muscles and submucosal layer. Different studies demonstrated that Bapx1 (for bagpipe homeobox homolog 1) negatively regulates the BMP pathway, but its precise expression pattern during the development and the differentiation of the GI tract mesenchyme actually remains to be examined. Here, we present the spatio-temporal expression profile of Bapx1 in the chick GI tract. We show that Bapx1 is first expressed in the undifferentiated mesenchyme of the gizzard and the colon. After the differentiation of the digestive mesenchyme, we found Bapx1 strongly expressed in the gizzard smooth muscle and in the submucosa layer of the colon. This expression pattern provides new insights into the roles of Bapx1 during the regionalization of the GI tract and the differentiation of the digestive mesenchyme of the colon and the stomach.

The RNA-binding protein RBPMS2 regulates development of gastrointestinal smooth muscle.

BACKGROUND & AIMS: Gastrointestinal development requires regulated differentiation of visceral smooth muscle cells (SMCs) and their contractile activities; alterations in these processes might lead to gastrointestinal neuromuscular disorders. Gastrointestinal SMC development and remodeling involves post-transcriptional modification of messenger RNA. We investigated the function of the RNA-binding protein for multiple splicing 2 (RBPMS2) during normal development of visceral smooth muscle in chicken and expression of its transcript in human pathophysiological conditions. METHODS: We used avian replication-competent retroviral misexpression approaches to analyze the function of RBPMS2 in vivo and in primary cultures of chicken SMCs. We analyzed levels of RBPMS2 transcripts in colon samples from pediatric patients with Hirschsprung's disease and patients with chronic pseudo obstruction syndrome (CIPO) with megacystis. RESULTS: RBPMS2 was expressed strongly during the early stage of visceral SMC development and quickly down-regulated in differentiated and mature SMCs. Misexpression of RBPMS2 in differentiated visceral SMCs induced their dedifferentiation and reduced their contractility by up-regulating expression of Noggin, which reduced activity of bone morphogenetic protein. Visceral smooth muscles from pediatric patients with CIPO expressed high levels of RBPMS2 transcripts, compared with smooth muscle from patients without this disorder. CONCLUSIONS: Expression of RBPMS2 is present in visceral SMC precursors. Sustained expression of RBPMS2 inhibits the expression of markers of SMC differentiation by inhibiting bone morphogenetic protein activity, and stimulates SMC proliferation. RBPMS2 transcripts are up-regulated in patients with CIPO; alterations in RBPMS2 function might be involved in digestive motility disorders, particularly those characterized by the presence of muscular lesions (visceral myopathies).

From snout to beak: the loss of teeth in birds.

All living birds are toothless, constituting by far the most diverse toothless vertebrate clade, and are striking examples of evolutionary success following tooth loss. In recent years, an unprecedented number of Mesozoic birds have been described, illustrating the evolution of dentition reductions. Simultaneously, major advances in experimental embryology have yielded new results concerning avian edentulism. Reviewing these lines of evidence, we propose hypotheses for its causes, with a prominent role for the horny beak during development. A horny beak and a muscular gizzard functionally 'replaced' dentition for food acquisition and processing, respectively. Together with edentulism itself, these features and others contributed to the later success of birds, as a result of their high performance or additional functionality working in concert in these complex organisms.

Ontogeny of ghrelin mRNA expression and identification of ghrelin-immunopositive cells in the gastrointestinal tract of the Peking duck, Anas platyrhynchos.

Ghrelin is an acylated peptide and an endogenous ligand for the growth hormone secretagogue receptor (GHS-R), and stimulates growth hormone release and food intake in mammals. Peking duck is a very fast growing species of poultry. Although the sequence and structure of ghrelin have recently been determined, the expression of ghrelin in Peking duck has not been studied. Here, we investigated the tissue expression and distribution of ghrelin by RT-PCR and immunohistochemistry, respectively, in Peking duck at different stages of development. Ghrelin mRNA expression was mainly detected in the proventriculus and proventriculus-gizzard junction. It was first expressed, but weakly, on embryonic day 14 (E14); the expression increased by embryonic day 21 (E21), and was maintained at high levels between post-hatching-day 1 (P1) and post-hatching-day 60 (P60). Weak expression of ghrelin mRNA was also found in the gizzard and duodenum. In the gastrointestinal tract of growing Peking duck in P60, the largest number of ghrelin-ip cells was detected in the epithelium of the compound tubular glands in the proventriculus and the next largest number was in the proventriculus-gizzard junction. Very few ghrelin-ip cells were located in the epithelium of the simple tubular glands adjacent to the gizzard. No ghrelin-ip cells were observed elsewhere in the gastrointestinal tract. Ghrelin-ip cells were found in embryos as early as day E21; at the same time, the compound tubular glands in the proventriculus had formed. The numbers of ghrelin-ip cells on P1 were similar to those of E21 embryos. However, on P60, high numbers of strongly stained ghrelin-ip cells were found to be scattered in the epithelium of the compound tubular glands in the proventriculus. The density of ghrelin-ip cells (cells/mm(2)) in the proventriculus on P60 was significantly greater than those of P1 and E21 embryos. These results demonstrate that ghrelin is expressed in the Peking duck gastrointestinal tract, especially in the proventriculus, from mid-late-stage embryos to growing period and suggested an involvement of ghrelin in the development and biology of the gastrointestinal tract of the Peking duck.

Sox9 and Nkx2.5 determine the pyloric sphincter epithelium under the control of BMP signaling.

The organs of the digestive tract are specified by coordinated signaling between the endoderm and mesoderm during development. These epithelial-mesenchymal interactions lead to the organ-specific morphogenesis and differentiation of regions along the gut tube. In this paper, we show that in the chick, the SRY-related transcription factor Sox9 is a marker for the posterior gizzard. Viral misexpression of Sox9 in the gizzard mesoderm is sufficient to specify epithelium characteristic of the pyloric sphincter. Sox9 expression is normally limited to the region of the posterior gizzard under the regulation of BMP signaling from the adjacent midgut. Misexpression of an activated form of BMPR1b in the gizzard upregulates Sox9 expression, while the BMP antagonist noggin down-regulates Sox9 expression in the gizzard mesoderm. Previously, Nkx2.5 was identified as a marker for the mesoderm of the pyloric sphincter. As with Sox9, BMP signaling appears to regulate Nkx2.5 and its ability to determine the pyloric epithelium. Despite these similarities, our evidence suggests that Sox9 and Nkx2.5 are regulated independently by BMP signaling, and act coordinately to specify the pyloric sphincter.

SOX9 specifies the pyloric sphincter epithelium through mesenchymal-epithelial signals.

Gastrointestinal (GI) development is highly conserved across vertebrates. Although several transcription factors and morphogenic proteins are involved in the molecular controls of GI development, the interplay between these factors is not fully understood. We report herein the expression pattern of Sox9 during GI development, and provide evidence that it functions, in part, to define the pyloric sphincter epithelium. SOX9 is expressed in the endoderm of the GI tract (with the exclusion of the gizzard) and its derivate organs, the lung and pancreas. Moreover, SOX9 is also expressed at the mesoderm of the pyloric sphincter, a structure that demarcates the gizzard from the duodenum. Using retroviral misexpression technique, we show that Sox9 expression in the pyloric sphincter is under the control of the BMP signaling pathway, known to play a key role in the development of this structure. By misexpressing SOX9 in the mesoderm of the gizzard, we show that SOX9 is able to transdifferentiate the adjacent gizzard epithelium into pyloric sphincter-like epithelium through the control of mesodermal-epithelial signals mediated in part by Gremlin (a modulator of the BMP pathway). Our results suggest that SOX9 is necessary and sufficient to specify the pyloric sphincter epithelial properties.

Gastric gross and microscopic lesions caused by the UNAM-97 variant strain of infectious bronchitis virus after the eighth passage in specific pathogen-free chicken embryos.

Herein we report a description of gross and microscopic lesions found in specific pathogen-free chicken embryos caused by UNAM-97 infectious bronchitis virus (IBV) variant strain after the eighth passage. Embryos were divided into three groups and were inoculated in the chorioallantoic sac with 0.2 mL of UNAM-97, Mass 41 IBV (positive control), or sterile PBS (negative control). Forty-eight hours later the allatoic fluid was taken and used to start a cycle of eight passages through 9-d-old embryos. Seven days after the last passage, embryos were harvested and macroscopic lesions in all organs were recorded. Proventriculus and gizzard samples were obtained from all embryos and routinely processed for microscopic and ultrastructural examinations. The UNAM-97 IBV variant strain caused two macroscopic lesions uncommon for Mexican strains: thin-walled proventriculus and gizzard, as well as urate accumulation within an extra-embryonic peritoneal sac, leaving the body through the umbilical duct and accompanied by the yolk sac. At microscopic level, two relevant findings were observed to be produced by this variant. In the proventriculus, there was a decrease in the gland papillary branching, while the gizzard showed a significant reduction in mucosa thickness and tubular-to-proliferative-cell ratio, as well as an absence of hyaline secretion in the lumen. Electrodense material scattered in proventricular and gizzard cells was observed, with a structure consistent with that of coronaviruses. These pathological chicken embryo findings have not been reported as being caused by other IBV strains in Mexico.

Versatile roles for sonic hedgehog in gut development.

Sonic hedgehog (Shh) is a gene encoding a protein that can be secreted and act as a morphogen. The protein exerts versatile and important effects on the surrounding cells by binding a specific receptor, named patched. So far Shh has been shown to be involved in the morphogenesis and cytodifferentiation of many organ systems, such as notochord, floor plate, limb, pancreas, and pituitary gland, to mention only a few examples. Shh is also involved in the determination of left-right asymmetry, at least in the chicken embryo. Here we present evidence that Shh is one of the key genes whose activity is pivotal for the normal morphogenesis and differentiation of digestive organs. Epithelial Shh regulates the formation of stomach glands and stratification of the mesenchyme into connective tissue and smooth muscle. It exerts its effect often through the induction of bone morphogenetic protein (BMP) genes in the mesenchyme. Thus, Shh is a key player in the epithelial-mesenchymal interactions in the development of the gut.

Prominent expression of transforming growth factor beta2 gene in the chicken embryonic gonad as revealed by suppressive subtraction cloning.

cDNA cloning from chicken embryonic gonad subtracted from tissues of the brain, heart, liver, gizzard, mesonephros, and muscle was performed to identify growth factor genes with expression unique to embryonic ovary and testis. We obtained several cDNA clones encoding known and many unknown genes. We found for the first time that the transforming growth factor beta2 (TGF-beta2) is preferentially expressed in the chicken embryonic ovary and testis. cDNA subtraction cloning with respect to the selective expression of TGF-beta2 in the ovary and testis was further analyzed by reverse transcription-polymerase chain reaction analyses of other embryonic tissues. The ontogeny of TGF-beta2 was evaluated in chicken embryonic ovary and testis. In both testis and ovary, the levels of TGF-beta2 transcripts were high during the early period of embryonic development (E7), gradually decreased until the late embryonic days (E14--E17), and then slightly increased at the last embryonic day (E21). There was no difference in the TGF-beta2 transcripts per RNA between the left and the right ovaries. TGF-beta2 may have a critical role in the regulation of the development of chicken ovarian and testicular germ cells during the embryonic period.

Differentiation of smooth muscle cells in chicken gizzard. Self-catalytic mechanism for the production of smooth muscle cells.

During development of the chicken gizzard, a thick layer of undifferentiated cells (mesenchymal cells) is constructed, and the cells differentiate into smooth muscle cells or connective tissues. We found that the differentiation of smooth muscle cells occurred first near the outer surface of the gizzard and the differentiated area spread to the inside of the gizzard. Therefore, we assumed that the differentiation of most of the smooth muscle cells in the gizzard is induced by differentiated smooth muscle itself. When undifferentiated cells from gizzard of 7-day-old embryo (Hamburger and Hamilton's stages 26-27) were cultured on a coverglass coated with extract of gizzard that contained differentiated smooth muscle cells, the cells attached to the coverglass and differentiated into smooth muscle cells. On the other hand, extract of gizzard from 7-day-old embryo did not induce the differentiation of smooth muscle cells, though it induced the attachment of cells. We found that activity for the differentiation of smooth muscle cells appeared when differentiated smooth muscle cells appeared in developing gizzard. Gizzard contained higher activity for the differentiation of smooth muscle cells than the other tissues. Transforming growth factor-beta (TGF-beta), which induces the differentiation of vascular smooth muscle cells, did not induce the differentiation of smooth muscle cells in gizzard, though extract of aorta induced the differentiation of smooth muscle cells in gizzard. The results obtained here support evidence that the differentiation of most of the smooth muscle cells in gizzard is induced by a self-catalytic mechanism in which differentiated smooth muscle itself induces the differentiation of smooth muscle cells.

Gizzard formation and the role of Bapx1.

The anterior-posterior gut pattern is formed from three broad domains: fore-, mid-, and hindgut that have distinct functional, morphological, and molecular boundaries. The stomach demarcates the posterior boundary of the foregut. Avian stomachs are composed of two chambers: the anterior chamber (proventriculus) and the thick muscular posterior chamber (gizzard). Expression of candidate pattern formation control factors are restricted in the chick stomach regions such that Bmp4 and Wnt5a are not expressed in the gizzard. We previously implicated Bmp4 as controlling growth and differentiation of the gut musculature. Bmp4 is not expressed in the developing gizzard but is expressed in the rest of the gut including the adjacent proventriculus and midgut. Bapx1 (Nkx3.2) is expressed in the gizzard musculature but not in the proventriculus or midgut. We show ectopic expression of Bapx1 in the proventriculus results in a gizzard-like morphology and inhibits the normal proventricular expression of Bmp4 and Wnt5a. Overexpression of a reverse-function Bapx1 construct can result in a small stomach and ectopic extension of Bmp4 and Wnt5a expression into the gizzard. We suggest the role of Bapx1 is to regulate the expression of Bmp4 and Wnt5a to pattern the avian stomach.

Can gastric endoderm change the regionally specific inducing ability of presumptive small intestinal mesoderm?

This study was designed to establish the source of gut mesoderm's ability to induce regional pattern in the endoderm. The most obvious possibility is induction by the endoderm through epithelial-mesenchymal interaction. To test this experimentally, reciprocal quail/chick combinations were prepared of early proventricular endoderm (that is already known to be regionally determined) and presumptive small intestinal mesoderm. The combinations were cultured for 7 days to allow for 'programming' of the mesoderm by the endoderm. After removal of the proventricular endoderm the mesoderm was combined with young gizzard endoderm. It is known that gizzard endoderm can be provoked to develop in either a proventricular or a small intestinal direction by association with the appropriate mesoderm. Thus, by combining intestinal mesoderm 'programmed' by association with proventricular endoderm with gizzard endoderm, the subsequent differentiation of the gizzard endoderm would indicate whether or not the inducing ability of the intestinal mesenchyme had been altered. In addition to such experimental grafts, three types of control graft were prepared. The results of the experiment, based on the morphology of the grafts and the immunocytochemical analysis of selected endocrine cell types, showed that in the majority of cases the gizzard endoderm developed the features of small intestine, not those of proventriculus. This indicates that at the stages studied, endoderm does not act to program mesoderm with which it is associated. If this does occur, it must take place at an earlier stage, i.e., before the time of explantation of the presumptive small intestinal mesoderm (1.25 days of incubation).

Determinants of the contractile properties in the embryonic chicken gizzard and aorta.

Smooth muscle is generally grouped into two classes of differing contractile properties. Tonic smooth muscles show slow rates of force activation and relaxation and slow speeds of shortening (V(max)) but force maintenance, whereas phasic smooth muscles show poor force maintenance but have fast V(max) and rapid rates of force activation and relaxation. We characterized the development of gizzard and aortic smooth muscle in embryonic chicks to identify the cellular determinants that define phasic (gizzard) and tonic (aortic) contractile properties. Early during development, tonic contractile properties are the default for both tissues. The gizzard develops phasic contractile properties between embryonic days (ED) 12 and 20, characterized primarily by rapid rates of force activation and relaxation compared with the aorta. The rapid rate of force activation correlates with expression of the acidic isoform of the 17-kDa essential myosin light chain (MLC(17a)). Previous data from in vitro motility assays (Rover AS, Frezon Y, and Trybus KM. J Muscle Res Cell Motil 18: 103-110, 1997) have postulated that myosin heavy chain (MHC) isoform expression is a determinant for V(max) in intact tissues. In the current study, differences in V(max) did not correlate with previously published differences in MHC or MLC(17a) isoforms. Rather, V(max) was increased with thiophosphorylation of the 20-kDa regulatory myosin light chain (MLC(20)) in the gizzard, suggesting that a significant internal load exists. Furthermore, V(max) in the gizzard increased during postnatal development without changes in MHC or MLC(17) isoforms. Although the rate of MLC(20) phosphorylation was similar at ED 20, the rate of MLC(20) dephosphorylation was significantly higher in the gizzard versus the aorta, correlating with expression of the M130 isoform of the myosin binding subunit in the myosin light chain phosphatase (MLCP) holoenzyme. These results indicate that unique MLCP and MLC(17) isoform expression marks the phasic contractile phenotype.

Roles of BMP signaling and Nkx2.5 in patterning at the chick midgut-foregut boundary.

Patterning of the gut into morphologically distinct regions results from the appropriate factors being expressed in strict spatial and temporal patterns to assign cells their fates in development. Often, the boundaries of gene expression early in development correspond to delineations between different regions of the adult gut. For example, Bmp4 is expressed throughout the hindgut and midgut, but is not expressed in the early gizzard. Ectopic BMP4 in the gizzard caused a thinning of the muscularis. To understand this phenotype we examined the expression of the receptors transducing BMP signaling during gut development. We find that the BMP receptors are differentially expressed in distinct regions of the chicken embryonic gut. By using constitutively activated versions of the BMP type I receptors, we find that the BMP receptors act similarly to BMP4 in the gizzard when ectopically expressed. We show that the mesodermal thinning seen upon ectopic BMP signaling is due to an increase in apoptosis and a decrease in proliferation within the gizzard mesoderm. The mesodermal thinning is characterized by a disorganization and lack of differentiation of smooth muscle in the gizzard mesoderm. Further, ectopic BMP receptors cause an upregulation of Nkx2.5, the pyloric sphincter marker, similar to that seen with ectopic BMP4. This upregulation of Nkx2.5 is a cell-autonomous event within the mesoderm of the gizzard. We also find that Nkx2.5 is necessary and sufficient for establishing aspects of pyloric sphincter differentiation.

BMPs are necessary for stomach gland formation in the chicken embryo: a study using virally induced BMP-2 and Noggin expression.

Epithelial-mesenchymal interactions are necessary for the normal development of various digestive organs. In chicken proventriculus (glandular stomach), morphogenesis and differentiation of the epithelium depend upon the inductive signals coming from underlying mesenchyme. However, the nature of such signals is still unclear despite extensive analyses carried out using experimental tissue recombinations. In this study we have examined the possible involvement of bone morphogenetic proteins (BMPs) in the formation of stomach glands in the chicken embryo. Analysis of the expression patterns of BMP-2, -4 and -7 showed that these BMPs were present in the proventricular mesenchyme prior to the initiation of the proventricular gland formation. BMP-2 expression, in particular, was restricted to the proventriculus among anterior digestive organs. Virus-mediated BMP-2 overexpression resulted in an increase in the number of glands formed. Moreover, ectopic expression of Noggin, which antagonizes the effect of BMPs, in the proventricular mesenchyme or epithelium, led to the complete inhibition of gland formation, indicating that BMP signals are necessary for the proventricular gland formation. These findings suggest that BMPs are of prime importance as mesenchymal signals for inducing proventricular glands.

Phenotype-dependent expression of alpha-smooth muscle actin in visceral smooth muscle cells.

Alpha-Smooth muscle actin is one of the molecular markers for a phenotype of vascular smooth muscle cells, because the actin is a major isoform expressed in vascular smooth muscle cells and its expression is upregulated during differentiation. Here, we first demonstrate that the phenotype-dependent expression of this actin in visceral smooth muscles is quite opposite to that in vascular smooth muscles. This actin isoform is not expressed in adult chicken visceral smooth muscles including gizzard, trachea, and intestine except for the inner layer of intestinal muscle layers, whereas its expression is clearly detected in these visceral smooth muscles at early stages of the embryo (10-day-old embryo) and is developmentally downregulated. In cultured gizzard smooth muscle cells maintaining a differentiated phenotype, alpha-smooth muscle actin is not detected while its expression dramatically increases during serum-induced dedifferentiation. Promoter analysis reveals that a sequence (-238 to -219) in the promoter region of this actin gene acts as a novel negative cis-element. In conclusion, the phenotype-dependent expression of alpha-smooth muscle actin would be regulated by the sum of the cooperative contributions of the negative element and well-characterized positive elements, purine-rich motif, and CArG boxes and their respective transacting factors.

Ontogeny of galanin-immunoreactive elements in the intrinsic nervous system of the chicken gut.

Galanin is a brain-gut peptide that is present in the central and peripheral nervous systems. In the gut, it is contained exclusively in intrinsic and extrinsic nerve supplies, and it is involved overall in the regulation of gut motility. To obtain information about the ontogeny of galanin, we undertook an immunohistochemical study of chicken embryos. The time of first appearance and the distribution patterns of galanin were investigated with fluorescence and streptavidin-biotin-peroxidase (ABC) immunohistochemical protocols by using a galanin polyclonal antiserum. The various regions of the gut and the pancreas were obtained from chicken embryos aged from 3 days of incubation to hatching. All specimens were fixed in buffered picric acid-paraformaldehyde, frozen, and cut with a cryostat. Galanin-immunoreactive neuroblasts were first detected at 4 days in the mesenchyme of the proventriculus/gizzard primordium and within the Remak ganglion. They then extended cranially and caudally, reaching all of the other gut regions at 6.5 days. Galanin-immunoreactive nerve elements mainly occupied the sites of myenteric and submucous plexuses. From day 15, galanin-immunoreactive nerve fibers tended to invade the circular muscular layer and part of the lamina propria of the mucosa. In the pancreas, weak galanin-immunoreactive nerve elements were detected at 5.5 days. They tended to be distributed among the glandular lobules according to the organ differentiation. The widespread distribution during the earlier embryonic stages represents evidence indicating that the neuropeptide galanin may have a role as a differentiating or growth factor. From late embryonic life, its predominant presence in sympathetic nerves and in muscular layers fits with the functions demonstrated previously in adults of other vertebrates for galanin as a modulator of intestinal motility.