A neural crest cell isotropic-to-nematic phase transition in the developing mammalian gut.

While the colonization of the embryonic gut by neural crest cells has been the subject of intense scrutiny over the past decades, we are only starting to grasp the morphogenetic transformations of the enteric nervous system happening in the fetal stage. Here, we show that enteric neural crest cell transit during fetal development from an isotropic cell network to a square grid comprised of circumferentially-oriented cell bodies and longitudinally-extending interganglionic fibers. We present ex-vivo dynamic time-lapse imaging of this isotropic-to-nematic phase transition and show that it occurs concomitantly with circular smooth muscle differentiation in all regions of the gastrointestinal tract. Using conditional mutant embryos with enteric neural crest cells depleted of ß1-integrins, we show that cell-extracellular matrix anchorage is necessary for ganglia to properly reorient. We demonstrate by whole mount second harmonic generation imaging that fibrous, circularly-spun collagen I fibers are in direct contact with neural crest cells during the orientation transition, providing an ideal orientation template. We conclude that smooth-muscle associated extracellular matrix drives a critical reorientation transition of the enteric nervous system in the mammalian fetus.

The origin and mechanisms of smooth muscle cell development in vertebrates.

Smooth muscle cells (SMCs) represent a major structural and functional component of many organs during embryonic development and adulthood. These cells are a crucial component of vertebrate structure and physiology, and an updated overview of the developmental and functional process of smooth muscle during organogenesis is desirable. Here, we describe the developmental origin of SMCs within different tissues by comparing their specification and differentiation with other organs, including the cardiovascular, respiratory and intestinal systems. We then discuss the instructive roles of smooth muscle in the development of such organs through signaling and mechanical feedback mechanisms. By understanding SMC development, we hope to advance therapeutic approaches related to tissue regeneration and other smooth muscle-related diseases.

Development of the smooth muscle layer in the ileum of mouse embryos.

The smooth muscle layer (SML) comprises a significant portion of the intestines and other tubular organs. Whereas epithelial development has recently been extensively studied, SML development has drawn relatively less attention. Previous morphological reports revealed that the inner circular layer (IC) differentiates earlier than the outer longitudinal layer (OL), but detailed development of the SML, including chronological changes in the cell layer number, precise cell orientation, and regional differences in relation to the mesentery, has not been reported. We here observed the development of the SML in the C57BL/6J mouse ileum near the ileocecal junction at embryonic day (E) 13.5, 15.5, and 17.5. By histo-morphometric analyses, in IC, smooth muscle cells (SMCs) were oval-shaped and irregularly arranged in 3-4 layers at E13.5, then adopted an elongated spindle shape and decreased to two cell layers at E15.5 and E17.5. The IC SMC nuclear angle was not vertical, but oriented at 60-80° against the mid-axis of the intestinal lumen. The single SMC layer in OL was observed at E17.5, and the SMC nuclear angle was parallel to the luminal mid-axis. No clear regional difference against the mesentery was observed. Collectively, the findings suggest that development and differentiation of the ileal SML is not simple but regulated in a complex manner and possibly related to the macroscopic organogenesis.

Shifting into high gear: how interstitial cells of Cajal change the motility pattern of the developing intestine.

The first contractile waves in the developing embryonic gut are purely myogenic; they only involve smooth muscle. Here, we provide evidence for a transition from smooth muscle to interstitial cell of Cajal (ICC)-driven contractile waves in the developing chicken gut. In situ hybridization staining for anoctamin-1 (ANO1), a known ICC marker, shows that ICCs are already present throughout the gut, as from embryonic day (E)7. We devised a protocol to reveal ICC oscillatory and propagative calcium activity in embryonic gut whole mount and found that the first steady calcium oscillations in ICCs occur on (E14). We show that the activation of ICCs leads to an increase in contractile wave frequency, regularity, directionality, and velocity between E12 and E14. We finally demonstrate that application of the c-KIT antagonist imatinib mesylate in organ culture specifically depletes the ICC network and inhibits the transition to a regular rhythmic wave pattern. We compare our findings to existing results in the mouse and predict that a similar transition should take place in the human fetus between 12 and 14 wk of development. Together, our results point to an abrupt physiological transition from smooth muscle mesenchyme self-initiating waves to ICC-driven motility in the fetus and clarify the contribution of ICCs to the contractile wave pattern.NEW & NOTEWORTHY We reveal a sharp transition from smooth muscle to interstitial cell of Cajal (ICC)-driven motility in the chicken embryo, leading to higher-frequency, more rhythmic contractile waves. We predict the transition to happen between 12 and 14 embryonic wk in humans. We image for the first time the onset of ICC activity in an embryonic gut by calcium imaging. We show the first KIT and anoctamin-1 (ANO1) in situ hybridization micrographs in the embryonic chicken gut.

Hydrogen sulfide, oxygen, and calcium regulation in developing human airway smooth muscle.

Preterm infants can develop airway hyperreactivity and impaired bronchodilation following supplemental O2 (hyperoxia) in early life, making it important to understand mechanisms of hyperoxia effects. Endogenous hydrogen sulfide (H2 S) has anti-inflammatory and vasodilatory effects with oxidative stress. There is little understanding of H2 S signaling in developing airways. We hypothesized that the endogenous H2 S system is detrimentally influenced by O2 and conversely H2 S signaling pathways can be leveraged to attenuate deleterious effects of O2 . Using human fetal airway smooth muscle (fASM) cells, we investigated baseline expression of endogenous H2 S machinery, and effects of exogenous H2 S donors NaHS and GYY4137 in the context of moderate hyperoxia, with intracellular calcium regulation as a readout of contractility. Biochemical pathways for endogenous H2 S generation and catabolism are present in fASM, and are differentially sensitive to O2 toward overall reduction in H2 S levels. H2 S donors have downstream effects of reducing [Ca2+ ]i responses to bronchoconstrictor agonist via blunted plasma membrane Ca2+ influx: effects blocked by O2 . However, such detrimental O2 effects are targetable by exogenous H2 S donors such as NaHS and GYY4137. These data provide novel information regarding the potential for H2 S to act as a bronchodilator in developing airways in the context of oxygen exposure.

Smooth muscle contractility causes the gut to grow anisotropically.

The intestine is the most anisotropically shaped organ, but, when grown in culture, embryonic intestinal stem cells form star- or sphere-shaped organoids. Here, we present evidence that spontaneous tonic and phasic contractions of the circular smooth muscle of the embryonic gut cause short-timescale elongation of the organ by a purely mechanical, self-squeezing effect. We present an innovative culture set-up to achieve embryonic gut growth in culture and demonstrate by three different methods (embryological, pharmacological and microsurgical) that gut elongational growth is compromised when smooth muscle contractions are inhibited. We conclude that the cumulated short-term mechanical deformations induced by circular smooth muscle lead to long-term anisotropic growth of the gut, thus demonstrating a self-consistent way by which the function of this organ (peristalsis) directs its shape (morphogenesis). Our model correctly predicts that longitudinal smooth muscle differentiation later in embryogenesis slows down elongation, and that several mice models with defective gut smooth muscle contractility also exhibit gut growth defects. We lay out a comprehensive scheme of forces acting on the gut during embryogenesis and of their role in the morphogenesis of this organ. This knowledge will help design efficient in vitro organ growth protocols and handle gut growth pathologies such as short bowel syndrome.

Epigenetic factors Dnmt1 and Uhrf1 coordinate intestinal development.

Intestinal tract development is a coordinated process involving signaling among the progenitors and developing cells from all three germ layers. Development of endoderm-derived intestinal epithelium has been shown to depend on epigenetic modifications, but whether that is also the case for intestinal tract cell types from other germ layers remains unclear. We found that functional loss of a DNA methylation machinery component, ubiquitin-like protein containing PHD and RING finger domains 1 (uhrf1), leads to reduced numbers of ectoderm-derived enteric neurons and severe disruption of mesoderm-derived intestinal smooth muscle. Genetic chimeras revealed that Uhrf1 functions both cell-autonomously in enteric neuron precursors and cell-non-autonomously in surrounding intestinal cells, consistent with what is known about signaling interactions between these cell types that promote one another's development. Uhrf1 recruits the DNA methyltransferase Dnmt1 to unmethylated DNA during replication. Dnmt1 is also expressed in enteric neurons and smooth muscle progenitors. dnmt1 mutants have fewer enteric neurons and disrupted intestinal smooth muscle compared to wildtypes. Because dnmt1;uhrf1 double mutants have a similar phenotype to dnmt1 and uhrf1 single mutants, Dnmt1 and Uhrf1 must function together during enteric neuron and intestinal muscle development. This work shows that genes controlling epigenetic modifications are important to coordinate intestinal tract development, provides the first demonstration that these genes influence development of the ENS, and advances uhrf1 and dnmt1 as potential new Hirschsprung disease candidates.

Análise histológica do estômago da prole de ratas Wistar submetidas ao consumo crônico de álcool durante a prenhez / Histological analysis of stomach of offspring of Wistar rats submitted to chronic alcohol consumption during pregnancy

O consumo de bebidas alcoólicas na gravidez consiste em um importante problema de saúde pública, visto que, pode causar prejuízos na organogênese de diversos órgãos, incluindo o estômago, entretanto, poucos estudos avaliam o efeito da exposição pré-natal ao álcool nesse órgão. O objetivo deste estudo foi analisar histologicamente o estômago da prole de ratas submetidas ao consumo crônico de álcool durante a prenhez. Utilizou-se 10 ratas prenhes divididas nos grupos: Controle - ratas que receberam água destilada durante todo período gestacional e Álcool ­ ratas que receberam álcool etílico absoluto (3g/kg/dia) durante todo período gestacional. Logo após o nascimento, 12 neonatos (6 machos e 6 fêmeas) de cada grupo foram anestesiados e os estômagos coletados. Posteriormente, os órgãos foram fixados e processados seguindo a técnica histológica de rotina. Foram feitas análises histomorfométricas das camadas mucosa, muscular e da parede total do estômago. Observou-se que as proles macho e fêmea expostas ao etanol apresentaram diminuição da área de epitélio, contudo, os machos também mostraram redução significativa do número de células epiteliais. Demonstrou-se ainda redução na espessura das camadas mucosa, muscular e da parede total do estômago da prole fêmea do grupo Álcool. No entanto, a camada muscular apresentou aumento significativo em sua espessura no grupo de neonatos machos expostos ao etanol. Assim, concluímos que a exposição pré-natal ao álcool provoca efeitos nocivos sobre o estômago dos neonatos, contudo, estudos futuros são necessários para melhor elucidar os mecanismos envolvidos na patogênese e possíveis consequências para os animais na fase adulta.

Consumption of alcoholic beverages during pregnancy is a significant public health issue since it can damage the organogenesis of several organs, including the stomach; however, few studies evaluate the effect of prenatal exposure to alcohol in this organ. The objective of this study was to analyze the histology of the stomach of offspring of rats submitted to chronic alcohol consumption during pregnancy. Ten pregnant rats were divided into two groups: Control - rats receiving distilled water throughout the gestation period, and Alcohol - rats receiving absolute ethyl alcohol (3g/kg/day) throughout the gestation period. After birth, 12 newborn rats (6 males and 6 females) from each group were anesthetized and their stomachs were collected. Subsequently, the organs were fixed and processed following the routine histological technique. The mucosa, muscle and total stomach were submitted to histomorphometric analyses. It was observed that the male and female offspring exposed to ethanol had a decrease in the epithelium area. However, males also showed a significant reduction in the number of epithelial cells. There was also a reduction in the layer's thickness mucosa, muscle and total stomach wall of the female offspring from the alcohol group. Additionally, the muscular layer presented a significant increase in its thickness in the group of male neonates exposed to ethanol. It can be concluded that prenatal exposure to alcohol causes harmful effects on neonates' stomachs; however, future studies are necessary to better elucidate the mechanisms involved in the pathogenesis and possible consequences for the animals in adulthood.

Alteration of cystic airway mesenchyme in congenital pulmonary airway malformation.

Congenital pulmonary airway malformation (CPAM) is the most common congenital lesion detected in the neonatal lung, which may lead to respiratory distress, infection, and pneumothorax. CPAM is thought to result from abnormal branching morphogenesis during fetal lung development, arising from different locations within the developing respiratory tract. However, the pathogenic mechanisms are unknown, and previous studies have focused on abnormalities in airway epithelial cells. We have analyzed 13 excised lung specimens from infants (age < 1 year) with a confirmed diagnosis of type 2 CPAM, which is supposed to be derived from abnormal growth of intrapulmonary distal airways. By examining the mesenchymal components including smooth muscle cells, laminin, and elastin in airway and cystic walls using immunofluorescence staining, we found that the thickness and area of the smooth muscle layer underlining the airway cysts in these CPAM tissue sections were significantly decreased compared with those in bronchiolar walls of normal controls. Extracellular elastin fibers were also visually reduced or absent in airway cystic walls. In particular, a layer of elastin fibers seen in normal lung between airway epithelia and underlying smooth muscle cells was missing in type 2 CPAM samples. Thus, our data demonstrate for the first time that airway cystic lesions in type 2 CPAM occur not only in airway epithelial cells, but also in adjacent mesenchymal tissues, including airway smooth muscle cells and their extracellular protein products. This provides a new direction to study the molecular and cellular mechanisms of CPAM pathogenesis in human.

Striated-for-smooth muscle replacement in the developing mouse esophagus.

The esophagus is a muscular tube which transports swallowed content from the oral cavity and the pharynx to the stomach. Early in mouse development, an entire layer of the esophagus, the muscularis externa, consists of differentiated smooth muscle cells. Starting shortly after mid-gestation till about two weeks after birth, the muscularis externa almost entirely consists of striated muscle. This proximal-to-distal replacement of smooth muscle by the striated muscle depends on a number of factors. To identify the nature of the hypothetical "proximal" (mainly striated muscle originating) and "distal" (mainly smooth muscle originating) signals that govern the striated-for-smooth muscle replacement, we compared the esophagus of Myf5:MyoD null fetuses completely lacking striated muscle to the normal control using cDNA microarray analysis, followed by a comprehensive database search. Here we provide an insight into the nature of "proximal" and "distal" signals that govern the striated-for-smooth muscle replacement in the esophagus.

Fibrillin-2 is a key mediator of smooth muscle extracellular matrix homeostasis during mouse tracheal tubulogenesis.

Epithelial tubes, comprised of polarised epithelial cells around a lumen, are crucial for organ function. However, the molecular mechanisms underlying tube formation remain largely unknown. Here, we report on the function of fibrillin (FBN)2, an extracellular matrix (ECM) glycoprotein, as a critical regulator of tracheal tube formation.We performed a large-scale forward genetic screen in mouse to identify regulators of respiratory organ development and disease. We identified Fbn2 mutants which exhibit shorter and narrowed tracheas as well as defects in tracheal smooth muscle cell alignment and polarity.We found that FBN2 is essential for elastic fibre formation and Fibronectin accumulation around tracheal smooth muscle cells. These processes appear to be regulated at least in part through inhibition of p38-mediated upregulation of matrix metalloproteinases (MMPs), as pharmacological decrease of p38 phosphorylation or MMP activity partially attenuated the Fbn2 mutant tracheal phenotypes. Analysis of human tracheal tissues indicates that a decrease in ECM proteins, including FBN2 and Fibronectin, is associated with tracheomalacia.Our findings provide novel insights into the role of ECM homeostasis in mesenchymal cell polarisation during tracheal tubulogenesis.

Smooth muscle: a stiff sculptor of epithelial shapes.

Smooth muscle is increasingly recognized as a key mechanical sculptor of epithelia during embryonic development. Smooth muscle is a mesenchymal tissue that surrounds the epithelia of organs including the gut, blood vessels, lungs, bladder, ureter, uterus, oviduct and epididymis. Smooth muscle is stiffer than its adjacent epithelium and often serves its morphogenetic function by physically constraining the growth of a proliferating epithelial layer. This constraint leads to mechanical instabilities and epithelial morphogenesis through buckling. Smooth muscle stiffness alone, without smooth muscle cell shortening, seems to be sufficient to drive epithelial morphogenesis. Fully understanding the development of organs that use smooth muscle stiffness as a driver of morphogenesis requires investigating how smooth muscle develops, a key aspect of which is distinguishing smooth muscle-like tissues from one another in vivo and in culture. This necessitates a comprehensive appreciation of the genetic, anatomical and functional markers that are used to distinguish the different subtypes of smooth muscle (for example, vascular versus visceral) from similar cell types (including myofibroblasts and myoepithelial cells). Here, we review how smooth muscle acts as a mechanical driver of morphogenesis and discuss ways of identifying smooth muscle, which is critical for understanding these morphogenetic events.This article is part of the Theo Murphy meeting issue 'Mechanics of Development'.

Morphogenesis and motility of the Astyanax mexicanus gastrointestinal tract.

Through the course of evolution, the gastrointestinal (GI) tract has been modified to maximize nutrient absorption, forming specialized segments that are morphologically and functionally distinct. Here we show that the GI tract of the Mexican tetra, Astyanax mexicanus, has distinct regions, exhibiting differences in morphology, motility, and absorption. We found that A. mexicanus populations adapted for life in subterranean caves exhibit differences in the GI segments compared to those adapted to surface rivers. Cave-adapted fish exhibit bi-directional churning motility in the stomach region that is largely absent in river-adapted fish. We investigated how this motility pattern influences intestinal transit of powdered food and live prey. We found that powdered food is more readily emptied from the cavefish GI tract. In contrast, the transit of live rotifers from the stomach region to the midgut occurs more slowly in cavefish compared to surface fish, consistent with the presence of churning motility. Differences in intestinal motility and transit likely reflect adaptation to unique food sources available to post-larval A. mexicanus in the cave and river environments. We found that cavefish grow more quickly than surface fish when fed ad libitum, suggesting that altered GI function may aid in nutrient consumption or absorption. We did not observe differences in enteric neuron density or smooth muscle organization between cavefish and surface fish. Altered intestinal motility in cavefish could instead be due to changes in the activity or patterning of the enteric nervous system. Exploring this avenue will lead to a better understanding of how the GI tract evolves to maximize energy assimilation from novel food sources.

Loss of smooth muscle myosin heavy chain results in the bladder and stomach developing lesion during foetal development in mice.

Smooth muscle myosin heavy chain (SM-MHC) is exclusively expresses in smooth muscle, which takes part in smooth muscle cell contraction. Here, we used an insertional mutation mouse whose heavy polypeptide 11 (Myh11) gene has been disrupted and no SM-MHC protein has been detected. Compared to the wild-type and SM-MHC+/- mice, the SM-MHC-/- neonates had large round bellies, thin-walled giant bladders, and large stomachs with huge gas bubbles. Most of it died within 10 h and the rest within 20 h after birth. Further analysis of the developing foetuses from 16.5 days postcoitum (dpc) stage to newborn showed no significant (P<0.05) difference in the ratio of Mendelian inheritance and average body weight among SM-MHC+/+ , SM-MHC+/- and SM-MHC-/- mice, whereas the abnormal exterior appearance was observed in each SM-MHC-/- bladders from 16.5 dpc. Histological analysis showed no difference in stomach tissues but evidently thin-walled smooth muscle layer and a giant cavity in bladders of SM-MHC-/- foetuses at various stages from 15.5 dpc to newborn. The results indicated that the defect of SM-MHC lead to the bladder developing lesions initially at 15.5 dpc stage in mouse and also implied that the SM-MHC loss might result in the gas bubbles in stomach. The study should facilitate further detailed analyses of the potential role of SM-MHC in bladder and stomach development.

Development of contractile properties in the fetal porcine urinary bladder.

BackgroundIn early fetal life, the bladder is merely a conduit allowing urine to pass through freely into the amniotic cavity. As the striated external urethral sphincter evolves, the bladder acquires its reservoir and voiding functions. We characterized the myogenic and neurogenic contractions of the normal fetal porcine bladder from midterm until close to full-term gestation.MethodsContractile responses were measured in vitro using bladder strips from fetuses at 60 (N=23) and 100 days (N=21) of gestation. Spontaneous activity, and the responses to potassium chloride (KCl) solution, electrical field stimulation (EFS), and receptor activation were recorded. The smooth muscle content was evaluated histologically.ResultsHistological studies revealed that the fractional content of smooth muscle doubled between the two time points, and passive tension was adjusted to take that into account. Spontaneous activity was regular at 60 days, changing toward an irregular pattern at 100 days. Contractile force elicited by KCl and carbachol increased significantly with gestational age, while contractions to the purinergic agonist, α-ß-methylene adenosine 5'-triphosphate did not. The responses to EFS were almost completely blocked by atropine.ConclusionSpontaneous myogenic contractions become irregular and contractile responses to muscarinic receptor stimulation increase during gestation, as the bladder reservoir and voiding functions develop.

Microfluidic chest cavities reveal that transmural pressure controls the rate of lung development.

Mechanical forces are increasingly recognized to regulate morphogenesis, but how this is accomplished in the context of the multiple tissue types present within a developing organ remains unclear. Here, we use bioengineered 'microfluidic chest cavities' to precisely control the mechanical environment of the fetal lung. We show that transmural pressure controls airway branching morphogenesis, the frequency of airway smooth muscle contraction, and the rate of developmental maturation of the lungs, as assessed by transcriptional analyses. Time-lapse imaging reveals that branching events are synchronized across distant locations within the lung, and are preceded by long-duration waves of airway smooth muscle contraction. Higher transmural pressure decreases the interval between systemic smooth muscle contractions and increases the rate of morphogenesis of the airway epithelium. These data reveal that the mechanical properties of the microenvironment instruct crosstalk between different tissues to control the development of the embryonic lung.

Embryonic cholecystitis and defective gallbladder contraction in the Sox17-haploinsufficient mouse model of biliary atresia.

The gallbladder excretes cytotoxic bile acids into the duodenum through the cystic duct and common bile duct system. Sox17 haploinsufficiency causes biliary atresia-like phenotypes and hepatitis in late organogenesis mouse embryos, but the molecular and cellular mechanisms underlying this remain unclear. In this study, transcriptomic analyses revealed the early onset of cholecystitis in Sox17+/- embryos, together with the appearance of ectopic cystic duct-like epithelia in their gallbladders. The embryonic hepatitis showed positive correlations with the severity of cholecystitis in individual Sox17+/- embryos. Embryonic hepatitis could be induced by conditional deletion of Sox17 in the primordial gallbladder epithelia but not in fetal liver hepatoblasts. The Sox17+/- gallbladder also showed a drastic reduction in sonic hedgehog expression, leading to aberrant smooth muscle formation and defective contraction of the fetal gallbladder. The defective gallbladder contraction positively correlated with the severity of embryonic hepatitis in Sox17+/- embryos, suggesting a potential contribution of embryonic cholecystitis and fetal gallbladder contraction in the early pathogenesis of congenital biliary atresia.

Colonic mesenchyme differentiates into smooth muscle before its colonization by vagal enteric neural crest-derived cells in the chick embryo.

During development, the gastrointestinal (GI) tract arises from a primary tube composed of mesoderm and endoderm. The mesoderm gives rise to the digestive mesenchyme, which in turn differentiates into multiple tissues, namely the submucosa, the interstitial cells of Cajal and the smooth muscle cells (SMCs). Concomitant with these early patterning events, the primitive GI tract is colonized by vagal enteric neural crest-derived cells (vENCDCs), a population of cells that gives rise to the enteric nervous system, the intrinsic innervation of the GI tract. Reciprocal neuro-mesenchymal interactions are essential for the coordinated development of GI musculature. The aim of this study is to examine and compare the kinetics of mesenchymal cell differentiation into SMCs along the anterior-posterior axis to the pattern of vENCDCs migration using whole-mount in situ hybridization and paraffin section immunofluorescence analyses on chick embryonic GI tracts from E4-Stage 23 to E7-Stages 30-31. We confirmed that gastric and pre-umbilical intestine mesenchyme differentiation into SMCs occurs after vENCDCs colonization. However, we found that colonic and post-umbilical intestine mesenchyme differentiation occurs before vENCDCs colonization. These findings suggest that regional-specific mechanisms are involved in the mesenchyme differentiation into SMCs along the GI anterior-posterior axis.

Emergence and development of gut motility in the chicken embryo.

The gastrointestinal tract transports the food bolus by peristalsis. Gut motility starts at an early age in the developing embryo, well before it is required for nutrition of the organism. We present a comprehensive kinematic study of the emergence and physiological development of gut motility in all regions of the lower digestive tract of the chicken embryo from embryonic days E5 through E9. We characterized motility emergence time, propagation patterns, speed, frequency and amplitude of peristalsis waves. We found that the emergence of an uninterrupted circular ring of smooth muscle correlated with the appearance of propagative contractile waves, at E6 in the hindgut and midgut, and at E9 in the caecal appendix. We show that peristalsis at these stages is critically dependent on calcium and is not mediated by neurons as gut motility is insensitive to tetrodotoxin and takes place in the hindgut in the absence of neurons. We further demonstrate that motility also matures in ex-vivo organ culture. We compare our results to existing literature on zebrafish, mouse and human motility development, and discuss their chronological relationship with other major developmental events occurring in the chicken embryonic gut at these stages. Our work sets a baseline for further investigations of motility development in this important animal model.