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Intestinal smooth muscle differentiation is a complex physico-biological process involving several different pathways. Here, we investigate the properties of Ca 2+ waves in the developing intestinal mesenchyme using GCamp6f expressing mouse embryos and investigate their relationship with smooth muscle differentiation. We find that Ca 2+ waves are absent in the pre-differentiation mesenchyme and start propagating immediately following α-SMA expression. Ca 2+ waves are abrogated by Ca V 1.2 and gap-junction blockers, but are independent of the Rho pathway. The myosine light-chain kinase inhibitor ML-7 strongly disorganized or abolished Ca 2+ waves, showing that perturbation of the contractile machinery at the myosine level also affected the upstream Ca 2+ handling chain. Inhibiting Ca 2+ waves and contractility with Ca V 1.2 blockers did not perturb circular smooth muscle differentiation at early stages. At later stages, Ca V 1.2 blockers abolished intestinal elongation and differentiation of the longitudinal smooth muscle, leading instead to the emergence of KIT-expressing interstitial cells of Cajal at the gut periphery. Ca V 1.2 blockers also drove apoptosis of already differentiated, Ca V 1.2-expressing smooth muscle and enteric neural cells. We provide fundamental new data on Ca 2+ waves in the developing murine gut and their relation to myogenesis in this organ., (© 2024. The Author(s).)

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 β1-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.

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

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.

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.

Preterm infants can develop airway hyperreactivity and impaired bronchodilation following supplemental O 2 (hyperoxia) in early life, making it important to understand mechanisms of hyperoxia effects. Endogenous hydrogen sulfide (H 2 S) has anti-inflammatory and vasodilatory effects with oxidative stress. There is little understanding of H 2 S signaling in developing airways. We hypothesized that the endogenous H 2 S system is detrimentally influenced by O 2 and conversely H 2 S signaling pathways can be leveraged to attenuate deleterious effects of O 2 . Using human fetal airway smooth muscle (fASM) cells, we investigated baseline expression of endogenous H 2 S machinery, and effects of exogenous H 2 S donors NaHS and GYY4137 in the context of moderate hyperoxia, with intracellular calcium regulation as a readout of contractility. Biochemical pathways for endogenous H 2 S generation and catabolism are present in fASM, and are differentially sensitive to O 2 toward overall reduction in H 2 S levels. H 2 S donors have downstream effects of reducing [Ca 2+ ] i responses to bronchoconstrictor agonist via blunted plasma membrane Ca 2+ influx: effects blocked by O 2 . However, such detrimental O 2 effects are targetable by exogenous H 2 S donors such as NaHS and GYY4137. These data provide novel information regarding the potential for H 2 S to act as a bronchodilator in developing airways in the context of oxygen exposure., (© 2020 Federation of American Societies for Experimental Biology.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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.

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.

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.

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., Competing Interests: Conflict of interest: W. Yin has nothing to disclose. Conflict of interest: H-T. Kim has nothing to disclose. Conflict of interest: S. Wang has nothing to disclose. Conflict of interest: F. Gunawan has nothing to disclose. Conflict of interest: R. Li has nothing to disclose. Conflict of interest: C. Buettner has nothing to disclose. Conflict of interest: B. Grohmann has nothing to disclose. Conflict of interest: G. Sengle has nothing to disclose. Conflict of interest: D. Sinner has nothing to disclose. Conflict of interest: S. Offermanns has nothing to disclose. Conflict of interest: D.Y.R. Stainier has nothing to disclose., (Copyright ©ERS 2019.)

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'., (© 2018 The Author(s).)

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., (Copyright © 2018. Published by Elsevier Inc.)

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.

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-β-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.

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

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.

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

The development of the enteric nervous system (ENS) and intestinal smooth muscle occurs in a spatially and temporally correlated manner, but how they influence each other is unknown. In the developing mid-gut of the chick embryo, we find that α-smooth muscle actin expression, indicating early muscle differentiation, occurs after the arrival of migrating enteric neural crest-derived cells (ENCCs). In contrast, hindgut smooth muscle develops prior to ENCC arrival. Smooth muscle development is normal in experimentally aganglionic hindguts, suggesting that proper development and patterning of the muscle layers does not rely on the ENS. However, inhibiting early smooth muscle development severely disrupts ENS patterning without affecting ENCC proliferation or apoptosis. Our results demonstrate that early intestinal smooth muscle differentiation is required for patterning the developing ENS., (© 2017 Anatomical Society.)

While urothelial signals, including sonic hedgehog (Shh), drive bladder mesenchyme differentiation, it is unclear which pathways within the mesenchyme are critical for its development. Studies have shown that fibroblast growth factor receptor 2 (Fgfr2) is necessary for kidney and ureter mesenchymal development. Our objective was to determine the role of Fgfr2 in bladder mesenchyme. We used Tbx18cre mice to delete Fgfr2 in bladder mesenchyme ( Fgfr2 BM -/- ). We performed three-dimensional reconstructions, quantitative real-time PCR, in situ hybridization, immunolabeling, ELISAs, immunoblotting, void stain on paper, ex vivo bladder sheet assays, and in vivo decerebrated cystometry. Compared with controls, embryonic ( E ) day 16.5 ( E16.5 ) Fgfr2 BM -/- bladders have thin muscle layers with reduced α-smooth muscle actin levels and thickened lamina propria with increased collagen expression that intrudes into muscle. From postnatal ( P ) day 1 ( P1 ) to P30 , Fgfr2 BM -/- bladders demonstrate progressive muscle loss and increased collagen expression. Postnatal Fgfr2 BM -/- bladder sheets exhibit decreased contractility and increased passive stretch tension compared with controls. In vivo cystometry revealed high baseline and threshold pressures and shortened intercontractile intervals in Fgfr2 BM -/- bladders compared with controls. Mechanistically, while Shh expression appears normal, mRNA and protein readouts of hedgehog activity are increased in E16.5 Fgfr2 BM -/- bladders compared with controls. Moreover, E16.5 Fgfr2 BM -/- bladders exhibit higher levels of Cdo and Boc , hedgehog coreceptors that enhance sensitivity to Shh, than controls. Fgfr2 is critical for bladder mesenchyme patterning by virtue of its role in modulation of hedgehog signaling., (Copyright © 2017 the American Physiological Society.)

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.

The esophagus functions to transport food from the oropharyngeal region to the stomach via waves of peristalsis and transient relaxation of the lower esophageal sphincter. The gastrointestinal tract, including the esophagus, is ensheathed by the muscularis externa (ME). However, while the ME of the gastrointestinal tract distal to the esophagus is exclusively smooth muscle, the esophageal ME of many vertebrate species comprises a variable amount of striated muscle. The esophageal ME is initially composed only of smooth muscle, but its developmental maturation involves proximal-to-distal replacement of smooth muscle with striated muscle. This fascinating phenomenon raises two important questions: what is the developmental origin of the striated muscle precursor cells, and what are the cellular and morphogenetic mechanisms underlying the process? Studies addressing these questions have provided controversial answers. In this review, we discuss the development of ideas in this area and recent work that has shed light on these issues. A working model has emerged that should permit deeper understanding of the role of ME development and maturation in esophageal disorders and in the functional and evolutionary underpinnings of the variable degree of esophageal striated myogenesis in vertebrate species.

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Digestion is facilitated by coordinated contractions of the intestinal muscularis externa, a bilayered smooth muscle structure that is composed of inner circular muscles (ICM) and outer longitudinal muscles (OLM). We performed transcriptome analysis of intestinal mesenchyme tissue at E14.5, when the ICM, but not the OLM, is present, to investigate the transcriptional program of the ICM., Results: We identified 3967 genes enriched in E14.5 intestinal mesenchyme. The gene expression profiles were clustered and annotated to known muscle genes, identifying a muscle-enriched subcluster. Using publically available in situ data, 127 genes were verified as expressed in ICM. Examination of the promoter and regulatory regions for these co-expressed genes revealed enrichment for cJUN transcription factor binding sites, and cJUN protein was enriched in ICM. cJUN ChIP-seq, performed at E14.5, revealed that cJUN regulatory regions contain characteristics of muscle enhancers. Finally, we show that cJun is a target of Hedgehog (Hh), a signaling pathway known to be important in smooth muscle development, and identify a cJun genomic enhancer that is responsive to Hh., Conclusions: This work provides the first transcriptional catalog for the developing ICM and suggests that cJun regulates gene expression in the ICM downstream of Hh signaling. Developmental Dynamics 245:614-626, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Background: Epithelial-mesenchymal cross talk is centerpiece in the development of many branched organs, including the lungs. The embryonic lung mesoderm provides instructional information not only for lung architectural development, but also for patterning, commitment and differentiation of its many highly specialized cell types. The mesoderm also serves as a reservoir of progenitors for generation of differentiated mesenchymal cell types that include αSMA-expressing fibroblasts, lipofibroblasts, endothelial cells and others. Transforming Growth Factor β (TGFβ) is a key signaling pathway in epithelial-mesenchymal cross talk. Using a cre-loxP approach we have elucidated the role of the TGFβ type I receptor tyrosine kinase, ALK5, in epithelial-mesenchymal cross talk during lung morphogenesis., Results: Targeted early inactivation of Alk5 in mesodermal progenitors caused abnormal development and maturation of the lung that included reduced physical size of the sub-mesothelial mesoderm, an established source of specific mesodermal progenitors. Abrogation of mesodermal ALK5-mediated signaling also inhibited differentiation of cell populations in the epithelial and endothelial lineages. Importantly, Alk5 mutant lungs contained a reduced number of αSMA(pos) cells and correspondingly increased lipofibroblasts. Elucidation of the underlying mechanisms revealed that through direct and indirect modulation of target signaling pathways and transcription factors, including PDGFRα, PPARγ, PRRX1, and ZFP423, ALK5-mediated TGFβ controls a process that regulates the commitment and differentiation of αSMA(pos) versus lipofibroblast cell populations during lung development., Conclusion: ALK5-mediated TGFβ signaling controls an early pathway that regulates the commitment and differentiation of αSMA(pos) versus LIF cell lineages during lung development.

In the intestine, finger-like villi provide abundant surface area for nutrient absorption. During murine villus development, epithelial Hedgehog (Hh) signals promote aggregation of subepithelial mesenchymal clusters that drive villus emergence. Clusters arise first dorsally and proximally and spread over the entire intestine within 24 h, but the mechanism driving this pattern in the murine intestine is unknown. In chick, the driver of cluster pattern is tensile force from developing smooth muscle, which generates deep longitudinal epithelial folds that locally concentrate the Hh signal, promoting localized expression of cluster genes. By contrast, we show that in mouse, muscle-induced epithelial folding does not occur and artificial deformation of the epithelium does not determine the pattern of clusters or villi. In intestinal explants, modulation of Bmp signaling alters the spatial distribution of clusters and changes the pattern of emerging villi. Increasing Bmp signaling abolishes cluster formation, whereas inhibiting Bmp signaling leads to merged clusters. These dynamic changes in cluster pattern are faithfully simulated by a mathematical model of a Turing field in which an inhibitor of Bmp signaling acts as the Turing activator. In vivo, genetic interruption of Bmp signal reception in either epithelium or mesenchyme reveals that Bmp signaling in Hh-responsive mesenchymal cells controls cluster pattern. Thus, unlike in chick, the murine villus patterning system is independent of muscle-induced epithelial deformation. Rather, a complex cocktail of Bmps and Bmp signal modulators secreted from mesenchymal clusters determines the pattern of villi in a manner that mimics the spread of a self-organizing Turing field., (© 2016. Published by The Company of Biologists Ltd.)

MicroRNAs (miRs) are a group of small RNAs that play a major role in post-transcriptional regulation of gene expression. In animals, many of the miRs are expressed in a conserved spatiotemporal manner. Muscle tissues, the major cellular systems involved in the locomotion and physiological functions of animals, have been one of the main sites for verification of miR targets and analysis of their developmental functions. During the determination and differentiation of muscle cells, numerous miRs bind to and repress target mRNAs in a highly specific but redundant manner. Interspecific comparisons of the sequences and expression of miRs have suggested that miR regulation became increasingly important during the course of vertebrate evolution. However, the detailed molecular interactions that have led to the highly complex morphological structures still await investigation. In this review, we will summarize the recent findings on the functional and developmental characteristics of miRs that have played major roles in vertebrate myogenesis, and discuss how the evolution of miRs is related to the morphological complexity of the vertebrates., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The airway epithelium develops into a tree-like structure via branching morphogenesis. Here, we show a critical role for localized differentiation of airway smooth muscle during epithelial bifurcation in the embryonic mouse lung. We found that during terminal bifurcation, changes in the geometry of nascent buds coincided with patterned smooth muscle differentiation. Evaluating spatiotemporal dynamics of α-smooth muscle actin (αSMA) in reporter mice revealed that αSMA-expressing cells appear at the basal surface of the future epithelial cleft prior to bifurcation and then increase in density as they wrap around the bifurcating bud. Disrupting this stereotyped pattern of smooth muscle differentiation prevents terminal bifurcation. Our results reveal stereotyped differentiation of airway smooth muscle adjacent to nascent epithelial buds and suggest that localized smooth muscle wrapping at the cleft site is required for terminal bifurcation during airway branching morphogenesis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Exposure to moderate hyperoxia in prematurity contributes to subsequent airway dysfunction and increases the risk of developing recurrent wheeze and asthma. The nitric oxide (NO)-soluble guanylate cyclase (sGC)-cyclic GMP (cGMP) axis modulates airway tone by regulating airway smooth muscle (ASM) intracellular Ca(2+) ([Ca(2+)]i) and contractility. However, the effects of hyperoxia on this axis in the context of Ca(2+)/contractility are not known. In developing human ASM, we explored the effects of novel drugs that activate sGC independent of NO on alleviating hyperoxia (50% oxygen)-induced enhancement of Ca(2+) responses to bronchoconstrictor agonists. Treatment with BAY 41-2272 (sGC stimulator) and BAY 60-2770 (sGC activator) increased cGMP levels during exposure to 50% O2. Although 50% O2 did not alter sGCα1 or sGCβ1 expression, BAY 60-2770 did increase sGCβ1 expression. BAY 41-2272 and BAY 60-2770 blunted Ca(2+) responses to histamine in cells exposed to 50% O2. The effects of BAY 41-2272 and BAY 60-2770 were reversed by protein kinase G inhibition. These novel data demonstrate that BAY 41-2272 and BAY 60-2770 stimulate production of cGMP and blunt hyperoxia-induced increases in Ca(2+) responses in developing ASM. Accordingly, sGC stimulators/activators may be a useful therapeutic strategy in improving bronchodilation in preterm infants., (Copyright © 2015 the American Physiological Society.)

Diverse functions of the homeodomain transcription factor BARX1 include Wnt-dependent, non-cell autonomous specification of the stomach epithelium, tracheo-bronchial septation, and Wnt-independent expansion of the spleen primordium. Tight spatio-temporal regulation of Barx1 levels in the mesentery and stomach mesenchyme suggests additional roles. To determine these functions, we forced constitutive BARX1 expression in the Bapx1 expression domain, which includes the mesentery and intestinal mesenchyme, and also examined Barx1(-/)(-) embryos in further detail. Transgenic embryos invariably showed intestinal truncation and malrotation, in part reflecting abnormal left-right patterning. Ectopic BARX1 expression did not affect intestinal epithelium, but intestinal smooth muscle developed with features typical of the stomach wall. BARX1, which is normally restricted to the developing stomach, drives robust smooth muscle expansion in this organ by promoting proliferation of myogenic progenitors at the expense of other sub-epithelial cells. Undifferentiated embryonic stomach and intestinal mesenchyme showed modest differences in mRNA expression and BARX1 was sufficient to induce much of the stomach profile in intestinal cells. However, limited binding at cis-regulatory sites implies that BARX1 may act principally through other transcription factors. Genes expressed ectopically in BARX1(+) intestinal mesenchyme and reduced in Barx1(-/-) stomach mesenchyme include Isl1, Pitx1, Six2 and Pitx2, transcription factors known to control left-right patterning and influence smooth muscle development. The sum of evidence suggests that potent BARX1 functions in intestinal rotation and stomach myogenesis occur through this small group of intermediary transcription factors., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Congenital anomalies of the kidneys and urinary tract (CAKUT) are the most common cause of chronic kidney disease in the first three decades of life. Identification of single-gene mutations that cause CAKUT permits the first insights into related disease mechanisms. However, for most cases the underlying defect remains elusive. We identified a kindred with an autosomal-dominant form of CAKUT with predominant ureteropelvic junction obstruction. By whole exome sequencing, we identified a heterozygous truncating mutation (c.1010delG) of T-Box transcription factor 18 (TBX18) in seven affected members of the large kindred. A screen of additional families with CAKUT identified three families harboring two heterozygous TBX18 mutations (c.1570C>T and c.487A>G). TBX18 is essential for developmental specification of the ureteric mesenchyme and ureteric smooth muscle cells. We found that all three TBX18 altered proteins still dimerized with the wild-type protein but had prolonged protein half life and exhibited reduced transcriptional repression activity compared to wild-type TBX18. The p.Lys163Glu substitution altered an amino acid residue critical for TBX18-DNA interaction, resulting in impaired TBX18-DNA binding. These data indicate that dominant-negative TBX18 mutations cause human CAKUT by interference with TBX18 transcriptional repression, thus implicating ureter smooth muscle cell development in the pathogenesis of human CAKUT., (Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

In vertebrates, the digestive tract develops from a uniform structure where reciprocal epithelial-mesenchymal interactions pattern this complex organ into regions with specific morphologies and functions. Concomitant with these early patterning events, the primitive GI tract is colonized by the vagal enteric neural crest cells (vENCCs), a population of cells that will give rise to the enteric nervous system (ENS), the intrinsic innervation of the GI tract. The influence of vENCCs on early patterning and differentiation of the GI tract has never been evaluated. In this study, we report that a crucial number of vENCCs is required for proper chick stomach development, patterning and differentiation. We show that reducing the number of vENCCs by performing vENCC ablations induces sustained activation of the BMP and Notch pathways in the stomach mesenchyme and impairs smooth muscle development. A reduction in vENCCs also leads to the transdifferentiation of the stomach into a stomach-intestinal mixed phenotype. In addition, sustained Notch signaling activity in the stomach mesenchyme phenocopies the defects observed in vENCC-ablated stomachs, indicating that inhibition of the Notch signaling pathway is essential for stomach patterning and differentiation. Finally, we report that a crucial number of vENCCs is also required for maintenance of stomach identity and differentiation through inhibition of the Notch signaling pathway. Altogether, our data reveal that, through the regulation of mesenchyme identity, vENCCs act as a new mediator in the mesenchymal-epithelial interactions that control stomach development., (© 2015. Published by The Company of Biologists Ltd.)

The gastric ligaments are superficial cord-like structures, located on the lesser curvature of the stomach, that extend from the pylorus to the esophagus. These ligaments have been documented in a wide variety of mammalian species, including humans, but their composition and ontogeny is unexplored. Here, we demonstrate that, during ontogeny, the gastric ligaments are first visible as extensions from a C-shaped domain of Gata3-expressing cells that surround the future pylorus; this domain will later give rise to the pyloric outer longitudinal muscle (OLM). The open ends of the C are located ventrally, and, beginning at embryonic day (E) 13.5, the ligaments grow anteriorly from these points. Whereas most other ligaments of the stomach are neurovascular in nature, the gastric ligaments are composed of smooth muscle cells that mature between E14.5 and E16.5. The gastric ligaments coexpress the transcription factors Gata3, Nkx2-5, and Sox9, and germline loss of Gata3 or conditional deletion of Nkx2-5 abrogates Sox9 expression and impairs gastric ligament smooth muscle development; similar phenotypes were previously seen in the OLM. In accord with this molecular contiguity between the OLM and gastric ligaments, three-dimensional image reconstruction highlights physical contiguity between these smooth muscle structures, suggesting that they may work together as a unit to control flexure of the pyloric region, a function similar to the ligament of Treitz at the duodenojejunal junction. These findings may have implications for our understanding of normal pyloric sphincter function, as well as the common human congenital pathology idiopathic hypertrophic pyloric stenosis., (Copyright © 2014 the American Physiological Society.)

Background: Lineage segregation from multipotent epithelia is a central theme in development and in adult stem cell plasticity. Previously, we demonstrated that striated and smooth muscle cells share a common progenitor within their epithelium of origin, the lateral domain of the somite-derived dermomyotome. However, what controls the segregation of these muscle subtypes remains unknown. We use this in vivo bifurcation of fates as an experimental model to uncover the underlying mechanisms of lineage diversification from bipotent progenitors., Results: Using the strength of spatio-temporally controlled gene missexpression in avian embryos, we report that Notch harbors distinct pro-smooth muscle activities depending on the duration of the signal; short periods prevent striated muscle development and extended periods, through Snail1, promote cell emigration from the dermomyotome towards a smooth muscle fate. Furthermore, we define a Muscle Regulatory Network, consisting of Id2, Id3, FoxC2 and Snail1, which acts in concert to promote smooth muscle by antagonizing the pro-myogenic activities of Myf5 and Pax7, which induce striated muscle fate. Notch and BMP closely regulate the network and reciprocally reinforce each other¿s signal. In turn, components of the network strengthen Notch signaling, while Pax7 silences this signaling. These feedbacks augment the robustness and flexibility of the network regulating muscle subtype segregation., Conclusions: Our results demarcate the details of the Muscle Regulatory Network, underlying the segregation of muscle sublineages from the lateral dermomyotome, and exhibit how factors within the network promote the smooth muscle at the expense of the striated muscle fate. This network acts as an exemplar demonstrating how lineage segregation occurs within epithelial primordia by integrating inputs from competing factors.

The embryonic endothelium is a known source of hematopoietic stem cells. Moreover, vessel-associated progenitors/stem cells with multilineage mesodermal differentiation potential, such as the 'embryonic mesoangioblasts', originate in vitro from the endothelium. Using a genetic lineage tracing approach, we show that early extra-embryonic endothelium generates, in a narrow time-window and prior to the hemogenic endothelium in the major embryonic arteries, hematopoietic cells that migrate to the embryo proper, and are subsequently found within the mesenchyme. A subpopulation of these cells, distinct from embryonic macrophages, co-expresses mesenchymal and hematopoietic markers. In addition, hemogenic endothelium-derived cells contribute to skeletal and smooth muscle, and to other mesodermal cells in vivo, and display features of embryonic mesoangioblasts in vitro. Therefore, we provide new insights on the distinctive characteristics of the extra-embryonic and embryonic hemogenic endothelium, and we identify the putative in vivo counterpart of embryonic mesoangioblasts, suggesting their identity and developmental ontogeny.

Interstitial cells of Cajal (ICCs) are located in various smooth muscle organs and act as pacemaker cells, or ensure neuromodulation or mechanosensory roles. The study aims to investigate functional states of human ICCs in morphogenesis, focusing on the anoctamin 1 phenotype. The investigation was performed in five late stage human embryos with lengths varying between 23 and 29 mm. Immunohistochemistry on paraffin embedded specimens was performed for a series of antibodies: a-smooth muscle actin (a-SMA), desmin, CD31, CD34, CD117/c-kit, DOG1, and nestin. Longitudinal and circular muscle layers were a-SMA1/desmin1/nestin1. An immature microvascular layer located in the inner submucosa was CD341/CD311/a-SMA1/ nestin1; endothelial tip cells were supporting active processes of sprouting angiogenesis. A CD341/CD31- mesenchymal network was found in the circular muscle layer. CD117/c-kit1 multipolar ICCs with dichotomizing processes were found mostly in the myenteric plexus layer; processes were configuring a network within the circular muscle layer where intramuscular ICCs were scarcely found. A strong DOG11 reaction was found for the ICCs of the myenteric plexus layer apposed on the outer surface of the circular muscle layer, and for the intramuscular ICCs. The evidence of a sublayer of DOG11 myenteric ICCs is suggestive for a subpopulation of ICCs being qualified for pacemaking at this early developmental stage., (Copyright © 2013 Wiley Periodicals, Inc.)

Background & Aims: Infantile hypertrophic pyloric stenosis is a common birth anomaly characterized by obstruction of the pyloric lumen. A genome-wide association study implicated NKX2-5, which encodes a transcription factor that is expressed in embryonic heart and pylorus, in the pathogenesis of infantile hypertrophic pyloric stenosis. However, the function of the NKX2-5 in pyloric smooth muscle development has not been examined directly. We investigated the pattern of Nkx2-5 during the course of murine pyloric sphincter development and examined coexpression of Nkx2-5 with Gata3 and Sox9-other transcription factors with pyloric-specific mesenchymal expression. We also assessed pyloric sphincter development in mice with disruption of Nkx2-5 or Gata3., Methods: We used immunofluorescence analysis to compare levels of NKX2-5, GATA3, and SOX9 in different regions of smooth muscle cells. Pyloric development was assessed in mice with conditional or germline deletion of Nkx2-5 or Gata3, respectively., Results: Gata3, Nkx2-5, and Sox9 are coexpressed in differentiating smooth muscle cells of a distinct fascicle of the pyloric outer longitudinal muscle. Expansion of this fascicle coincides with development of the pyloric sphincter. Disruption of Nkx2-5 or Gata3 causes severe hypoplasia of this fascicle and alters pyloric muscle shape. Although expression of Sox9 requires Nkx2-5 and Gata3, there is no apparent hierarchical relationship between Nkx2-5 and Gata3 during pyloric outer longitudinal muscle development., Conclusions: Nkx2-5 and Gata3 are independently required for the development of a pyloric outer longitudinal muscle fascicle, which is required for pyloric sphincter morphogenesis in mice. These data indicate that regulatory changes that alter Nkx2-5 or Gata3 expression could contribute to pathogenesis of infantile hypertrophic pyloric stenosis., (Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Purpose: Intestinal perforations are common in premature infants, leading to a diagnostic dilemma between necrotizing enterocolitis and isolated intestinal perforation (IIP). IIP is thought to result from a congenital or acquired absence of the muscularis propria. However, developmental events leading to IIP are not well understood. This study examines the relationship between corrected gestational age (CGA) and intestinal muscle development in controls and patients with IIP., Methods: Specimens from stillbirths and infants undergoing intestinal surgery from 8 to 48weeks' CGA were collected from 2005 to 2012. Twelve patients with IIP were identified. Control specimens were collected during 25 fetal autopsies and 39 bowel resections. In each case, three sections of intestine were examined histologically for muscularis mucosa, circular and longitudinal muscle thickness. Comparisons of control and perforated specimens were performed via linear regression and ANOVA., Results: Controls and adjacent normal segments in IIP showed a linear relationship between thickness of circular and longitudinal muscles with CGA. Circular and longitudinal muscles were thinner in perforated segments than in adjacent normals and CGA-matched controls (p<0.05)., Conclusion: Intestinal muscularis propria increases in thickness with CGA. Muscle thickness is focally attenuated in patients with isolated intestinal perforations, while the remaining intestine is normal, suggesting that primary repair is an appropriate treatment., (© 2014.)

Objective: To analyze the development and innervation of bladder smooth muscle and lesions of the spinal cord in fetal rats with meningomyelocele (MMC) at different gestational ages and to investigate interactions between spinal cord lesions and bladder., Method: Each fetus was assigned to the MMC group or the normal group. Each group was further divided into three subgroups by gestational age: E16, E18, and E20 (embryonic days 16, 18, and 20, respectively). α-Actin and neurotubulin-β-III were analyzed in the bladder, and GFAP and VAChT were analyzed in the lumbosacral spinal cord by immunohistochemistry. Photographs were taken to determine the integrated optical density of each sample., Results: Neurotubulin-β-III was significantly lower in the MMC group than in the normal group at all fetal ages. Abundant α-actin was detected in both groups at all fetal ages. No significant difference was found between the MMC group and the normal group at any fetal age. At E16 and E18, no GFAP-positive astrocyte was detected in the MMC group or the normal group. At E20, numerous GFAP-positive astrocytes were detected in the MMC group, with significant difference from the normal group. VAChT was detected less in the MMC group than in the normal group at all fetal ages with significant differences., Conclusion: Bladder smooth muscle of fetal MMC rat seems morphologically normal in development, while the innervation of the bladder smooth muscle is markedly decreased centrally and peripherally. Astrocytosis appears at a later embryonic stage, which could be a concern in the nerve repair of the spinal cord., (© 2013.)

In the trachea and bronchi of the mouse, airway smooth muscle (SM) and cartilage are localized to complementary domains surrounding the airway epithelium. Proper juxtaposition of these tissues ensures a balance of elasticity and rigidity that is critical for effective air passage. It is unknown how this tissue complementation is established during development. Here we dissect the developmental relationship between these tissues by genetically disrupting SM formation (through Srf inactivation) or cartilage formation (through Sox9 inactivation) and assessing the impact on the remaining lineage. We found that, in the trachea and main bronchi, loss of SM or cartilage resulted in an increase in cell number of the remaining lineage, namely the cartilage or SM, respectively. However, only in the main bronchi, but not in the trachea, did the loss of SM or cartilage lead to a circumferential expansion of the remaining cartilage or SM domain, respectively. In addition to SM defects, cartilage-deficient tracheas displayed epithelial phenotypes, including decreased basal cell number, precocious club cell differentiation, and increased secretoglobin expression. These findings together delineate the mechanisms through which a cell-autonomous disruption of one structural tissue can have widespread consequences on upper airway function.

Introduction: Latent TGFβ binding proteins (LTBPs) govern TGFβ presentation and activation and are important for elastogenesis. Although TGFβ is well-known as a tumor suppressor and metastasis promoter, and LTBP1 is elevated in two distinct breast cancer metastasis signatures, LTBPs have not been studied in the normal mammary gland., Methods: To address this we have examined Ltbp1 promoter activity throughout mammary development using an Ltbp1L-LacZ reporter as well as expression of both Ltbp1L and 1S mRNA and protein by qRT-PCR, immunofluorescence and flow cytometry., Results: Our data show that Ltbp1L is transcribed coincident with lumen formation, providing a rare marker distinguishing ductal from alveolar luminal lineages. Ltbp1L and Ltbp1S are silent during lactation but robustly induced during involution, peaking at the stage when the remodeling process becomes irreversible. Ltbp1L is also induced within the embryonic mammary mesenchyme and maintained within nipple smooth muscle cells and myofibroblasts. Ltbp1 protein exclusively ensheaths ducts and side branches., Conclusions: These data show Ltbp1 is transcriptionally regulated in a dynamic manner that is likely to impose significant spatial restriction on TGFβ bioavailability during mammary development. We hypothesize that Ltbp1 functions in a mechanosensory capacity to establish and maintain ductal luminal cell fate, support and detect ductal distension, trigger irreversible involution, and facilitate nipple sphincter function.

The villi of the human and chick gut are formed in similar stepwise progressions, wherein the mesenchyme and attached epithelium first fold into longitudinal ridges, then a zigzag pattern, and lastly individual villi. We find that these steps of villification depend on the sequential differentiation of the distinct smooth muscle layers of the gut, which restrict the expansion of the growing endoderm and mesenchyme, generating compressive stresses that lead to their buckling and folding. A quantitative computational model, incorporating measured properties of the developing gut, recapitulates the morphological patterns seen during villification in a variety of species. These results provide a mechanistic understanding of the formation of these elaborations of the lining of the gut, essential for providing sufficient surface area for nutrient absorption.

Recent efforts examining forces during morphogenesis suggest a role for mechanical crosstalk between epithelium and mesenchyme in tissue patterning. Reporting in Science, Shyer et al. (2013) show that differences between the mechanical properties of the developing intestinal epithelium and surrounding smooth muscle fold the epithelium into villi via mucosal buckling., (Copyright © 2013 Elsevier Inc. All rights reserved.)

To assess the development of the duodenal window in fetuses, we examined semiserial histological sections of 59 human fetuses with a crown-rump length of 27-156 mm (∼4-18 weeks of gestation). In 44 of the 54 fetuses with horizontal sections, the duodenal window was formed by interdigitation of the anterior and posterior muscle slips from the proper duodenal circular muscle coat. The anterior slips approached the common bile duct from the anterior side and wound around the bile duct from the right aspect, whereas the posterior slips approached the main pancreatic duct from the posterior side, reaching the left or outer aspect of the duct without winding. These slips may become longitudinal muscles in the ampulla after birth. Six specimens showed variations in this typical pattern, in that the posterior muscle slips as well as the duodenal longitudinal muscle coat wound around the bile duct. In the remaining four specimens, we observed an abnormal union of the bile and pancreatic ducts, with the duodenal circular muscles suddenly ending along the window or slightly inserted into the right side of the common duct after joining. In all later-stage fetuses, the common sphincter surrounded both the bile and pancreatic ducts in the ampulla. Consequently, at and along the duodenal window, the proper duodenal circular muscle seemed to contribute to fetal sphincter formation. The window was not a simple hiatus but a functional interface between the sphincter and the duodenal wall., (Copyright © 2012 Wiley Periodicals, Inc.)

Gastrointestinal stromal tumor (GIST) is believed to original from interstitial cells of (ICC) present in Auerbach's nerve plexus. GIST frequently shows gain-of-function mutations of KIT and PDGFRA. In practical pathology, GIST is diagnosed by positive immunostaining or KIT and/or CD34. The author herein demonstrates that human embryonic gastrointestinal tract smooth muscles (HEGITSM) and human embryonic stem gastrointestinal cells (HEGISC) consistently express KIT, CD34, NCAM, PDGFRA and other stem cell (SC) antigens NSE, synaptophysin, chromogranin, bcl-2, ErbB, and MET throughout the embryonic development of 7-40 gestational week (GW). CK14 was negative. The author examines 42 cases (7-40 GW) of embryonic GI tract (EGI). The HEGISM, HEGIST, and gall bladder smooth muscles (SM) were consistently positive for KIT, CD34, NCAM, PDGFRA, synaptophysin, chromogranin, NSE, bcl-2, ErbB2, and MET in foregut, stomach, GB, midgut, and hindgut throughout the fetal life (7-40 GW). The stem cells (SC) were seen to create the SM, nerves, ICC, and other all structures of GI tract. In adult gastrointestinal walls (n=30), KIT, CD34, PDGFRA, and S100 proteins were expressed in Auerbach's nerve plexus and ICC. The bronchial and vascular SM of embryos did not express these molecules. In GIST, frequent expressions of KIT (100%, 30/30), CD34 (90%, 27/30), and PDGFRA (83%, 25/30) were seen. In general, characteristics of tumors recapitulate their embryonic life. Therefore, it is strongly suggested that GIST may be originated from GI SM and/or GI SC in addition to ICC.

Purpose: It is still unclear whether the longitudinal anal muscles or conjoint longitudinal coats (CLCs) are attached to the vagina, although such an attachment, if present, would appear to make an important contribution to the integrated supportive system of the female pelvic floor., Materials and Methods: Using immunohistochemistry for smooth muscle actin, we examined semiserial frontal sections of 1) eleven female late-stage fetuses at 28-37 weeks of gestation, 2) two female middle-stage fetus (2 specimens at 13 weeks), and, 3) six male fetuses at 12 and 37 weeks as a comparison of the morphology., Results: In late-stage female fetuses, the CLCs consistently (11/11) extended into the subcutaneous tissue along the vaginal vestibule on the anterior side of the external anal sphincter. Lateral to the CLCs, the external anal sphincter also extended anteriorly toward the vaginal side walls. The anterior part of the CLCs originated from the perimysium of the levator ani muscle without any contribution of the rectal longitudinal muscle layer. However, in 2 female middle-stage fetuses, smooth muscles along the vestibulum extended superiorly toward the levetor ani sling. In male fetuses, the CLCs were separated from another subcutaneous smooth muscle along the scrotal raphe (posterior parts of the dartos layer) by fatty tissue., Conclusion: In terms of topographical anatomy, the female anterior CLCs are likely to correspond to the lateral extension of the perineal body (a bulky subcutaneous smooth muscle mass present in adult women), supporting the vaginal vestibule by transmission of force from the levator ani.

Literature on the development of the human vagina is abundant; however, contributions concerning the prenatal development of the entire utero-vaginal anlagen (UVA) are rare or carried out in rodents. The primary epithelial characteristics in the adult vagina and uterus are determined during prenatal development and depend on epithelio-mesenchymal stroma interaction; thus an investigation summarizing the spatiotemporal distribution of relevant molecular markers in the entire human UVA will be of current interest. We phenotyped epithelial and mesenchymal characteristics in sagittal sections from 24 female fetuses of 14-34 weeks of gestation and two female newborns by immunostaining with cytokeratins 8, 13, 14 and 17, p63, bcl-2, bmp4, HOX A13, CD31, VEGF, SMA, Pax2 and vimentin. Epithelial differentiation followed a caudal-to-cranial direction in the UVA. Due to the cytokeratin profile of cytokeratins 8, 13 and 14, the characteristics of the different epithelial zones in the UVA could already be recognized in middle-age fetuses. Vaginal epithelium originated from the urogenital sinus in the lower portion and initiated the transformation of vimentin-positive Müllerian epithelium in the upper vaginal portion. During prenatal development the original squamo-columnar junction was clearly detectable from week 24 onwards and was always found in the cervical canal. Early blc-2 positivity within the surrounding mesenchyme of the entire vagina including the portio region pointed to an organ-specific mesenchymal influence. Prenatal findings in human specimens clearly show that fornix epithelium up to the squamo-columnar junction is of vaginal Müllerian origin, and the cervical epithelium cranial to the squamo-columnar junction is of uterine Müllerian origin and includes cells with enough plasticity to transform into squamous epithelium., (© 2013 The Authors Journal of Anatomy © 2013 Anatomical Society.)

The article presents the results of evaluation of features of the structural organization of microvascular complexes of muscular fascicles and glands in the prostate of men of different ages. Autopsy material of 103 human prostate was used for examination. The data on the structural transformation of glands, muscle cells and connective tissue components in human prostate suggests that at different age periods the growth and differentiation of various structures occur unevenly, constantly changing relationships between components of organ and parts of its circulatory bed. Circulatory bed of prostate is developed and transformed according to the needs of structures perfused. Age involution processes in the prostate and circulatory bed develop irrespective of man's age and are individual in nature. The general trend of involutional changes of prostate histology suggests that the involution begins within paravasal zones. It seems that the reason is increase the hydrodynamic pressure on the venous bed of prostate.