Embryonic Mouse Gut Research Articles

Calcium wave dynamics in the embryonic mouse gut mesenchyme: impact on smooth muscle differentiation

Intestinal smooth muscle differentiation is a complex physico-biological process involving several different pathways. Here, we investigate the properties of Ca2+ waves in the developing intestinal mesenchyme using GCamp6f expressing mouse embryos and investigate their relationship with smooth muscle differentiation. We find that Ca2+ waves are absent in the pre-differentiation mesenchyme and start propagating immediately following α-SMA expression. Ca2+ waves are abrogated by CaV1.2 and gap-junction blockers, but are independent of the Rho pathway. The myosine light-chain kinase inhibitor ML-7 strongly disorganized or abolished Ca2+ waves, showing that perturbation of the contractile machinery at the myosine level also affected the upstream Ca2+ handling chain. Inhibiting Ca2+ waves and contractility with CaV1.2 blockers did not perturb circular smooth muscle differentiation at early stages. At later stages, CaV1.2 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. CaV1.2 blockers also drove apoptosis of already differentiated, CaV1.2-expressing smooth muscle and enteric neural cells. We provide fundamental new data on Ca2+ waves in the developing murine gut and their relation to myogenesis in this organ.

The Interplay between Nutrition, Innate Immunity, and the Commensal Microbiota in Adaptive Intestinal Morphogenesis.

The gastrointestinal tract is a functionally and anatomically segmented organ that is colonized by microbial communities from birth. While the genetics of mouse gut development is increasingly understood, how nutritional factors and the commensal gut microbiota act in concert to shape tissue organization and morphology of this rapidly renewing organ remains enigmatic. Here, we provide an overview of embryonic mouse gut development, with a focus on the intestinal vasculature and the enteric nervous system. We review how nutrition and the gut microbiota affect the adaptation of cellular and morphologic properties of the intestine, and how these processes are interconnected with innate immunity. Furthermore, we discuss how nutritional and microbial factors impact the renewal and differentiation of the epithelial lineage, influence the adaptation of capillary networks organized in villus structures, and shape the enteric nervous system and the intestinal smooth muscle layers. Intriguingly, the anatomy of the gut shows remarkable flexibility to nutritional and microbial challenges in the adult organism.

Isolation of Enteric Nervous System Progenitor Cells from the Aganglionic Gut of Patients with Hirschsprung's Disease.

Enteric nervous system progenitor cells isolated from postnatal human gut and cultured as neurospheres can then be transplanted into aganglionic gut to restore normal patterns of contractility. These progenitor cells may be of future use to treat patients with Hirschprung’s disease, a congenital condition characterized by hindgut dysmotility due to the lack of enteric nervous system ganglia. Here we demonstrate that progenitor cells can also be isolated from aganglionic gut removed during corrective surgery for Hirschsprung’s disease. Although the enteric nervous system marker calretinin is not expressed in the aganglionic gut region, de novo expression is initiated in cultured neurosphere cells isolated from aganglionic Hirschsprung bowel. Furthermore, expression of the neural markers NOS, VIP and GFAP also increased during culture of aganglionic gut neurospheres which we show can be transplantation into cultured embryonic mouse gut explants to restore a normal frequency of contractility. To determine the origin of the progenitor cells in aganglionic region, we used fluorescence-activated cell sorting to demonstrate that only p75-positive neural crest-derived cells present in the thickened nerve trunks characteristic of the aganglionic region of Hirschsprung gut gave rise to neurons in culture. The derivation of enteric nervous system progenitors in the aganglionic gut region of Hirschprung’s patients not only means that this tissue is a potential source of cells for future autologous transplantation, but it also raises the possibility of inducing the differentiation of these endogenous cells in situ to compensate for the aganglionosis.

Fractional models for the migration of biological cells in complex spatial domains

Travelling wave phenomena are observed in many biological applications. Mathematical theory of standard reaction-diffusion problems shows that simple partial differential equations exhibit travelling wave solutions with constant wavespeed and such models are used to describe, for example, waves of chemical concentrations, electrical signals, cell migration, waves of epidemics and population dynamics. However, as in the study of cell motion in complex spatial geometries, experimental data are often not consistent with constant wavespeed. Non-local spatial models are successfully used to model anomalous diffusion and spatial heterogeneity in different physical contexts. We develop a fractional model based on the Fisher--Kolmogoroff equation, analyse it for its wavespeed properties, and relate the numerical results obtained from our simulations to experimental data describing enteric neural crest-derived cells migrating along the intact gut of mouse embryos. The model proposed essentially combines fractional and standard diffusion in different regions of the spatial domain and qualitatively reproduces the behaviour of neural crest-derived cells observed in the caecum and the hindgut of mouse embryos during in vivo experiments. References R. J. Adler, R. E. Feldman and M. S. Taqqu. A practical guide to heavy tails: Statistical techniques and applications. Birkauser, 1998. I. J. Allan and D. F. Newgreen. The origin and differentiation of enteric neurons of the intestine of the fowl embryo. The American Journal of Anatomy , 157, 137--154, 1980. doi:10.1002/aja.1001570203 B. J. Binder, K. A. Landman, M. J. Simpson, M. Mariani and D. F. Newgreen. Modeling proliferative tissue growth: A general approach and an avian case study. Physical Review E (Statistical, Nonlinear, and Soft Matter Physics) , 78(3), 1--13, 2008. doi:10.1103/PhysRevE.78.031912 M. A. Breau, A. Dahmani, F. Broders--Bondon, J. P. Thiery and S. Dufour. $\beta 1$ integrins are required for the invasion of the caecum and proximal hindgut by enteric neural crest cells. Development , 136, 2791--2801, 2009. doi:10.1242/dev.031419 K. Burrage, N. Hale and D. Kay. An efficient implementation of an implicit FEM scheme for fractional-in-space reaction-diffusion equations. SIAM Journal on Scientific Computing , 34(4), A2145--A2172. doi:10.1137/110847007 N. R. Druckenbrod and M. L. Epstein. The patterns of neural crest advance in the cecum and colon. Developmental Biology , 287, 125--133, 2005. doi:10.1016/j.ydbio.2005.08.040 H. Engler. On the speed of spread for fractional reaction-diffusion equations. International Journal of Differential Equations , 2010, Article ID 315421, 2010. doi:10.1155/2010/315421 M. Ilic, F. Liu, I. Turner and V. Anh. Numerical approximation of a fractional-in-space diffusion equation (II)--with nonhomogeneous boundary conditions. Fractional Calculus and Applied Analysis , 9, 333--349, 2006. http://eprints.qut.edu.au/23835/ P. K. Maini, D. L. S. McElwain and D. Leavesley. Travelling waves in a wound healing assay. Applied Mathematics Letters , 17, 575--580, 2004. doi:10.1016/S0893-9659(04)90128-0 R. McLennan, L. Dyson, K. W. Prather, J. A. Morrison, R. E. Baker, P. K. Maini and P. M. Kulesa. Multiscale mechanisms of cell migration during development: theory and experiment. Development , 139, 2935--2944, 2012. doi:10.1242/dev.081471 J. D. Murray. Mathematical Biology I and II. Springer Verlag, 2003. M. J. Simpson, D. C. Zhang, M. Mariani, K. A. Landman and D. F. Newgreen. Cell proliferation drives neural crest cell invasion of the intestine. Developmental Biology , 302, 553--568, 2007. doi:10.1016/j.ydbio.2006.10.017 H. M. Young, A. J. Bergner, R. B. Anderson, H. Enomoto, J. Milbrandt, D. F. Newgreen and P. M. Whitington. Dynamics of neural crest-derived cell migration in the embryonic mouse gut. Developmental Biology , 270, 455--473, 2004. doi:10.1016/j.ydbio.2004.03.015 P. Zhuang, F. Liu, V. Anh and I. Turner. Numerical methods for the variable-order fractional advection-diffusion equation with a nonlinear source term. SIAM Journal on Numerical Analysis , 47(3), 1760--1781, 2009. doi:10.1137/080730597

Prospective Identification and Isolation of Enteric Nervous System Progenitors Using Sox2

The capacity to identify and isolate lineage-specific progenitor cells from developing and mature tissues would enable the development of cell replacement therapies for disease treatment. The enteric nervous system (ENS) regulates important gut functions, including controlling peristaltic muscular contractions, and consists of interconnected ganglia containing neurons and glial cells. Hirschsprung's disease (HSCR), one of the most common and best understood diseases affecting the ENS, is characterized by absence of enteric ganglia from the distal gut due to defects in gut colonization by neural crest progenitor cells and is an excellent candidate for future cell replacement therapies. Our previous microarray experiments identified the neural progenitor and stem cell marker SRY-related homoebox transcription factor 2 (Sox2) as expressed in the embryonic ENS. We now show that Sox2 is expressed in the ENS from embryonic to adult stages and constitutes a novel marker of ENS progenitor cells and their glial cell derivatives. We also show that Sox2 expression overlaps significantly with SOX10, a well-established marker of ENS progenitors and enteric glial cells. We have developed a strategy to select cells expressing Sox2, by using G418 selection on cultured gut cells derived from Sox2βgeo/+ mouse embryos, thus allowing substantial enrichment and expansion of neomycin-resistant Sox2-expressing cells. Sox2βgeo cell cultures are enriched for ENS progenitors. Following transplantation into embryonic mouse gut, Sox2βgeo cells migrate, differentiate, and colocalize with the endogenous ENS plexus. Our studies will facilitate development of cell replacement strategies in animal models, critical to develop human cell replacement therapies for HSCR. Stem Cells 2011;29:128–140

S3.5 Induction of neural crest stem cells from human ES-derived neural progenitor cells: Migration and differentiation in avian embryos and explants of embryonic mouse gut

S3.5 Induction of neural crest stem cells from human ES-derived neural progenitor cells: Migration and differentiation in avian embryos and explants of embryonic mouse gut

Effects of pharmacological inhibition of small GTPases on axon extension and migration of enteric neural crest-derived cells

In the developing enteric nervous system, there is a close association between migrating neural crest-derived cells and the axons of early differentiating neurons. We used pharmacological inhibitors of small GTPases to determine if crest cell migration and axon growth could be uncoupled in cultured intact explants of embryonic mouse gut and slices of embryonic gut grown on collagen gels containing GDNF. Inhibition of the Rho effectors, ROCKI/II, or Rac/Cdc42 inhibited both cell migration and neurite growth in intact explants of embryonic gut. The effects of both ROCKI/II and Rac/Cdc42 inhibitors were more severe on cell migration and axon extension in gut explants from Ret +/− mice than in explants from wildtype mice, indicating that Rho GTPases probably act downstream of the receptor tyrosine kinase, Ret. Inhibition of ROCKI/II had different effects on migration and axon extension in gut slices grown on collagen gels containing GDNF from that seen in intact explants of gut. We conclude that ROCKI/II and Rac/Cdc42 are required for both neural crest-derived cell migration and axon growth in the developing gut.

The Cell Adhesion Molecule L1 Is Required for Chain Migration of Neural Crest Cells in the Developing Mouse Gut

During development, the enteric nervous system is derived from neural crest cells that emigrate from the hindbrain, enter the foregut, and colonize the gut. Defects in neural crest migration can result in intestinal aganglionosis. Hirschsprung's disease (congenital aganglionosis) is a human condition in which enteric neurons are absent from the distal bowel. A number of clinical studies have implicated the cell adhesion molecule L1 in Hirschsprung's disease. We examined the role of L1 in the migration of neural crest cells through the developing mouse gut. A variety of in vitro and in vivo assays were used to examine: (1) the effect of L1 blocking antibodies or exogenous soluble L1 protein known to compromise L1 function on the rate of crest cell migration, (2) the effect of blocking L1 activity on the dynamic behavior of crest cells using time-lapse microscopy, and (3) whether the colonization of the gut by crest cells in L1-deficient mice differs from control mice. We show that L1 is expressed by neural crest cells as they colonize the gut. Perturbation studies show that disrupting L1 activity retards neural crest migration and increases the number of solitary neural crest cells. L1-deficient mice show a small but significant reduction in neural crest cell migration at early developmental stages, but the entire gastrointestinal tract is colonized. L1 is important for the migration of neural crest cells through the developing gut and is likely to be involved in the etiology of Hirschsprung's disease.

Dynamics of neural crest-derived cell migration in the embryonic mouse gut

Neural crest-derived cells that form the enteric nervous system undergo an extensive migration from the caudal hindbrain to colonize the entire gastrointestinal tract. Mice in which the expression of GFP is under the control of the Ret promoter were used to visualize neural crest-derived cell migration in the embryonic mouse gut in organ culture. Time-lapse imaging revealed that GFP + crest-derived cells formed chains that displayed complicated patterns of migration, with sudden and frequent changes in migratory speed and trajectories. Some of the leading cells and their processes formed a scaffold along which later cells migrated. To examine the effect of population size on migratory behavior, a small number of the most caudal GFP + cells were isolated from the remainder of the population. The isolated cells migrated slower than cells in large control populations, suggesting that migratory behavior is influenced by cell number and cell–cell contact. Previous studies have shown that neurons differentiate among the migrating cell population, but it is unclear whether they migrate. The phenotype of migrating cells was examined. Migrating cells expressed the neural crest cell marker, Sox10, but not neuronal markers, indicating that the majority of migratory cells observed did not have a neuronal phenotype.

Analysis of the effects of endothelin-3 on the development of neural crest cells in the embryonic mouse gut

Background/Purpose: Mutations in the endothelin-3 (ET-3) and endothelin-B receptor (EDNR-B) genes cause terminal colonic aganglionosis in mice and are linked to Hirschsprung’s disease. These experiments are designed to determine if the development of terminal enteric ganglia depends on changes in proliferation, apoptosis, or differentiation of enteric neural crest (NC) cells in response to ET-3. Methods: Gut from embryonic lethal-spotted mice (lacking ET-3) and controls were investigated in vivo. NC-derived cells were identified immunohistochemically and their proliferation, apoptosis and differentiation monitored by bromodeoxyuridine incorporation, the terminal deoxytransferase poly dU nick end labelling (TUNEL) reaction, and appearance of neuronal nitric oxide synthase (NOS), respectively. Results: No differences in apoptosis or proliferation of NC cells were apparent between lethal-spotted embryos and controls. Although no temporal differences in the differentiation of NOS neurones were evident, these cells appeared more cranially in the gut in the absence of ET-3 than in controls. Conclusions: ET-3 has no detectable influence on proliferation, apoptosis, or timing of differentiation of NC-derived cells in the gut. However, the more proximal location of differentiated neurones in the absence of ET-3 is consistent with a restricted role in migration of NC-derived cells.

How to approach the ENS: various ways to analyse motility disorders in situ and in vitro.

Motility disorders of the human intestine are so variable that they cannot be diagnosed by just one technique. Their aetiology is obviously so varied that they have to be approached with a broad range of technical methods. These reach from the simple haematoxylin-stained section to the isolation of stem or precursor cells. In this study, various methods to investigate the enteric nervous system and its surrounding tissue are demonstrated. While sections from paraffin-embedded material or cryostat sections provide only a two-dimensional perspective of the ENS, the whole-mount method yields three-dimensional perspectives of large areas of the gut wall. The three-dimensional impression can even be enhanced by electron microscopy of the isolated ENS. Dynamical aspects of ENS development can be tackled by in vitro studies. The myenteric plexus can be isolated and cultivated under the influence of the microenvironment (protein extracts). Although the postnatal myenteric plexus is not fully developed, the choice of embryological neuronal cells seems to be more effective for certain approaches. They can be isolated from the embryonic mouse gut and cultivated under the influence of various factors. This method seems to us a valuable tool for the investigation of the aetiology of motility disorders, although only a "complete" approach which considers all available methods will yield at the end a clear understanding which might lead to new therapeutical concepts.

Enteric neural crest‐derived cells: Origin, identification, migration, and differentiation

Enteric neural crest‐derived cells: Origin, identification, migration, and differentiation

EGF regulates early embryonic mouse gut development in chemically defined organ culture.

The profound intestinal epithelial defects in the newborn epidermal growth factor receptor (EGFR) knockout mouse suggests that EGFR signaling plays important roles in embryonic gut development. Herein, we further elucidated the function of EGFR signaling on early embryonic gut development by comparing the effects of 1-10 ng/mL of exogenous epidermal growth factor (EGF) or 10-25 microM of the tyrphostin 3,4,5 trihydroxybenzene malononitrile, a specific inhibitor of EGFR tyrosine kinase, on intact E12 Swiss-Webster mouse midgut grown in chemically defined organ culture using Fitton-Jackson BGJb medium for 4 or 6 d. Intestinal development during culture was assayed by morphometry, histology, reverse transcription/competitive PCR for villin and intestinal fatty acid binding protein mRNA, and immunohistochemistry for epithelial proliferative markers. During organ culture, control specimens grew in length, developed smooth muscle, simple columnar epithelial and goblet cell phenotypes, showed early villus formation in the proximal intestine, and increased expression of villin and intestinal fatty acid binding protein mRNA. EGF failed to significantly alter small intestinal lengthening, whereas EGF 10 ng/mL inhibited colonic length growth. Tyrphostin 25 microM resulted in regional losses of stromal and smooth muscle cells in the small intestine and absent colonic goblet cells. In controls, cellular proliferation initially occurred throughout the small intestinal epithelium but became increasingly localized to the intervillus crypt regions. This sequestration of epithelial proliferation into crypts was much more apparent in EGF-treated versus tyrphostin-treated specimens. EGFR activation, therefore, appears to accelerate the maturation rate of goblet cells and the differential crypt/villus proliferation pattern in early embryonic mouse gut.

Expression of Ret-, p75(NTR)-, Phox2a-, Phox2b-, and tyrosine hydroxylase-immunoreactivity by undifferentiated neural crest-derived cells and different classes of enteric neurons in the embryonic mouse gut.

Cells of the enteric nervous system are derived from the neural crest. Probes to a number of molecules identify neural crest-derived cells within the gastrointestinal tract of embryonic mice prior to their differentiation into neurons and glial cells. However, it is unclear whether the different markers are identifying all neural crest-derived cells. In this study the distribution of p75(NTR)-immunoreactivity was compared with that of Ret-, Phox2a-, Phox2b-, and tyrosine hydroxylase (TH) in undifferentiated neural crest-derived cells in the E10.5-E13.5 mouse intestine. Neural crest-derived cells colonise the embryonic mouse gut in a rostral-to-caudal wave between E9.5-E14, and differentiation into enteric neurons also occurs in a rostral-to-caudal wave. Thus, the most caudal neural crest-derived cells within the gut are undifferentiated. These most caudal neural crest-derived cells co-expressed p75(NTR)-, Phox2b- and Ret-immunoreactivity; at E10.5 a sub-population was also TH-positive. The most caudal cells did not show Phox2a-immunoreactivity at any stage. However, a sub-population of cells, which was rostral to the undifferentiated neural crest-derived cells, was Phox2a-positive, and these are likely to be cells beginning to differentiate along a neuronal lineage. The expression of Ret-, Phox2a-, Phox2b- and p75(NTR)-immunoreactivity by two classes of enteric neurons that differentiate prior to birth was also examined. Nitric oxide synthase (NOS) neurons showed Phox2b and Ret immunoreactivity at all ages, and Phox2a and p75(NTR) immunoreactivity only transiently. Calcitonin gene-related peptide (CGRP) neurons showed Phox2b and Ret-immunoreactivity, but not Phox2a immunoreactivity. It is concluded that all undifferentiated neural crest-derived cells initially express Phox2b, Ret, and p75(NTR); a sub-population of these cells also expresses TH transiently. Those cells that are beginning to differentiate along a neuronal lineage maintain their expression of Phox2b and Ret, and they start to express Phox2a, but down-regulate p75(NTR); those cells that differentiate along a glial lineage down-regulate Ret and maintain their expression of p75(NTR). Dev Dyn 1999;216:137-152.