Control of the collective migration of enteric neural crest cells by the Complement anaphylatoxin C3a and N-cadherin.

We analyzed the cellular and molecular mechanisms governing the adhesive and migratory behavior of enteric neural crest cells (ENCCs) during their collective migration within the developing mouse gut. We aimed to decipher the role of the complement anaphylatoxin C3a during this process, because this well-known immune system attractant has been implicated in cephalic NCC co-attraction, a process controlling directional migration. We used the conditional Ht-PA-cre transgenic mouse model allowing a specific ablation of the N-cadherin gene and the expression of a fluorescent reporter in migratory ENCCs without affecting the central nervous system. We performed time-lapse videomicroscopy of ENCCs from control and N-cadherin mutant gut explants cultured on fibronectin (FN) and micropatterned FN-stripes with C3a or C3aR antagonist, and studied cell migration behavior with the use of triangulation analysis to quantify cell dispersion. We performed ex vivo gut cultures with or without C3aR antagonist to determine the effect on ENCC behavior. Confocal microscopy was used to analyze the cell-matrix adhesion properties. We provide the first demonstration of the localization of the complement anaphylatoxin C3a and its receptor on ENCCs during their migration in the embryonic gut. C3aR receptor inhibition alters ENCC adhesion and migration, perturbing directionality and increasing cell dispersion both in vitro and ex vivo. N-cadherin-null ENCCs do not respond to C3a co-attraction. These findings indicate that C3a regulates cell migration in a N-cadherin-dependent process. Our results shed light on the role of C3a in regulating collective and directional cell migration, and in ganglia network organization during enteric nervous system ontogenesis. The detection of an immune system chemokine in ENCCs during ENS development may also shed light on new mechanisms for gastrointestinal disorders.

Sox10 and Itgb1 interaction in enteric neural crest cell migration.

SOX10 involvement in syndromic form of Hirschsprung disease (intestinal aganglionosis, HSCR) in humans as well as developmental defects in animal models highlight the importance of this transcription factor in control of the pool of enteric progenitors and their differentiation. Here, we characterized the role of SOX10 in cell migration and its interactions with ß1-integrins. To this end, we crossed the Sox10(lacZ/+) mice with the conditional Ht-PA::Cre; beta1(neo/+) and beta1(fl/fl) mice and compared the phenotype of embryos of different genotypes during enteric nervous system (ENS) development. The Sox10(lacZ/+); Ht-PA::Cre; beta1(neo/fl) double mutant embryos presented with increased intestinal aganglionosis length and more severe neuronal network disorganization compared to single mutants. These defects, detected by E11.5, are not compensated after birth, showing that a coordinated and balanced interaction between these two genes is required for normal ENS development. Use of video-microscopy revealed that defects observed result from reduced migration speed and altered directionality of enteric neural crest cells. Expression of ß1-integrins upon SOX10 overexpression or in Sox10(lacZ/+) mice was also analyzed. The modulation of SOX10 expression altered ß1-integrins, suggesting that SOX10 levels are critical for proper expression and function of this adhesion molecule. Together with previous studies, our results strongly indicate that SOX10 mediates ENCC adhesion and migration, and contribute to the understanding of the molecular and cellular basis of ENS defects observed both in mutant mouse models and in patients carrying SOX10 mutations.

N-cadherin and ß1-integrins cooperate during the development of the enteric nervous system.

Cell adhesion controls various embryonic morphogenetic processes, including the development of the enteric nervous system (ENS). Ablation of ß1-integrin (ß1-/-) expression in enteric neural crest cells (ENCC) in mice leads to major alterations in the ENS structure caused by reduced migration and increased aggregation properties of ENCC during gut colonization, which gives rise to a Hirschsprung's disease-like phenotype. In the present study, we examined the role of N-cadherin in ENS development and the interplay with ß1 integrins during this process. The Ht-PA-Cre mouse model was used to target gene disruption of N-cadherin and ß1 integrin in migratory NCC and to produce single- and double-conditional mutants for these two types of adhesion receptors. Double mutation of N-cadherin and ß1 integrin led to embryonic lethality with severe defects in ENS development. N-cadherin-null (Ncad-/-) ENCC exhibited a delayed colonization in the developing gut at E12.5, although this was to a lesser extent than in ß1-/- mutants. This delay of Ncad-/- ENCC migration was recovered at later stages of development. The double Ncad-/-; ß1-/- mutant ENCC failed to colonize the distal part of the gut and there was more severe aganglionosis in the proximal hindgut than in the single mutants for N-cadherin or ß1-integrin. This was due to an altered speed of locomotion and directionality in the gut wall. The abnormal aggregation defect of ENCC and the disorganized ganglia network in the ß1-/- mutant was not observed in the double mutant. This indicates that N-cadherin enhances the effect of the ß1-integrin mutation and demonstrates cooperation between these two adhesion receptors during ENS ontogenesis. In conclusion, our data reveal that N-cadherin is not essential for ENS development but it does modulate the modes of ENCC migration and acts in concert with ß1-integrin to control the proper development of the ENS.

Beta1 integrins are required for the invasion of the caecum and proximal hindgut by enteric neural crest cells.

Integrins are the major adhesive receptors for extracellular matrix and have various roles in development. To determine their role in cell migration, the gene encoding the beta1 integrin subunit (Itgb1) was conditionally deleted in mouse neural crest cells just after their emigration from the neural tube. We previously identified a major defect in gut colonisation by conditional Itgb1-null enteric neural crest cells (ENCCs) resulting from their impaired migratory abilities and enhanced aggregation properties. Here, we show that the migration defect occurs primarily during the invasion of the caecum, when Itgb1-null ENCCs stop their normal progression before invading the caecum and proximal hindgut by becoming abnormally aggregated. We found that the caecum and proximal hindgut express high levels of fibronectin and tenascin-C, two well-known ligands of integrins. In vitro, tenascin-C and fibronectin have opposite effects on ENCCs, with tenascin-C decreasing migration and adhesion and fibronectin strongly promoting them. Itgb1-null ENCCs exhibited an enhanced response to the inhibitory effect of tenascin-C, whereas they were insensitive to the stimulatory effect of fibronectin. These findings suggest that beta1 integrins are required to overcome the tenascin-C-mediated inhibition of migration within the caecum and proximal hindgut and to enhance fibronectin-dependent migration in these regions.

Mechano-biochemical marine stimulation of inversion, gastrulation, and endomesoderm specification in multicellular Eukaryota.

The evolutionary emergence of the primitive gut in Metazoa is one of the decisive events that conditioned the major evolutionary transition, leading to the origin of animal development. It is thought to have been induced by the specification of the endomesoderm (EM) into the multicellular tissue and its invagination (i.e., gastrulation). However, the biochemical signals underlying the evolutionary emergence of EM specification and gastrulation remain unknown. Herein, we find that hydrodynamic mechanical strains, reminiscent of soft marine flow, trigger active tissue invagination/gastrulation or curvature reversal via a Myo-II-dependent mechanotransductive process in both the metazoan Nematostella vectensis (cnidaria) and the multicellular choanoflagellate Choanoeca flexa. In the latter, our data suggest that the curvature reversal is associated with a sensory-behavioral feeding response. Additionally, like in bilaterian animals, gastrulation in the cnidarian Nematostella vectensis is shown to participate in the biochemical specification of the EM through mechanical activation of the ß-catenin pathway via the phosphorylation of Y654-ßcatenin. Choanoflagellates are considered the closest living relative to metazoans, and the common ancestor of choanoflagellates and metazoans dates back at least 700 million years. Therefore, the present findings using these evolutionarily distant species suggest that the primitive emergence of the gut in Metazoa may have been initiated in response to marine mechanical stress already in multicellular pre-Metazoa. Then, the evolutionary transition may have been achieved by specifying the EM via a mechanosensitive Y654-ßcatenin dependent mechanism, which appeared during early Metazoa evolution and is specifically conserved in all animals.

Ret kinase-mediated mechanical induction of colon stem cells by tumor growth pressure stimulates cancer progression in vivo.

How mechanical stress actively impacts the physiology and pathophysiology of cells and tissues is little investigated in vivo. The colon is constantly submitted to multi-frequency spontaneous pulsatile mechanical waves, which highest frequency functions, of 2 s period, remain poorly understood. Here we find in vivo that high frequency pulsatile mechanical stresses maintain the physiological level of mice colon stem cells (SC) through the mechanosensitive Ret kinase. When permanently stimulated by a magnetic mimicking-tumor growth analogue pressure, we find that SC levels pathologically increase and undergo mechanically induced hyperproliferation and tumorigenic transformation. To mimic the high frequency pulsatile mechanical waves, we used a generator of pulsed magnetic force stimulation in colonic tissues pre-magnetized with ultra-magnetic liposomes. We observed the pulsatile stresses using last generation ultra-wave dynamical high-resolution imaging. Finally, we find that the specific pharmacological inhibition of Ret mechanical activation induces the regression of spontaneous formation of SC, of CSC markers, and of spontaneous sporadic tumorigenesis in Apc mutated mice colons. Consistently, in human colon cancer tissues, Ret activation in epithelial cells increases with tumor grade, and partially decreases in leaking invasive carcinoma. High frequency pulsatile physiological mechanical stresses thus constitute a new niche that Ret-dependently fuels mice colon physiological SC level. This process is pathologically over-activated in the presence of permanent pressure due to the growth of tumors initiated by pre-existing genetic alteration, leading to mechanotransductive self-enhanced tumor progression in vivo, and repressed by pharmacological inhibition of Ret.

Mechanotransduction in tumor progression: The dark side of the force.

Cancer has been characterized as a genetic disease, associated with mutations that cause pathological alterations of the cell cycle, adhesion, or invasive motility. Recently, the importance of the anomalous mechanical properties of tumor tissues, which activate tumorigenic biochemical pathways, has become apparent. This mechanical induction in tumors appears to consist of the destabilization of adult tissue homeostasis as a result of the reactivation of embryonic developmental mechanosensitive pathways in response to pathological mechanical strains. These strains occur in many forms, for example, hypervascularization in late tumors leads to high static hydrodynamic pressure that can promote malignant progression through hypoxia or anomalous interstitial liquid and blood flow. The high stiffness of tumors directly induces the mechanical activation of biochemical pathways enhancing the cell cycle, epithelial-mesenchymal transition, and cell motility. Furthermore, increases in solid-stress pressure associated with cell hyperproliferation activate tumorigenic pathways in the healthy epithelial cells compressed by the neighboring tumor. The underlying molecular mechanisms of the translation of a mechanical signal into a tumor inducing biochemical signal are based on mechanically induced protein conformational changes that activate classical tumorigenic signaling pathways. Understanding these mechanisms will be important for the development of innovative treatments to target such mechanical anomalies in cancer.

Endothelin-3 stimulates cell adhesion and cooperates with ß1-integrins during enteric nervous system ontogenesis.

Endothelin-3 (EDN3) and ß1-integrins are required for the colonization of the embryonic gut by enteric neural crest cells (ENCCs) to form the enteric nervous system (ENS). ß1-integrin-null ENCCs exhibit migratory defects in a region of the gut enriched in EDN3 and in specific extracellular matrix (ECM) proteins. We investigated the putative role of EDN3 on ENCC adhesion properties and its functional interaction with ß1-integrins during ENS development. We show that EDN3 stimulates ENCC adhesion to various ECM components in vitro. It induces rapid changes in ENCC shape and protrusion dynamics favouring sustained growth and stabilization of lamellipodia, a process coincident with the increase in the number of focal adhesions and activated ß1-integrins. In vivo studies and ex-vivo live imaging revealed that double mutants for Itgb1 and Edn3 displayed a more severe enteric phenotype than either of the single mutants demonstrated by alteration of the ENS network due to severe migratory defects of mutant ENCCs taking place early during the ENS development. Altogether, our results highlight the interplay between the EDN3 and ß1-integrin signalling pathways during ENS ontogenesis and the role of EDN3 in ENCC adhesion.

Wnt11r is required for cranial neural crest migration.

wnt11r is a recently identified member of the Wnt family of genes, which has been proposed to be the true Xenopus homologue to the mammalian wnt11 gene. In this study we have examined the role of wnt11r on neural crest development. Expression analysis of wnt11r and comparison with the neural crest marker snail2 and the noncanonical Wnt, wnt11, shows wnt11r is expressed at the medial or neural plate side of the neural crest while wnt11 is expressed at the lateral or epidermal side. Injection of wnt11r morpholino leads to strong inhibition of neural crest migration with no effect on neural crest induction or maintenance. This effect can be rescued by co-injection of Wnt11r but not by Wnt11 mRNA, demonstrating the specificity of the loss of function treatment. Finally, neural crest graft experiments show that wnt11r is required in a non-cell-autonomous manner to control neural crest migration.

Regulation of XSnail2 expression by Rho GTPases.

We analyzed the effects of Rho GTPases on XSnail2 expression during neural crest (NC) ontogeny in Xenopus laevis embryos. The ectopic expression of both dominant-negative (N-) and constitutively active (V-) Rho GTPase mutants after RNA or DNA microinjection disrupted the endogenous expression of XSnail2, XFoxD3, and XSnail1. V14RhoA and N17Rac1 were inhibitory, whereas N19RhoA and V12Rac1 increased NC marker gene expression. In reporter assays using a XSnail2 promoter-green fluorescent protein (GFP) construct (alpha700BA-GFP), the ectopic expression of V14RhoA, N17Rac1, or the Rac1 inhibitor NSC 23766 decreased reporter expression in NC-neural plate, whereas N19RhoA or the RhoA inhibitor Y27632 and V12Rac1 enhanced it. Similarly, transgenic embryos expressing Rho GTPase mutants and GFP under control of the alpha700BA promoter displayed variations similar to those observed for ectopic RNA and DNA expression. These results show that Rho GTPases can regulate the expression of XSnail2 during NC ontogeny.