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

Abstract

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.

Retinoic acid temporally orchestrates colonization of the gut by vagal neural crest cells

“…Nonetheless, Seiler et al discovered that differentiation of smooth muscle in zebrafish occurred after enteric neuron terminal differentiation (Seiler et al, 2010), suggesting that enteric colonization and differentiation does not depend upon differentiated gut mesenchyme. Corroborating these data, previous studies in amniotes have shown that differentiation of foregut (stomach) mesenchyme depends upon and follows the arrival of enteric neural crest cells (Faure et al, 2016; Bourret et al, 2017); loss of enteric neural crest cells via tissue-specific ablation impairs the Notch signaling pathway in stomach mesenchyme and perturbs smooth muscle development (Faure et al, 2016). Thus, it may be difficult to parse whether effects on the mesenchyme are direct or indirect via alterations to the neural crest.…”

Chick midgut morphogenesis

“…Therefore, there may be compartment-specific dependencies of smooth muscle differentiation on the neural crest. Consistent with this idea, hindgut smooth muscle differentiation occurs prior to the arrival of neural crest cells, whereas the foregut is colonized concomitant with smooth muscle differentiation (Bourret et al , 2017, Faure et al , 2015). It is important to note that the same signaling pathways, Hh and Bmp, that pattern the smooth muscle also pattern the enteric nervous system.…”

“…In the avian intestine, the circumferential layer of muscle can further be subdivided into an inner layer that stains highly for αSMA and a thicker outer layer that has low αSMA expression (Gabella, 2002, Thomason et al , 2012, Yamamoto et al , 1996). While it was classically believed that muscle differentiation proceeds in a proximal-to-distal wave, recent analysis re-evaluates this hypothesis and suggests that a concurrent distal-to-proximal wave of differentiation initiates in the hindgut (Bourret et al , 2017, Graham et al , 2017). Despite the difference in timing, the general pattern of muscle layers is conserved along the length of the gut.…”

PROX1 is a specific and dynamic marker of sacral neural crest cells in the chicken intestine

“…Whole‐mount in situ hybridization was carried out as previously described using antisense SOX10 (Bourret, Chauvet, de Santa Barbara, & Faure, ; Moniot et al, ) and PROX1 riboprobes. For this, E6 GI tract total RNA and primers specific for chick PROX1 (Gene Bank ID: NM_001005616.1; forward: 5′‐TGT AAA GTT CAA CAG ATG CAT TAC C‐3′, reverse: 5′‐ATG TTA AGG GTC TCG GGC AA‐3′) were used to amplify chick PROX1 cDNA.…”

“…The anti‐HNK‐1 antibody (LabVision/Neo‐Markers, CA; mouse monoclonal, cat. #MS‐1163‐P1, ) recognizes a N‐linked carbohydrate (Abo & Balch, ) and is a well‐known avian neural crest marker (Bourret et al, ; Bronner‐Fraser, ; Faure et al, ; Moniot et al, ; Nagy et al, ). The anti‐HUC/D antibody (Invitrogen; mouse monoclonal, clone 16A11, cat.…”