Single continuous lumen formation in the zebrafish gut is mediated by smoothened-dependent tissue remodeling

Abstract

The formation of a single lumen during tubulogenesis is crucial for the development and function of many organs. Although 3D cell culture models have identified molecular mechanisms controlling lumen formation in vitro, their function during vertebrate organogenesis is poorly understood. Using light sheet microscopy and genetic approaches we have investigated single lumen formation in the zebrafish gut. Here we show that during gut development multiple lumens open and enlarge to generate a distinct intermediate, which consists of two adjacent unfused lumens separated by basolateral contacts. We observed that these lumens arise independently from each other along the length of the gut and do not share a continuous apical surface. Resolution of this intermediate into a single, continuous lumen requires the remodeling of contacts between adjacent lumens and subsequent lumen fusion. We show that lumen resolution, but not lumen opening, is impaired in smoothened (smo) mutants, indicating that fluid-driven lumen enlargement and resolution are two distinct processes. Furthermore, we show that smo mutants exhibit perturbations in the Rab11 trafficking pathway and demonstrate that Rab11-mediated trafficking is necessary for single lumen formation. Thus, lumen resolution is a distinct genetically controlled process crucial for single, continuous lumen formation in the zebrafish gut.

P114RhoGEF governs cell motility and lumen formation during tubulogenesis via ROCK-myosin II pathway

“…Jiang et al have described that tubulogenesis of notochord cells involved the remodeling of cell shape and tissue configuration in the absence of cell proliferation (Denker and Jiang, 2012;Dong et al, 2009;Sehring et al, 2014). In the zebrafish gut, lumen fusion into a single, continuous lumen occurs through both luminal membrane expansion and the loss of adhesion at the fusion site (Alvers et al, 2014). We found similarities in both systems in that multiple lumens were consolidated into a single lumen in an anterior-to-posterior manner without apoptosis.…”

“…The lumen in the developing mouse pancreas arises as multiple microlumens, which coalesce into a continuous luminal network (Kesavan et al, 2009). In the zebrafish gut, mutation of MODY5 or the gene tcf2 causes accumulation of multiple small lumens, rather than a single central lumen (Bagnat et al, 2007), and mutations in the protein smoothened cause impaired single lumen resolution (Alvers et al, 2014). In development of the mammalian intestine, multiple secondary lumens form within the stratified epithelium; these eventually fuse with the central lumen.…”

Epigenetic control of intestinal barrier function and inflammation in zebrafish

“…Zebrafish were maintained at 28°C and mated as described (42). The following zebrafish lines were used for this work: AB/TL, EK, Tg(lysC:dsRed)nz50 (21), uhrf1 hi3020 (17, 18) Tg(NFkB:EGFP)nc1 (43), Tg(−4.5ifabp:DsRed)pd1000 (43), TgBAC(anxa2b-RFP)pd1113, TgBAC(cldn15la-GFP)pd1034 (qPCR) (44), TgBAC(tnfa:GFP)pd1028, and aa51.3 pd1092 (this study) and cc39.3 pd1093 (this study).…”

“…Embryo dissociation and FACS was performed as described (44) RNA Isolation and qPCR. RNA isolation and cDNA synthesis for qPCR was performed as described (44). qPCR was performed by using a Roche LightCycler carousel-based system and the Roche LightCycler FastStart DNA Master PLUS SYBR Green I.…”

“…For mouse qPCR experiments, RNA was isolated from epithelial sheets from the mouse proximal, medial and distal small intestine, and the colon, and cDNA was synthesized as previously described (44).…”