Cell volume changes contribute to epithelial morphogenesis in zebrafish Kupffer's vesicle.

How epithelial cell behaviors are coordinately regulated to sculpt tissue architecture is a fundamental question in biology. Kupffer's vesicle (KV), a transient organ with a fluid-filled lumen, provides a simple system to investigate the interplay between intrinsic cellular mechanisms and external forces during epithelial morphogenesis. Using 3-dimensional (3D) analyses of single cells we identify asymmetric cell volume changes along the anteroposterior axis of KV that coincide with asymmetric cell shape changes. Blocking ion flux prevents these cell volume changes and cell shape changes. Vertex simulations suggest cell shape changes do not depend on lumen expansion. Consistent with this prediction, asymmetric changes in KV cell volume and shape occur normally when KV lumen growth fails due to leaky cell adhesions. These results indicate ion flux mediates cell volume changes that contribute to asymmetric cell shape changes in KV, and that these changes in epithelial morphology are separable from lumen-generated forces.

Mosaic Labeling and 3-Dimensional Morphological Analysis of Single Cells in the Zebrafish Left-right Organizer.

A transient epithelial structure called the left-right organizer (LRO) establishes left-right asymmetry in vertebrate embryos. Developmental defects that alter LRO formation result in left-right patterning errors that often lead to congenital heart malformations. However, little is known about mechanisms that regulate individual cell behaviors during LRO formation. To address this, we developed a Cre-loxP based method to mosaically label precursor cells, called dorsal forerunner cells, that give rise to the zebrafish LRO known as Kupffer's vesicle. This methodology allows lineage tracing, 3-dimensional (3D) reconstruction and morphometric analysis of single LRO cells in living embryos. The ability to visualize and quantify individual LRO cell dynamics provides an opportunity to advance our understanding of LRO development, and in a broader sense, investigate the interplay between intrinsic biochemical mechanisms and extrinsic mechanical forces that drive morphogenesis of epithelial tissues.

Paxillin genes and actomyosin contractility regulate myotome morphogenesis in zebrafish.

Paxillin (Pxn) is a key adapter protein and signaling regulator at sites of cell-extracellular matrix (ECM) adhesion. Here, we investigated the role of Pxn during vertebrate development using the zebrafish embryo as a model system. We have characterized two Pxn genes, pxna and pxnb, in zebrafish that are maternally supplied and expressed in multiple tissues. Gene editing and antisense gene knockdown approaches were used to uncover Pxn functions during zebrafish development. While mutation of either pxna or pxnb alone did not cause gross embryonic phenotypes, double mutants lacking maternally supplied pxna or pxnb displayed defects in cardiovascular, axial, and skeletal muscle development. Transient knockdown of Pxn proteins resulted in similar defects. Irregular myotome shape and ECM composition were observed, suggesting an "inside-out" signaling role for Paxillin genes in the development of myotendinous junctions. Inhibiting non-muscle Myosin-II during somitogenesis altered the subcellular localization of Pxn protein and phenocopied pxn gene loss-of-function. This indicates that Paxillin genes are effectors of actomyosin contractility-driven morphogenesis of trunk musculature in zebrafish. Together, these results reveal new functions for Pxn during muscle development and provide novel genetic models to elucidate Pxn functions.

Evolution and Expression of Paxillin Genes in Teleost Fish.

BACKGROUND: Paxillin family proteins regulate intracellular signaling downstream of extracellular matrix adhesion. Tissue expression patterns and cellular functions of Paxillin proteins during embryo development remain poorly understood. Additionally, the evolution of this gene family has not been thoroughly investigated. RESULTS: This report characterizes the evolution and expression of a novel Paxillin gene, called Paxillin-b, in Teleosts. Alignments indicate that Teleost Paxillin-a and Paxillin-b proteins are highly homologous to each other and to human Paxillin. Phylogenetic and synteny analyses suggest that these genes originated from the duplication of an ancestral Paxillin gene that was in a common ancestor of Teleosts and Tetrapods. Analysis of the spatiotemporal expression profiles of Paxillin-a and Paxillin-b using zebrafish revealed both overlapping and distinct domains for Paxillin-a and Paxillin-b during embryo development. Localization of zebrafish Paxillin orthologs expressed in mammalian cells demonstrated that both proteins localize to focal adhesions, similar to mammalian Paxillin. This suggests these proteins regulate adhesion-dependent processes in their endogenous tissues. CONCLUSION: Paxillin-a and Paxillin-b were generated by duplication in Teleosts. These genes likely play similar roles as Paxillin genes in other organisms. This work provides a framework for functional investigation of Paxillin family members during development using the zebrafish as an in vivo model system.