Mutations in zebrafish leucine-rich repeat-containing six-like affect cilia motility and result in pronephric cysts, but have variable effects on left-right patterning.

Cilia defects have been implicated in a variety of human diseases and genetic disorders, but how cilia motility contributes to these phenotypes is still unknown. To further our understanding of how cilia function in development, we have cloned and characterized two alleles of seahorse, a zebrafish mutation that results in pronephric cysts. seahorse encodes Lrrc6l, a leucine-rich repeat-containing protein that is highly conserved in organisms that have motile cilia. seahorse is expressed in zebrafish tissues known to contain motile cilia. Although mutants do not affect cilia structure and retain the ability to interact with Disheveled, both alleles of seahorse strongly affect cilia motility in the zebrafish pronephros and neural tube. Intriguingly, although seahorse mutations variably affect fluid flow in Kupffer's vesicle, they can have very weak effects on left-right patterning. Combined with recently published results, our alleles suggest that the function of seahorse in cilia motility is separable from its function in other cilia-related phenotypes.

Direct and indirect roles for Nodal signaling in two axis conversions during asymmetric morphogenesis of the zebrafish heart.

The Nodal signaling pathway plays a conserved role in determining left-sided identity in vertebrates with this early left-right (L/R) patterning influencing the asymmetric development and placement of visceral organs. We have studied the role of Nodal signaling in asymmetric cardiac morphogenesis in zebrafish and describe two distinct rotations occurring within the heart. The first is driven by an asymmetric migration of myocardial cells during cardiac jogging, resulting in the conversion of the L/R axis to the dorsal-ventral (D/V) axis of the linear heart. This first rotation is directly influenced by the laterality of asymmetric gene expression. The second rotation occurs before cardiac looping and positions the original left cells exposed to Nodal signaling back to the left of the wild-type (WT) heart by 48 hours postfertilization (hpf). The direction of this second rotation is determined by the laterality of cardiac jogging and is not directly influenced by asymmetric gene expression. Finally, we have identified a role for Nodal signaling in biasing the location of the inner ventricular and outer atrial curvature formations. These results suggest that Nodal signaling directs asymmetric cardiac morphogenesis through establishing and subsequently reinforcing laterality information over the course of cardiac development.

SIX2 and BMP4 mutations associate with anomalous kidney development.

Renal hypodysplasia (RHD) is characterized by reduced kidney size and/or maldevelopment of the renal tissue following abnormal organogenesis. Mutations in renal developmental genes have been identified in a subset of affected individuals. Here, we report the first mutations in BMP4 and SIX2 identified in patients with RHD. We detected 3 BMP4 mutations in 5 RHD patients, and 3 SIX2 mutations in 5 different RHD patients. Overexpression assays in zebrafish demonstrated that these mutations affect the function of Bmp4 and Six2 in vivo. Overexpression of zebrafish six2.1 and bmp4 resulted in dorsalization and ventralization, respectively, suggesting opposing roles in mesendoderm formation. When mutant constructs containing the identified human mutations were overexpressed instead, these effects were attenuated. Morpholino knockdown of bmp4 and six2.1 affected glomerulogenesis, suggesting specific roles for these genes in the formation of the pronephros. In summary, these studies implicate conserved roles for Six2 and Bmp4 in the development of the renal system. Defects in these proteins could affect kidney development at multiple stages, leading to the congenital anomalies observed in patients with RHD.