Nucleoporin NUP205 plays a critical role in cilia and congenital disease.

Ciliopathies affect a variety of tissues during development including the heart, kidneys, respiratory tract, and retina. Though an increasing number of monogenic causes of ciliopathies have been described, many remain unexplained. Recently, recessive variants in NUP93 and NUP205 encoding two proteins of the inner ring of the nuclear pore complex were implicated as causes of steroid resistant nephrotic syndrome. In addition, we previously found that the inner ring nucleoporins NUP93 and NUP188 function in proper left-right patterning in developing embryos via a role at the cilium. Here, we describe the role of an additional inner ring nucleoporin NUP205 in cilia biology and establishment of normal organ situs. Using knockdown in Xenopus, we show that Nup205 depletion results in loss of cilia and abnormal cardiac morphology. Furthermore, by transmission electron microscopy, we observe a loss of cilia and mispositioning of intracellular ciliary structures such as basal bodies and rootlets upon depleting inner ring nucleoporins. We describe a model wherein NUP93 interacting with either NUP188 or NUP205 is necessary for cilia. We thus provide evidence that dysregulation of inner ring nucleoporin genes that have been identified in patients may contribute to pathogenesis through cilia dysfunction.

CRISPR/Cas9: An inexpensive, efficient loss of function tool to screen human disease genes in Xenopus.

Congenital malformations are the major cause of infant mortality in the US and Europe. Due to rapid advances in human genomics, we can now efficiently identify sequence variants that may cause disease in these patients. However, establishing disease causality remains a challenge. Additionally, in the case of congenital heart disease, many of the identified candidate genes are either novel to embryonic development or have no known function. Therefore, there is a pressing need to develop inexpensive and efficient technologies to screen these candidate genes for disease phenocopy in model systems and to perform functional studies to uncover their role in development. For this purpose, we sought to test F0 CRISPR based gene editing as a loss of function strategy for disease phenocopy in the frog model organism, Xenopus tropicalis. We demonstrate that the CRISPR/Cas9 system can efficiently modify both alleles in the F0 generation within a few hours post fertilization, recapitulating even early disease phenotypes that are highly similar to knockdowns from morpholino oligos (MOs) in nearly all cases tested. We find that injecting Cas9 protein is dramatically more efficacious and less toxic than cas9 mRNA. We conclude that CRISPR based F0 gene modification in X. tropicalis is efficient and cost effective and readily recapitulates disease and MO phenotypes.

Exon capture and bulk segregant analysis: rapid discovery of causative mutations using high-throughput sequencing.

BACKGROUND: Exome sequencing has transformed human genetic analysis and may do the same for other vertebrate model systems. However, a major challenge is sifting through the large number of sequence variants to identify the causative mutation for a given phenotype. In models like Xenopus tropicalis, an incomplete and occasionally incorrect genome assembly compounds this problem. To facilitate cloning of X. tropicalis mutants identified in forward genetic screens, we sought to combine bulk segregant analysis and exome sequencing into a single step. RESULTS: Here we report the first use of exon capture sequencing to identify mutations in a non-mammalian, vertebrate model. We demonstrate that bulk segregant analysis coupled with exon capture sequencing is not only able to identify causative mutations but can also generate linkage information, facilitate the assembly of scaffolds, identify misassembles, and discover thousands of SNPs for fine mapping. CONCLUSION: Exon capture sequencing and bulk segregant analysis is a rapid, inexpensive method to clone mutants identified in forward genetic screens. With sufficient meioses, this method can be generalized to any model system with a genome assembly, polished or unpolished, and in the latter case, it also provides many critical genomic resources.

A novel approach to quantifying ciliary physiology: microfluidic mixing driven by a ciliated biological surface.

From the lungs to the central nervous system, cilia-driven fluid flow plays a fundamental role in many facets of life. Yet, there are few quantitative methods for analysing the function of ciliated surfaces. Here, we report a novel microfluidic approach for quantifying the performance of a ciliated surface using mixing performance as an integrated readout.

Microfluidic characterization of cilia-driven fluid flow using optical coherence tomography-based particle tracking velocimetry.

Motile cilia are cellular organelles that generate directional fluid flow across various epithelial surfaces including the embryonic node and respiratory mucosa. The proper functioning of cilia is necessary for normal embryo development and, for the respiratory system, the clearance of mucus and potentially harmful particulate matter. Here we show that optical coherence tomography (OCT) is well-suited for quantitatively characterizing the microfluidic-scale flow generated by motile cilia. Our imaging focuses on the ciliated epithelium of Xenopus tropicalis embryos, a genetically manipulable and experimentally tractable animal model of human disease. We show qualitative flow profile characterization using OCT-based particle pathline imaging. We show quantitative, two-dimensional, two-component flow velocity field characterization using OCT-based particle tracking velocimetry. Quantitative imaging and phenotyping of cilia-driven fluid flow using OCT will enable more detailed research in ciliary biology and in respiratory medicine.

Rapid gynogenetic mapping of Xenopus tropicalis mutations to chromosomes.

Pilot forward genetic screens in Xenopus tropicalis have isolated over 60 recessive mutations. Here we present a simple method for mapping mutations to chromosomes using gynogenesis and centromeric markers. When coupled with available genomic resources, gross mapping facilitates evaluation of candidate genes as well as higher resolution linkage studies. Using gynogenesis, we have mapped the genetic locations of the 10 X. tropicalis centromeres, and performed fluorescence in situ hybridization to validate these locations cytologically. We demonstrate the use of this very small set of centromeric markers to map mutations efficiently to specific chromosomes. Developmental Dynamics 238:1398-1406, 2009. (c) 2009 Wiley-Liss, Inc.