Congress Abstracts - Society for Developmental Biology

activity may determine the orientation of tissue structures. Indeed, chimeric analysis, which would disrupt these gradients, affects
medio-lateral migration. This clearly demonstrates that Fat-PCP signalling is conserved in vertebrates and also implicates for the first
time gradients of Fat4/Dchs1 activity in the establishment of PCP. This data also define Fat-PCP as novel neuronal guidance cues and
show a novel system where Fz-PCP and Fat-PCP determine the polarisation of tissue structures along orthogonal axes.
Program/Abstract # 13
Systematic discovery of novel cilia and ciliopathy genes through functional genomics in the zebrafish
Semil P. Choksi, Deepak Babu (IMCB, Singapore); Malgorzata Szczepaniak, Rim Hjeij (Univ. Hosp. Muenster, Germany); Doreen
Lau, Xianwen Yu (IMCB, Singapore); Petra Pennekamp, Claudius Werner, Niki T. Loges (Univ. Hosp. Muenster, Germany); Karsten
Häffner (Univ. Hosp. Freiburg, Germany); Shunzhen Chen, Kangli Noel Wong (IMCB, Singapore); Gerard Dougherty (Univ. Hosp.
Muenster, Germany); Ronald Roepman (Radboud Univ., Netherlands); Heymut Omran (Univ. Hosp. Muenster, Germany); Sudipto
Roy (IMCB, Singapore)
Cilia are microtubule-based hair-like organelles that play many important roles in animal development and physiology. Ciliary
dysfunction is implicated in a rapidly expanding spectrum of diseases, the ciliopathies. Primary ciliary dyskinesia (PCD), one of the
most prevalent ciliopathies, is a genetically heterogeneous disease that arises from faulty motile cilia. As causative mutations in
relatively few genes have been described, we sought to systematically identify new ciliopathy genes using functional genomics
coupled with rapid phenotype screening in the zebrafish embryo. With this approach, we identified hundreds of cilia genes, a majority
of which have never been associated with cilia before. We selected 50 genes at random and, remarkably, found that more than 60%
are required for ciliary differentiation or function. In addition, we find that proteins encoded by 14 of the genes localize to motile cilia.
By sequencing a cohort of PCD patients for mutations in these genes, we show that several of the novel candidates, including ZBBX
and MNS1, are mutated in patients suffering from PCD. We find that these genes affect ciliary motility by disrupting the nexin-dynein
regulatory complex and the outer dynein arm assembly, respectively. This large collection of functionally-validated motile cilia genes
will be invaluable for the study of ciliary biology, and in the identification of new mutations causing ciliopathies like PCD.
Program/Abstract # 14
Sox10 regulates enteric neural crest cell migration in the developing gut
Mai Har Sham, Carly Leung, Mei Zhang, Hon Man Sit (U Hong Kong, China)
Sox10 is a HMG-domain containing transcription factor which plays important roles in neural crest cell survival and differentiation.
Mutations of Sox10 have been identified in patients with Waardenburg-Hirschsprung syndrome, who suffer from deafness,
pigmentation defects and intestinal aganglionosis. Enteric neural crest cells (ENCCs) with Sox10 mutation undergo premature
differentiation and fail to colonize the distal hindgut. It is unclear, however, whether Sox10 plays a role in the migration of ENCCs. To
visualize the migration behaviour of mutant ENCCs, we generated a Sox10 NGFP mouse model where EGFP is fused to the N-terminal
domain of Sox10. Using time-lapse imaging, we found that ENCCs in Sox10 NGFP/+ mutants displays lower migration speed and altered
trajectories compared to normal controls. This behaviour was cell-autonomous, as shown by organotypic grafting of Sox10 NGFP/+ gut
segments onto control guts and vice versa. ENCCs encounter different extracellular matrix (ECM) molecules along the developing
gut. We performed gut explant culture on various ECM and found that Sox10 NGFP/+ ENCCs tend to form aggregates, particularly on
fibronectin. Time-lapse imaging of single cells in gut explant culture indicated that the tightly-packed Sox10 mutant cells failed to
exhibit contact inhibition of locomotion. We determined the expression of adhesion molecule families by qPCR analysis, and found
integrin expression unaffected while L1-cam and selected cadherins were altered, suggesting that Sox10 mutation affects cell adhesion
properties of ENCCs. Our findings identify a de novo role of Sox10 in regulating the migration behaviour of ENCCs, which has
important implications for the treatment of Hirschsprung disease.
Program/Abstract # 15
Epigenetic Regulation of Intestinal Inflammation in Zebrafish
Lindsay Marjoram, Michel Bagnat (Duke, USA)
Our lab recently performed a forward genetic screen in zebrafish and identified mutants with perturbed epithelial integrity in the
intestine. One of these mutants, aa51.3, shows excess epithelial cell shedding and apoptosis in the intestine. This phenotype is
accompanied by loss of epithelial folds and nonpolarized distribution of cadherin. The observed defects are consistent with an
inflammatory bowel disease-like phenotype. Tumor necrosis factor, a pro-inflammatory cytokine, plays a prominent role in
inflammation and its overexpression is correlated with inflammatory bowel disease and defects in intestinal epithelial integrity. To
monitor inflammation, we generated an inflammation-responsive tumor necrosis factor transgenic line, TgBAC(tnfa:GFP). In aa51.3
mutants, TgBAC(tnfa:GFP) is dramatically upregulated along the entire length of the intestine. Exome sequencing followed by
positional cloning identified a causative splice-site mutation in the gene encoding Ubiquitin-like, Containing PHD and Ring Finger
Domains 1 (Uhrf1), which plays a fundamental role in epigenetic regulation of gene expression by promoting DNA maintenance
methylation. Expression of uhrf1 is spatially restricted to the eye, liver and gut in five day post-fertilization larvae, a stage when we
observe elevated intestinal tnfa expression. Our data suggest that loss of maintenance methylation in the tnfa promoter leads to
elevated expression of tnfa in the intestine, which subsequently promotes the observed intestinal defects in aa51.3 mutants. This
research will provide insights into how changes in methylation may promote the development of inflammatory diseases and may
uncover new therapeutic targets.
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