Primary ciliary dyskinesia (PCD) is an autosomal recessive disease caused by defective cilia motility. The identified PCD genes account for about half of PCD incidences and the underlying mechanisms remain poorly understood. We demonstrate that Reptin/ Ruvbl2, a protein known to be involved in epigenetic and transcriptional regulation, is essential for cilia motility in zebrafish. We further show that Reptin directly interacts with the PCD protein Lrrc6/Seahorse and this interaction is critical for the in vivo function of Lrrc6/Seahorse in zebrafish. Moreover, whereas the expression levels of multiple dynein arm components remain unchanged or become elevated, the density of axonemal dynein arms is reduced in reptin hi2394 mutants. Furthermore, Reptin is highly enriched in the cytosol and colocalizes with Lrrc6/Seahorse. Combined, these results suggest that the Reptin-Lrrc6/Seahorse complex is involved in dynein arm formation. We also show that although the DNA damage response is induced in reptin hi2394 mutants, it remains unchanged in cilia biogenesis mutants and lrrc6/seahorse mutants, suggesting that increased DNA damage response is not intrinsic to ciliary defects and that in vertebrate development, Reptin functions in multiple processes, both cilia specific and cilia independent.ciliopathy | chromatin remodeling complex | dynein arm assembly factor A lthough the sensory function of the cilium has garnered significant attention only in the past decade, defective cilia motility was recognized nearly four decades ago as the cause of primary (genetic) ciliary dyskinesia (PCD) (1). PCD is a group of rare human genetic diseases characterized by recurrent infections of the respiratory system, male infertility, and frequently laterality defects, all of which are tightly linked to cilia motility abnormalities. Up to now, 19 PCD genes have been identified (2-20). However, combined they account for about 50% of all PCD cases, suggesting the existence of multiple additional causative genes. Despite such significant advances, our understanding of how cilia motility is regulated is limited and the list of genes involved in cilia motility remains incomplete.In motile cilia or flagella, dynein arms are large protein complexes powering cilia motility. Not surprisingly, multiple PCD genes encode dynein arm components, including DNAL1, DNAI1, DNAI2, DNAH5, and DNAH11 (2-6). In addition, it is understood that dynein arm subunits are preassembled in the cytosol, transported into cilia/flagellum, and docked onto the axoneme, although the underlying mechanisms are poorly understood (21). In recent years, three PCD genes, i.e., DNAAF1/LRRC50 (7, 8), DNAAF2/ KTU (9), and DNAAF3/PF22 (10) have been shown to be involved in the assembly of dynein arm subunits, highlighting the relevance and importance of this process in cilia motility and PCD.Very recently, mutations in human LRRC6/SEAHORSE were linked to PCD (18). Similar to the three known dynein arm assembly factors, mutations in LRRC6/SEAHORSE lead to the absence of both the outer...

The cilium is a signaling platform of the vertebrate cell. It has a critical role in polycystic kidney disease and nephronophthisis. Cilia have been detected on endothelial cells, but the function of these organelles in the vasculature remains incompletely defined. In this study, using genetic and chemical genetic tools in the model organism zebrafish, we reveal an essential role of cilia in developmental vascular integrity. Embryos expressing mutant intraflagellar transport genes, which are essential and specific for cilia biogenesis, displayed increased risk of developmental intracranial hemorrhage, whereas the morphology of the vasculature remained normal. Moreover, cilia were present on endothelial cells in the developing zebrafish vasculature. We further show that the involvement of cilia in vascular integrity is endothelial autonomous, because endothelial-specific re-expression of intraflagellar transport genes in respective mutants rescued the intracranial hemorrhage phenotype. Finally, whereas inhibition of Hedgehog signaling increased the risk of intracranial hemorrhage in ciliary mutants, activation of the pathway rescued this phenotype. In contrast, embryos expressing an inactivating mutation in pkd2, one of two autosomal dominant cystic kidney disease genes, did not show increased risk of developmental intracranial hemorrhage. These results suggest that Hedgehog signaling is a major mechanism for this novel role of endothelial cilia in establishing vascular integrity.

The multifunctional factors Imp-α and Imp-β are involved in nuclear protein import, mitotic spindle dynamics, and nuclear membrane formation. Furthermore, each of the three members of the Imp-α family exerts distinct tasks during development. In Drosophila melanogaster, the imp-α2 gene is critical during oogenesis for ring canal assembly; specific mutations, which allow oogenesis to proceed normally, were found to block early embryonic mitosis. Here, we show that imp-α2 and imp-β genetically interact during early embryonic development, and we characterize the pattern of defects affecting mitosis in embryos laid by heterozygous imp-α2D14 and imp-βKetRE34 females. Embryonic development is arrested in these embryos but is unaffected in combinations between imp-βKetRE34 and null mutations in imp-α1 or imp-α3. Furthermore, the imp-α2D14/imp-βKetRE34 interaction could only be rescued by an imp-α2 transgene, albeit not imp-α1 or imp-α3, showing the exclusive imp-α2 function with imp-β. Use of transgenes carrying modifications in the major Imp-α2 domains showed the critical requirement of the nuclear localization signal binding (NLSB) site in this process. In the mutant embryos, we found metaphase-arrested mitoses made of enlarged spindles, suggesting an unrestrained activity of factors promoting spindle assembly. In accordance with this, we found that Imp-βKetRE34 and Imp-βKetD bind a high level of RanGTP/GDP, and a deletion decreasing RanGTP level suppresses the imp-βKetRE34 phenotype. These data suggest that a fine balance among Imp-α2, Imp-β, RanGTP, and the NLS cargos is critical for mitotic progression during early embryonic development.

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