Ying Cao

Chemical modifier screen identifies HDAC inhibitors as suppressors of PKD models

Polycystic kidney disease (PKD) is a common human genetic disease with severe medical consequences. Although it is appreciated that the cilium plays a central role in PKD, the underlying mechanism for PKD remains poorly understood and no effective treatment is available. In zebrafish, kidney cyst formation is closely associated with laterality defects and body curvature. To discover potential drug candidates and dissect signaling pathways that interact with ciliary signals, we performed a chemical modifier screen for the two phenotypes using zebrafish pkd2hi4166 and ift172hi2211 models. pkd2 is a causal gene for autosomal dominant PKD and ift172 is essential for building and maintaining the cilium. We identified trichostatin A (TSA), a pan-HDAC (histone deacetylase) inhibitor, as a compound that affected both body curvature and laterality. Further analysis verified that TSA inhibited cyst formation in pkd2 knockdown animals. Moreover, we demonstrated that inhibiting class I HDACs, either by valproic acid (VPA), a class I specific HDAC inhibitor structurally unrelated to TSA, or by knocking down hdac1, suppressed kidney cyst formation and body curvature caused by pkd2 deficiency. Finally, we show that VPA was able to reduce the progression of cyst formation and slow the decline of kidney function in a mouse ADPKD model. Together, these data suggest body curvature may be used as a surrogate marker for kidney cyst formation in large-scale high-throughput screens in zebrafish. More importantly, our results also reveal a critical role for HDACs in PKD pathogenesis and point to HDAC inhibitors as drug candidates for PKD treatment.

Cystic Kidney Gene seahorse Regulates Cilia-Mediated Processes and Wnt Pathways

Recently the cilium has emerged as an important sensory organelle for a wide range of cell types in vertebrates. However, the signaling cascade that links ciliary signals to cellular events remains poorly understood. Here, we show that the zebrafish cystic kidney gene seahorse is closely associated with ciliary functions: seahorse is required for establishing left-right asymmetry and for preventing kidney cyst formation; seahorse transcript is highly enriched in heavily ciliated tissues; and seahorse genetically interacts with the ciliary gene inversin. Yet seahorse is dispensable for cilia assembly or motility and the Seahorse protein is cytoplasmic. We provide evidence that Seahorse associates with Dishevelled. Finally, we show that seahorse constrains the canonical Wnt pathway and promotes the noncanonical Wnt pathway during gastrulation. Together, these data suggest that Seahorse may provide a link between ciliary signals and Wnt pathways.

Intraflagellar Transport Proteins Are Essential for Cilia Formation and for Planar Cell Polarity

The highly conserved intraflagellar transport (IFT) proteins are essential for cilia formation in multiple organisms, but surprisingly, cilia form in multiple zebrafish ift mutants. Here, we detected maternal deposition of ift gene products in zebrafish and found that ciliary assembly occurs only during early developmental stages, supporting the idea that maternal contribution of ift gene products masks the function of IFT proteins during initial development. In addition, the basal bodies in multiciliated cells of the pronephric duct in ift mutants were disorganized, with a pattern suggestive of defective planar cell polarity (PCP). Depletion of pk1, a core PCP component, similarly led to kidney cyst formation and basal body disorganization. Furthermore, we found that multiple ift genes genetically interact with pk1. Taken together, these data suggest that IFT proteins play a conserved role in cilia formation and planar cell polarity in zebrafish.

An SP1-like transcription factor Spr2 acts downstream of Fgf signaling to mediate mesoderm induction.

Fgf signaling, mediated in part by the transcription factor Brachyury/Xbra/Ntl, plays important roles in mesoderm formation during the early development of vertebrate embryos. We have identified a zebrafish gene, spr2, which encodes a member of the Sp1‐like transcription factor family. spr2 is expressed in both hypoblast and epiblast cells during late blastulation/early gastrulation, and in some mesodermal and neural tissues at later stages. Injection with spr2 mRNA enhances ntl expression and alleviates the inhibitory effect on ntl of XFD, a Xenopus dominant‐negative FGF receptor. In contrast, morpholino‐ mediated knockdown of Spr2 activity inhibits ntl expression and reduces the inductive effect of Fgfs on ntl. We also demonstrate that Fgf signaling relays mesoderm induction activity of Nodal signaling and Spr2 is involved in this signal relay process. Furthermore, the correct spatial expression of spr2 requires Nodal, Fgf and Wnt signals. We suggest that expression of spr2 is an immediate‐early response to mesoderm induction by Fgfs, which in turn regulates the expression of effector genes involved in the development of mesodermal tissues.

Amotl2 is essential for cell movements in zebrafish embryo and regulates c-Src translocation.

Angiomotin (Amot), the founding member of the Motin family, is involved in angiogenesis by regulating endothelial cell motility, and is required for visceral endoderm movement in mice. However, little is known about biological functions of the other two members of the Motin family, Angiomotin-like1 (Amotl1) and Angiomotin-like2 (Amotl2). Here, we have identified zebrafish amotl2 as an Fgf-responsive gene. Zebrafish amotl2 is expressed maternally and in restricted cell types zygotically. Knockdown of amotl2 expression delays epiboly and impairs convergence and extension movement, and amotl2-deficient cells in mosaic embryos fail to migrate properly. This coincides with loss of membrane protrusions and disorder of F-actin. Amotl2 partially co-localizes with RhoB-or EEA1-positive endosomes and the non-receptor tyrosine kinase c-Src. We further demonstrate that Amotl2 interacts preferentially with and facilitates outward translocation of the phosphorylated c-Src, which may in turn regulate the membrane architecture. These data provide the first evidence that amotl2 is essential for cell movements in vertebrate embryos.

Reptin/Ruvbl2 is a Lrrc6/Seahorse interactor essential for cilia motility

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 reptinhi2394 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 reptinhi2394 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.

Characterization and expression pattern ofpouII1, a novel class II POU gene in zebrafish

POU domain transcription factors that share a conserved DNA-binding domain, POU domain, are important regulators for the development of embryos in various animal species. A novel zebrafish POU domain gene,pouII1 has been cloned. ThepouII1 cDNA is 2080 kb in length and encodes a putative polypeptide of 596 amino acids. It is placed into class II POU family since it shares a high degree of homology with the known members of this family. Northern hybridization identifies a major transcript of approximately 2.1 kb that was present in embryos at the single-cell stage throughout 24 h postfertilization. The whole mountin situ hybridization shows thatpouII1 transcripts are present in the single-cell embryos, strongly suggesting that these transcripts are of maternal origin. During early development of the embryos,pouII1 mRNA was ubiquitously distributed in all cells and tissues. The transcripts are gradually limited to brains and become completely undetectable by day 3. To our knowledge,pouII1 is the first class II POU gene identified in zebrafish.

Expression of zebrafish Lc3 synthase gene in embryonic lens requires hedgehog signaling

Glycosyltransferases are involved in synthesis of various glycolipids and glycoproteins that play important roles in many biological processes. We have identified a zebrafish gene encoding a member of β1,3‐N‐acetylglucosaminyltransferase family, the Lc3 synthase/bGn‐T5. Whole‐mount in situ hybridization reveals that the Lc3 synthase gene is expressed in two distinct phases during the zebrafish embryogenesis. The early phase extends from late blastulation to the completion of epiboly, during which the expression occurs in the superficial layer of the embryos. The second phase of expression starts during mid‐segmentation and persists until day 3, during which the expression occurs prominently in the developing lens. The expression of the Lc3 synthase gene in the lens is inhibited in you‐too (yot) mutant embryos that are defective in Hedgehog signaling. The expression in the lens also decreases in cyclops (cyc) and one‐eyed‐pinhead (oep) mutant embryos and lefty1‐injected embryos, which are deficient in Nodal signaling and lack Hedgehog activity in the ventral brain. These results suggest that Hedgehog signaling is required for the Lc3 synthase expression in embryonic lens. Developmental Dynamics, 2003.