The role of cilia during organogenesis in zebrafish.

Cilia are hair-like organelles that protrude from the surface of eukaryotic cells and are present on the surface of nearly all human cells. Cilia play a crucial role in signal transduction, organ development and tissue homeostasis. Abnormalities in the structure and function of cilia can lead to a group of human diseases known as ciliopathies. Currently, zebrafish serves as an ideal model for studying ciliary function and ciliopathies due to its relatively conserved structure and function of cilia compared to humans. In this review, we will summarize the different types of cilia that present in embryonic and adult zebrafish, and provide an overview of the advantages of using zebrafish as a vertebrate model for cilia research. We will specifically focus on the roles of cilia during zebrafish organogenesis based on recent studies. Additionally, we will highlight future prospects for ciliary research in zebrafish.

Ependymal polarity defects coupled with disorganized ciliary beating drive abnormal cerebrospinal fluid flow and spine curvature in zebrafish.

Idiopathic scoliosis (IS) is the most common spinal deformity diagnosed in childhood or early adolescence, while the underlying pathogenesis of this serious condition remains largely unknown. Here, we report zebrafish ccdc57 mutants exhibiting scoliosis during late development, similar to that observed in human adolescent idiopathic scoliosis (AIS). Zebrafish ccdc57 mutants developed hydrocephalus due to cerebrospinal fluid (CSF) flow defects caused by uncoordinated cilia beating in ependymal cells. Mechanistically, Ccdc57 localizes to ciliary basal bodies and controls the planar polarity of ependymal cells through regulating the organization of microtubule networks and proper positioning of basal bodies. Interestingly, ependymal cell polarity defects were first observed in ccdc57 mutants at approximately 17 days postfertilization, the same time when scoliosis became apparent and prior to multiciliated ependymal cell maturation. We further showed that mutant spinal cord exhibited altered expression pattern of the Urotensin neuropeptides, in consistent with the curvature of the spine. Strikingly, human IS patients also displayed abnormal Urotensin signaling in paraspinal muscles. Altogether, our data suggest that ependymal polarity defects are one of the earliest sign of scoliosis in zebrafish and disclose the essential and conserved roles of Urotensin signaling during scoliosis progression.

Zebrafish: an important model for understanding scoliosis.

Scoliosis is a common spinal deformity that considerably affects the physical and psychological health of patients. Studies have shown that genetic factors play an important role in scoliosis. However, its etiopathogenesis remain unclear, partially because of the genetic heterogeneity of scoliosis and the lack of appropriate model systems. Recently, the development of efficient gene editing methods and high-throughput sequencing technology has made it possible to explore the underlying pathological mechanisms of scoliosis. Owing to their susceptibility for developing scoliosis and high genetic homology with human, zebrafish are increasingly being used as a model for scoliosis in developmental biology, genetics, and clinical medicine. Here, we summarize the recent advances in scoliosis research on zebrafish and discuss the prospects of using zebrafish as a scoliosis model.

Cilia regulate meiotic recombination in zebrafish.

Meiosis is essential for evolution and genetic diversity in almost all sexual eukaryotic organisms. The mechanisms of meiotic recombination, such as synapsis, have been extensively investigated. However, it is still unclear whether signals from the cytoplasm or even from outside of the cell can regulate the meiosis process. Cilia are microtubule-based structures that protrude from the cell surface and function as signaling hubs to sense extracellular signals. Here, we reported an unexpected and critical role of cilia during meiotic recombination. During gametogenesis of zebrafish, cilia were specifically present in the prophase stages of both primary spermatocytes and primary oocytes. By developing a germ cell-specific CRISPR/Cas9 system, we demonstrated that germ cell-specific depletion of ciliary genes resulted in compromised double-strand break repair, reduced crossover formation, and increased germ cell apoptosis. Our study reveals a previously undiscovered role for cilia during meiosis and suggests that extracellular signals may regulate meiotic recombination via this particular organelle.

E2f4 is required for intestinal and otolith development in zebrafish.

E2f4 is a multifunctional transcription factor that is essential for many cellular processes. Although the role of E2f4 during cell cycle progression has been investigated in great detail, less is known about E2f4 during embryonic development. Here, we investigated the role of E2f4 during zebrafish development. Zebrafish e2f4 mutants displayed ectopic otolith formation due to abnormal ciliary beating in the otic vesicle. The beating defects of motile cilia were caused by abnormal expression of ciliary motility genes. The expression of two genes, lrrc23 and ccdc151, were significantly decreased in the absence of E2f4. In addition to that, e2f4 mutants also displayed growth retardation both in the body length and body weight and mostly died at around 6 months old. Although food intake was normal in the mutants, we found that the microvilli of the intestinal epithelia were significantly affected in the mutants. Finally, the intestinal epithelia of e2f4 mutants also displayed reduced cell proliferation, together with an increased level of cell apoptosis. Our data suggested a tissue-specific role of E2f4 during zebrafish development, which is distinct from the traditional views of this protein as a transcription repressor.

Heme-binding protein CYB5D1 is a radial spoke component required for coordinated ciliary beating.

Coordinated beating is crucial for the function of multiple cilia. However, the molecular mechanism is poorly understood. Here, we characterize a conserved ciliary protein CYB5D1 with a heme-binding domain and a cordon-bleu ubiquitin-like domain. Mutation or knockdown of Cyb5d1 in zebrafish impaired coordinated ciliary beating in the otic vesicle and olfactory epithelium. Similarly, the two flagella of an insertional mutant of the CYB5D1 ortholog in Chlamydomonas (Crcyb5d1) showed an uncoordinated pattern due to a defect in the cis-flagellum. Biochemical analyses revealed that CrCYB5D1 is a radial spoke stalk protein that binds heme only under oxidizing conditions. Lack of CrCYB5D1 resulted in a reductive shift in flagellar redox state and slowing down of the phototactic response. Treatment of Crcyb5d1 with oxidants restored coordinated flagellar beating. Taken together, these data suggest that CrCYB5D1 may integrate environmental and intraciliary signals and regulate the redox state of cilia, which is crucial for the coordinated beating of multiple cilia.

E2f5 is a versatile transcriptional activator required for spermatogenesis and multiciliated cell differentiation in zebrafish.

E2f5 is a member of the E2f family of transcription factors that play essential roles during many cellular processes. E2f5 was initially characterized as a transcriptional repressor in cell proliferation studies through its interaction with the Retinoblastoma (Rb) protein for inhibition of target gene transcription. However, the precise roles of E2f5 during embryonic and post-embryonic development remain incompletely investigated. Here, we report that zebrafish E2f5 plays critical roles during spermatogenesis and multiciliated cell (MCC) differentiation. Zebrafish e2f5 mutants develop exclusively as infertile males. In the mutants, spermatogenesis is arrested at the zygotene stage due to homologous recombination (HR) defects, which finally leads to germ cell apoptosis. Inhibition of cell apoptosis in e2f5;tp53 double mutants rescued ovarian development, although oocytes generated from the double mutants were still abnormal, characterized by aberrant distribution of nucleoli. Using transcriptome analysis, we identified dmc1, which encodes an essential meiotic recombination protein, as the major target gene of E2f5 during spermatogenesis. E2f5 can bind to the promoter of dmc1 to promote HR, and overexpression of dmc1 significantly increased the fertilization rate of e2f5 mutant males. Besides gametogenesis defects, e2f5 mutants failed to develop MCCs in the nose and pronephric ducts during early embryonic stages, but these cells recovered later due to redundancy with E2f4. Moreover, we demonstrate that ion transporting principal cells in the pronephric ducts, which remain intercalated with the MCCs, do not contain motile cilia in wild-type embryos, while they generate single motile cilia in the absence of E2f5 activity. In line with this, we further show that E2f5 activates the Notch pathway gene jagged2b (jag2b) to inhibit the acquisition of MCC fate as well as motile cilia differentiation by the neighboring principal cells. Taken together, our data suggest that E2f5 can function as a versatile transcriptional activator and identify novel roles of the protein in spermatogenesis as well as MCC differentiation during zebrafish development.

Ankrd45 Is a Novel Ankyrin Repeat Protein Required for Cell Proliferation.

Ankyrin repeats, the most common protein-protein interaction motifs in nature, are widely present in proteins of both eukaryotic and prokaryotic cells. Ankyrin repeat-containing proteins play diverse biological functions. Here, we identified the gene ankrd45, which encodes a novel, two ankyrin repeat-containing protein. Zebrafish ankrd45 displayed a tissue specific expression pattern during early development, with high expression in ciliated tissues, including otic vesicles, Kupffer's vesicles, pronephric ducts, and floor plates. Surprisingly, zebrafish ankrd45 mutants were viable and developed grossly normal cilia. In contrast, mutant larvae developed enlarged livers when induced with liver specific expression of KrasG12V, one of the common mutations of KRAS that leads to cancer in humans. Further, histological analysis suggested that multiple cysts developed in the mutant liver due to cell apoptosis. Similarly, knockdown of ANKRD45 expression with either siRNA or CRISPR/Cas9 methods induced apoptosis in cultured cells, similar to those in zebrafish ankrd45 mutant livers after induction. Using different cell lines, we show that the distribution of ANKRD45 protein was highly dynamic during mitosis. ANKRD45 is preferably localized to the midbody ring during cytokinesis. Together, our results suggest that Ankrd45 is a novel ankyrin repeat protein with a conserved role during cell proliferation in both zebrafish embryos and mammalian cells.

Primary cilia regulate hematopoietic stem and progenitor cell specification through Notch signaling in zebrafish.

Hematopoietic stem and progenitor cells (HSPCs) are capable of producing all mature blood lineages, as well as maintaining the self-renewal ability throughout life. The hairy-like organelle, cilium, is present in most types of vertebrate cells, and plays important roles in various biological processes. However, it is unclear whether and how cilia regulate HSPC development in vertebrates. Here, we show that cilia-specific genes, involved in primary cilia formation and function, are required for HSPC development, especially in hemogenic endothelium (HE) specification in zebrafish embryos. Blocking primary cilia formation or function by genetic or chemical manipulations impairs HSPC development. Mechanistically, we uncover that primary cilia in endothelial cells transduce Notch signal to the earliest HE for proper HSPC specification during embryogenesis. Altogether, our findings reveal a pivotal role of endothelial primary cilia in HSPC development, and may shed lights into in vitro directed differentiation of HSPCs.

The cytoplasmic tail of rhodopsin triggers rapid rod degeneration in kinesin-2 mutants.

Photoreceptor degeneration can lead to blindness and represents the most common form of neural degenerative disease worldwide. Although many genes involved in photoreceptor degeneration have been identified, the underlying mechanisms remain to be elucidated. Here we examined photoreceptor development in zebrafish kif3a and kif3b mutants, which affect two subunits of the kinesin-2 complex. In both mutants, rods degenerated quickly, whereas cones underwent a slow degeneration process. Notably, the photoreceptor defects were considerably more severe in kif3a mutants than in kif3b mutants. In the cone photoreceptors of kif3a mutants, opsin proteins accumulated in the apical region and formed abnormal membrane structures. In contrast, rhodopsins were enriched in the rod cell body membrane and represented the primary reason for rapid rod degeneration in these mutants. Moreover, removal of the cytoplasmic tail of rhodopsin to reduce its function, but not decreasing rhodopsin expression levels, prevented rod degeneration in both kif3a and kif3b mutants. Of note, overexpression of full-length rhodopsin or its cytoplasmic tail domain, but not of rhodopsin lacking the cytoplasmic tail, exacerbated rod degeneration in kif3a mutants, implying an important role of the cytoplasmic tail in rod degeneration. Finally, we showed that the cytoplasmic tail of rhodopsin might trigger rod degeneration through activating the downstream calcium signaling pathway, as drug treatment with inhibitors of intracellular calcium release prevented rod degeneration in kif3a mutants. Our results demonstrate a previously unknown function of the rhodopsin cytoplasmic domain during opsin-triggered photoreceptor degeneration and may open up new avenues for managing this disease.