WNT/RYK signaling functions as an antiinflammatory modulator in the lung mesenchyme.

A number of inflammatory lung diseases, including chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and pneumonia, are modulated by WNT/ß-catenin signaling. However, the underlying molecular mechanisms remain unclear. Here, starting with a forward genetic screen in mouse, we identify the WNT coreceptor Related to receptor tyrosine kinase (RYK) acting in mesenchymal tissues as a cell survival and antiinflammatory modulator. Ryk mutant mice exhibit lung hypoplasia and inflammation as well as alveolar simplification due to defective secondary septation, and deletion of Ryk specifically in mesenchymal cells also leads to these phenotypes. By analyzing the transcriptome of wild-type and mutant lungs, we observed the up-regulation of proapoptotic and inflammatory genes whose expression can be repressed by WNT/RYK signaling in vitro. Moreover, mesenchymal Ryk deletion at postnatal and adult stages can also lead to lung inflammation, thus indicating a continued role for WNT/RYK signaling in homeostasis. Our results indicate that RYK signaling through ß-catenin and Nuclear Factor kappa B (NF-κB) is part of a safeguard mechanism against mesenchymal cell death, excessive inflammatory cytokine production, and inflammatory cell recruitment and accumulation. Notably, RYK expression is down-regulated in the stromal cells of pneumonitis patient lungs. Altogether, our data reveal that RYK signaling plays critical roles as an antiinflammatory modulator during lung development and homeostasis and provide an animal model to further investigate the etiology of, and therapeutic approaches to, inflammatory lung diseases.

A Novel Anticancer Peptide Derived from Bryopsis plumosa Regulates Proliferation and Invasion in Non-Small Cell Lung Cancer Cells.

The discovery of new highly effective anticancer drugs with few side effects is a challenge for drug development research. Natural or synthetic anticancer peptides (ACPs) represent a new generation of anticancer agents with high selectivity and specificity. The rapid emergence of chemoradiation-resistant lung cancer has necessitated the discovery of novel anticancer agents as alternatives to conventional therapeutics. In this study, we synthesized a peptide containing 22 amino acids and characterized it as a novel ACP (MP06) derived from green sea algae, Bryopsis plumosa. Using the ACP database, MP06 was predicted to possess an alpha-helical secondary structure and functionality. The anti-proliferative and apoptotic effects of the MP06, determined using the cytotoxicity assay and Annexin V/propidium iodide staining kit, were significantly higher in non-small-cell lung cancer (NSCLC) cells than in non-cancerous lung cells. We confirmed that MP06 suppressed cellular migration and invasion and inhibited the expression of N-cadherin and vimentin, the markers of epithelial-mesenchymal transition. Moreover, MP06 effectively reduced the metastasis of tumor xenografts in zebrafish embryos. In conclusion, we suggest considering MP06 as a novel candidate for the development of new anticancer drugs functioning via the ERK signaling pathway.

WNT/RYK signaling restricts goblet cell differentiation during lung development and repair.

Goblet cell metaplasia and mucus hypersecretion are observed in many pulmonary diseases, including asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis. However, the regulation of goblet cell differentiation remains unclear. Here, we identify a regulator of this process in an N-ethyl-N-nitrosourea (ENU) screen for modulators of postnatal lung development; Ryk mutant mice exhibit lung inflammation, goblet cell hyperplasia, and mucus hypersecretion. RYK functions as a WNT coreceptor, and, in the developing lung, we observed high RYK expression in airway epithelial cells and moderate expression in mesenchymal cells as well as in alveolar epithelial cells. From transcriptomic analyses and follow-up studies, we found decreased WNT/ß-catenin signaling activity in the mutant lung epithelium. Epithelial-specific Ryk deletion causes goblet cell hyperplasia and mucus hypersecretion but not inflammation, while club cell-specific Ryk deletion in adult stages leads to goblet cell hyperplasia and mucus hypersecretion during regeneration. We also found that the airway epithelium of COPD patients often displays goblet cell metaplastic foci, as well as reduced RYK expression. Altogether, our findings reveal that RYK plays important roles in maintaining the balance between airway epithelial cell populations during development and repair, and that defects in RYK expression or function may contribute to the pathogenesis of human lung diseases.

Targeted knockout of a chemokine-like gene increases anxiety and fear responses.

Emotional responses, such as fear and anxiety, are fundamentally important behavioral phenomena with strong fitness components in most animal species. Anxiety-related disorders continue to represent a major unmet medical need in our society, mostly because we still do not fully understand the mechanisms of these diseases. Animal models may speed up discovery of these mechanisms. The zebrafish is a highly promising model organism in this field. Here, we report the identification of a chemokine-like gene family, samdori (sam), and present functional characterization of one of its members, sam2 We show exclusive mRNA expression of sam2 in the CNS, predominantly in the dorsal habenula, telencephalon, and hypothalamus. We found knockout (KO) zebrafish to exhibit altered anxiety-related responses in the tank, scototaxis and shoaling assays, and increased crh mRNA expression in their hypothalamus compared with wild-type fish. To investigate generalizability of our findings to mammals, we developed a Sam2 KO mouse and compared it to wild-type littermates. Consistent with zebrafish findings, homozygous KO mice exhibited signs of elevated anxiety. We also found bath application of purified SAM2 protein to increase inhibitory postsynaptic transmission onto CRH neurons of the paraventricular nucleus. Finally, we identified a human homolog of SAM2, and were able to refine a candidate gene region encompassing SAM2, among 21 annotated genes, which is associated with intellectual disability and autism spectrum disorder in the 12q14.1 deletion syndrome. Taken together, these results suggest a crucial and evolutionarily conserved role of sam2 in regulating mechanisms associated with anxiety.

WDR11-mediated Hedgehog signalling defects underlie a new ciliopathy related to Kallmann syndrome.

WDR11 has been implicated in congenital hypogonadotropic hypogonadism (CHH) and Kallmann syndrome (KS), human developmental genetic disorders defined by delayed puberty and infertility. However, WDR11's role in development is poorly understood. Here, we report that WDR11 modulates the Hedgehog (Hh) signalling pathway and is essential for ciliogenesis. Disruption of WDR11 expression in mouse and zebrafish results in phenotypic characteristics associated with defective Hh signalling, accompanied by dysgenesis of ciliated tissues. Wdr11-null mice also exhibit early-onset obesity. We find that WDR11 shuttles from the cilium to the nucleus in response to Hh signalling. WDR11 regulates the proteolytic processing of GLI3 and cooperates with the transcription factor EMX1 in the induction of downstream Hh pathway gene expression and gonadotrophin-releasing hormone production. The CHH/KS-associated human mutations result in loss of function of WDR11. Treatment with the Hh agonist purmorphamine partially rescues the WDR11 haploinsufficiency phenotypes. Our study reveals a novel class of ciliopathy caused by WDR11 mutations and suggests that CHH/KS may be a part of the human ciliopathy spectrum.

Fibrillin-2 is a key mediator of smooth muscle extracellular matrix homeostasis during mouse tracheal tubulogenesis.

Epithelial tubes, comprised of polarised epithelial cells around a lumen, are crucial for organ function. However, the molecular mechanisms underlying tube formation remain largely unknown. Here, we report on the function of fibrillin (FBN)2, an extracellular matrix (ECM) glycoprotein, as a critical regulator of tracheal tube formation.We performed a large-scale forward genetic screen in mouse to identify regulators of respiratory organ development and disease. We identified Fbn2 mutants which exhibit shorter and narrowed tracheas as well as defects in tracheal smooth muscle cell alignment and polarity.We found that FBN2 is essential for elastic fibre formation and Fibronectin accumulation around tracheal smooth muscle cells. These processes appear to be regulated at least in part through inhibition of p38-mediated upregulation of matrix metalloproteinases (MMPs), as pharmacological decrease of p38 phosphorylation or MMP activity partially attenuated the Fbn2 mutant tracheal phenotypes. Analysis of human tracheal tissues indicates that a decrease in ECM proteins, including FBN2 and Fibronectin, is associated with tracheomalacia.Our findings provide novel insights into the role of ECM homeostasis in mesenchymal cell polarisation during tracheal tubulogenesis.

Chd7 Is Critical for Early T-Cell Development and Thymus Organogenesis in Zebrafish.

Coloboma, heart defect, atresia choanae, retarded growth and development, genital hypoplasia, ear anomalies/deafness (CHARGE) syndrome is a congenital disorder affecting multiple organs and mainly caused by mutations in CHD7, a gene encoding a chromatin-remodeling protein. Immunodeficiency and reduced T cells have been noted in CHARGE syndrome. However, the mechanisms underlying T lymphopenia are largely unexplored. Herein, we observed dramatic decrease of T cells in both chd7knockdown and knockout zebrafish embryos. Unexpectedly, hematopoietic stem and progenitor cells and, particularly, lymphoid progenitor cells were increased peripherally in nonthymic areas in chd7-deficient embryos, unlikely to contribute to the T-cell decrease. Further analysis demonstrated that both the organogenesis and homing function of the thymus were seriously impaired. Chd7 might regulate thymus organogenesis through modulating the development of both neural crest cell-derived mesenchyme and pharyngeal endoderm-derived thymic epithelial cells. The expression of foxn1, a central regulator of thymic epithelium, was remarkably down-regulated in the pharyngeal region in chd7-deficient embryos. Moreover, the T-cell reduction in chd7-deficient embryos was partially rescued by overexpressing foxn1, suggesting that restoring thymic epithelium may be a potential therapeutic strategy for treating immunodeficiency in CHARGE syndrome. Collectively, the results indicated that chd7 was critical for thymic development and T-lymphopenia in CHARGE syndrome may be mainly attributed to the defects of thymic organogenesis. The current finding may benefit the diagnosis and therapy of T lymphopenia and immunodeficiency in CHARGE syndrome.

ZC4H2, an XLID gene, is required for the generation of a specific subset of CNS interneurons.

Miles-Carpenter syndrome (MCS) was described in 1991 as an XLID syndrome with fingertip arches and contractures and mapped to proximal Xq. Patients had microcephaly, short stature, mild spasticity, thoracic scoliosis, hyperextendable MCP joints, rocker-bottom feet, hyperextended elbows and knees. A mutation, p.L66H, in ZC4H2, was identified in a XLID re-sequencing project. Additional screening of linked families and next generation sequencing of XLID families identified three ZC4H2 mutations: p.R18K, p.R213W and p.V75in15aa. The families shared some relevant clinical features. In silico modeling of the mutant proteins indicated all alterations would destabilize the protein. Knockout mutations in zc4h2 were created in zebrafish and homozygous mutant larvae exhibited abnormal swimming, increased twitching, defective eye movement and pectoral fin contractures. Because several of the behavioral defects were consistent with hyperactivity, we examined the underlying neuronal defects and found that sensory neurons and motoneurons appeared normal. However, we observed a striking reduction in GABAergic interneurons. Analysis of cell-type-specific markers showed a specific loss of V2 interneurons in the brain and spinal cord, likely arising from mis-specification of neural progenitors. Injected human wt ZC4H2 rescued the mutant phenotype. Mutant zebrafish injected with human p.L66H or p.R213W mRNA failed to be rescued, while the p.R18K mRNA was able to rescue the interneuron defect. Our findings clearly support ZC4H2 as a novel XLID gene with a required function in interneuron development. Loss of function of ZC4H2 thus likely results in altered connectivity of many brain and spinal circuits.

Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases.

RNA-guided endonucleases (RGENs), derived from the prokaryotic Type II CRISPR-Cas system, enable targeted genome modification in cells and organisms. Here we describe the establishment of gene-knockout mice and zebrafish by the injection of RGENs as Cas9 protein:guide RNA complexes or Cas9 mRNA plus guide RNA into one-cell-stage embryos of both species. RGENs efficiently generated germline transmittable mutations in up to 93% of newborn mice with minimal toxicity. RGEN-induced mutations in the mouse Prkdc gene that encodes an enzyme critical for DNA double-strand break repair resulted in immunodeficiency both in F0 and F1 mice. We propose that RGEN-mediated mutagenesis in animals will greatly expedite the creation of genetically engineered model organisms, accelerating functional genomic research.

IFT46 plays an essential role in cilia development.

Cilia are microtubule-based structures that project into the extracellular space. Ciliary defects are associated with several human diseases, including polycystic kidney disease, primary ciliary dyskinesia, left-right axis patterning, hydrocephalus and retinal degeneration. However, the genetic and cellular biological control of ciliogenesis remains poorly understood. The IFT46 is one of the highly conserved intraflagellar transport complex B proteins. In zebrafish, ift46 is expressed in various ciliated tissues such as Kupffer׳s vesicle, pronephric ducts, ears and spinal cord. We show that ift46 is localized to the basal body. Knockdown of ift46 gene results in multiple phenotypes associated with various ciliopathies including kidney cysts, pericardial edema and ventral axis curvature. In ift46 morphants, cilia in kidney and spinal canal are shortened and abnormal. Similar ciliary defects are observed in otic vesicles, lateral line hair cells, olfactory pits, but not in Kupffer׳s vesicle. To explore the functions of Ift46 during mouse development, we have generated Ift46 knock-out mice. The Ift46 mutants have developmental defects in brain, neural tube and heart. In particular Ift46(-/-) homozygotes displays randomization of the embryo heart looping, which is a hallmark of defective left-right (L/R) axis patterning. Taken together, our results demonstrated that IFT46 has an essential role in vertebrate ciliary development.

Translocations disrupting PHF21A in the Potocki-Shaffer-syndrome region are associated with intellectual disability and craniofacial anomalies.

Potocki-Shaffer syndrome (PSS) is a contiguous gene disorder due to the interstitial deletion of band p11.2 of chromosome 11 and is characterized by multiple exostoses, parietal foramina, intellectual disability (ID), and craniofacial anomalies (CFAs). Despite the identification of individual genes responsible for multiple exostoses and parietal foramina in PSS, the identity of the gene(s) associated with the ID and CFA phenotypes has remained elusive. Through characterization of independent subjects with balanced translocations and supportive comparative deletion mapping of PSS subjects, we have uncovered evidence that the ID and CFA phenotypes are both caused by haploinsufficiency of a single gene, PHF21A, at 11p11.2. PHF21A encodes a plant homeodomain finger protein whose murine and zebrafish orthologs are both expressed in a manner consistent with a function in neurofacial and craniofacial development, and suppression of the latter led to both craniofacial abnormalities and neuronal apoptosis. Along with lysine-specific demethylase 1 (LSD1), PHF21A, also known as BHC80, is a component of the BRAF-histone deacetylase complex that represses target-gene transcription. In lymphoblastoid cell lines from two translocation subjects in whom PHF21A was directly disrupted by the respective breakpoints, we observed derepression of the neuronal gene SCN3A and reduced LSD1 occupancy at the SCN3A promoter, supporting a direct functional consequence of PHF21A haploinsufficiency on transcriptional regulation. Our finding that disruption of PHF21A by translocations in the PSS region is associated with ID adds to the growing list of ID-associated genes that emphasize the critical role of transcriptional regulation and chromatin remodeling in normal brain development and cognitive function.

Angiopoietin-like 3 regulates hepatocyte proliferation and lipid metabolism in zebrafish.

Loss-of-function mutations in angiopoietin-like 3 (ANGPTL3) cause familial hypobetalipoproteinemia type 2 (FHBL2) in humans. ANGPTL3 belongs to the angiopoietin-like family, the vascular endothelial growth factor family that is structurally similar to angiopoietins and is known for a regulator of lipid and glucose metabolism, although it is unclear how mutations in ANGPTL3 lead to defect in liver development in the vertebrates. We report here that angptl3 is primarily expressed in the zebrafish developing liver and that morpholino (MO) knockdown of Angptl3 reduces the size of the developing liver, which is caused by suppression of cell proliferation, but not by enhancement of apoptosis. However, MO knockdown of Angptl3 did not alter angiogenesis in the developing liver. Additionally, disruption of zebrafish Angptl3 elicits the hypocholesterolemia phenotype that is characteristic of FHBL2 in humans. Together, our findings propose a novel role for Angptl3 in liver cell proliferation and maintenance during zebrafish embryogenesis. Finally, angptl3 morphants will serve as a good model for understanding the pathophysiology of FHBL2.

Heterogenous effect of early adulthood stress on cognitive aging and synaptic function in the dentate gyrus.

Cognitive aging widely varies among individuals due to different stress experiences throughout the lifespan and vulnerability of neurocognitive mechanisms. To understand the heterogeneity of cognitive aging, we investigated the effect of early adulthood stress (EAS) on three different hippocampus-dependent memory tasks: the novel object recognition test (assessing recognition memory: RM), the paired association test (assessing episodic-like memory: EM), and trace fear conditioning (assessing trace memory: TM). Two-month-old rats were exposed to chronic mild stress for 6 weeks and underwent behavioral testing either 2 weeks or 20 months later. The results show that stress and aging impaired different types of memory tasks to varying degrees. RM is affected by combined effect of stress and aging. EM became less precise in EAS animals. TM, especially the contextual memory, showed impairment in aging although EAS attenuated the aging effect, perhaps due to its engagement in emotional memory systems. To further explore the neural underpinnings of these multi-faceted effects, we measured long-term potentiation (LTP), neural density, and synaptic density in the dentate gyrus (DG). Both stress and aging reduced LTP. Additionally, the synaptic density per neuron showed a further reduction in the stress aged group. In summary, EAS modulates different forms of memory functions perhaps due to their substantial or partial dependence on the functional integrity of the hippocampus. The current results suggest that lasting alterations in hippocampal circuits following EAS could potentially generate remote effects on individual variability in cognitive aging, as demonstrated by performance in multiple types of memory.

Impairments of cerebellar structure and function in a zebrafish KO of neuropsychiatric risk gene znf536.

Genetic variants in ZNF536 contribute to the risk for neuropsychiatric disorders such as schizophrenia, autism, and others. The role of this putative transcriptional repressor in brain development and function is, however, largely unknown. We generated znf536 knockout (KO) zebrafish and studied their behavior, brain anatomy, and brain function. Larval KO zebrafish showed a reduced ability to compete for food, resulting in decreased total body length and size. This phenotype can be rescued by segregating the homozygous KO larvae from their wild-type and heterozygous siblings, enabling studies of adult homozygous KO animals. In adult KO zebrafish, we observed significant reductions in anxiety-like behavior and social interaction. These znf536 KO zebrafish have decreased cerebellar volume, corresponding to decreased populations of specific neuronal cells, especially in the valvular cerebelli (Va). Finally, using a Tg[mbp:mgfp] line, we identified a previously undetected myelin structure located bilaterally within the Va, which also displayed a reduction in volume and disorganization in KO zebrafish. These findings indicate an important role for ZNF536 in brain development and implicate the cerebellum in the pathophysiology of neuropsychiatric disorders.

Characterization of exosomal microRNAs in preterm infants fed with breast milk and infant formula.

Breastfeeding not only reduces infection-related morbidity, but also increases growth of preterm infants. Advantages of breast milk (BM) for preterm infants are significant. They continue to be studied. However, because not all preterm infants can receive breastfeeding, bovine-based infant formula (IF) is used as an alternative, which may increase the risk of several preterm complications. Exosomes isolated from biofluids are emerging as biomarkers in research of various diseases. Here, we characterized miRNA contents of exosomes in urine and serum samples of preterm infants who were BM and IF fed and performed transcriptomic analysis of small RNA libraries. We identified significantly up-regulated 6 miRNAs and 10 miRNAs, respectively. Gene Ontology (GO) analysis revealed that target genes of these miRNAs might participate in neuronal development, immunity modulation, detoxification of reactive oxygen species, and transmembrane exchange. Our data suggest that exosome-based systemic screening for preterm infants with breastfeeding might be a screening tool for identifying target molecules involved in therapy for preterm infants in neonatal intensive care unit (NICU) and for future application as nutraceutical formulations or pharmaceuticals.

Therapeutic single-cell landscape: methotrexate exacerbates interstitial lung disease by compromising the stemness of alveolar epithelial cells under systemic inflammation.

BACKGROUND: Interstitial lung disease (ILD) poses a serious threat in patients with rheumatoid arthritis (RA). However, the impact of cornerstone drugs, including methotrexate (MTX) and TNF inhibitor, on RA-associated ILD (RA-ILD) remains controversial. METHODS: Using an SKG mouse model and single-cell transcriptomics, we investigated the effects of MTX and TNF blockade on ILD. FINDINGS: Our study revealed that MTX exacerbates pulmonary inflammation by promoting immune cell infiltration, Th17 activation, and fibrosis. In contrast, TNF inhibitor ameliorates these features and inhibits ILD progression. Analysis of data from a human RA-ILD cohort revealed that patients with ILD progression had persistently higher systemic inflammation than those without progression, particularly among the subgroup undergoing MTX treatment. INTERPRETATION: These findings highlight the need for personalized therapeutic approaches in RA-ILD, given the divergent outcomes of MTX and TNF inhibitor. FUNDING: This work was funded by GENINUS Inc., and the National Research Foundation of Korea, and Seoul National University Hospital.

WDR11, a WD protein that interacts with transcription factor EMX1, is mutated in idiopathic hypogonadotropic hypogonadism and Kallmann syndrome.

By defining the chromosomal breakpoint of a balanced t(10;12) translocation from a subject with Kallmann syndrome and scanning genes in its vicinity in unrelated hypogonadal subjects, we have identified WDR11 as a gene involved in human puberty. We found six patients with a total of five different heterozygous WDR11 missense mutations, including three alterations (A435T, R448Q, and H690Q) in WD domains important for ß propeller formation and protein-protein interaction. In addition, we discovered that WDR11 interacts with EMX1, a homeodomain transcription factor involved in the development of olfactory neurons, and that missense alterations reduce or abolish this interaction. Our findings suggest that impaired pubertal development in these patients results from a deficiency of productive WDR11 protein interaction.

Inhibition of MKK7-JNK by the TOR signaling pathway regulator-like protein contributes to resistance of HCC cells to TRAIL-induced apoptosis.

BACKGROUND & AIMS: The TOR signaling pathway regulator-like (TIPRL) protein, the mammalian ortholog of yeast TIP41, was identified in an expression profiling screen for factors that regulate human liver carcinogenesis. We investigated the role of human TIPRL protein in hepatocellular carcinoma (HCC). METHODS: We measured the level of TIPRL in HCC and adjacent nontumor tissues from patients. We used small interfering RNAs and zebrafish to study the function of TIPRL. We used annexin V propidium iodide staining and immunoblot analyses to measure apoptosis and activation of apoptotic signaling pathways. We used confocal microscopy, coimmunoprecipitation, and glutathione-S transferase pull-down analyses to determine interactions among mitogen-activated protein kinase kinase 7 (MKK7 or MAP2K7), TIPRL, and the protein phosphatase type 2A (PP2Ac). We studied the effects of TIPRL in tumor xenografts in mice. RESULTS: Levels of TIPRL were higher in HCC tissues and cell lines than nontumor tissues and primary hepatocytes. Knockdown of tiprl expression in zebrafish led to large amounts of apoptosis throughout the embryos. Incubation of HCC cells, but not primary human hepatocytes, with small interfering RNA against TIPRL (siTIPRL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) caused prolonged activation (phosphorylation) of MKK7 and c-Jun N-terminal kinase (JNK) and led to apoptosis, indicated by cleavage of procaspase-8,-3 and of poly-(adenosine diphosphate-ribose) polymerase. TIPRL bound to MKK7 and PP2Ac and promoted the interaction between MKK7 and PP2Ac. In mice, injection of HCC xenograft tumors with siTIPRL and TRAIL led to tumor apoptosis and regression. CONCLUSIONS: TIPRL is highly up-regulated in human HCC samples and cell lines, compared with noncancerous liver tissues. TIPRL prevents prolonged activation of MKK7 and JNK and TRAIL-induced apoptosis by mediating the interaction between MKK7 and PP2Ac.

Establishment of a transgenic zebrafish EF1α:Kaede for monitoring cell proliferation during regeneration.

Zebrafish is considered as a versatile experimental animal for various research models from development to diseases. In this study, we report the development of transgenic zebrafish line named as Tg(EF1α:Kaede) that expresses translation elongation factor 1 subunit alpha (EF1α) promoter linked to a fluorescent protein (FP), Kaede for monitoring proliferating cells in during regeneration. It was revealed that about 1.4 kb 5'-flanking region of the EF1α was sufficient for its promoter activity. Expression of Kaede with a property of photo-conversion from green to red was detected in different embryonic stages as well as various organs such as brain, heart, pancreas, intestine, ovary, and fins of adult fish. Cell proliferation pattern during fin regeneration was monitored after amputation of Tg(EF1α:Kaede) caudal fin and results shown that this system is simple and efficient method for detecting proliferating cells during tissue regeneration. Developed Tg(EF1α:Kaede) line has potential to investigate the cell proliferation, regeneration, wound healing capacities after tissue damage and evaluate the therapeutic power of wound healing drugs.

IFT46 gene promoter-driven ciliopathy disease model in zebrafish.

Ciliopathies are human genetic disorders caused by abnormal formation and dysfunction of cellular cilia. Cilia are microtubule-based organelles that project into the extracellular space and transduce molecular and chemical signals from the extracellular environment or neighboring cells. Intraflagellar transport (IFT) proteins are required for the assembly and maintenance of cilia by transporting proteins along the axoneme which consists of complexes A and B. IFT46, a core IFT-B protein complex, is required for cilium formation and maintenance during vertebrate embryonic development. Here, we introduce transgenic zebrafish lines under the control of ciliated cell-specific IFT46 promoter to recapitulate human ciliopathy-like phenotypes. We generated a Tg(IFT46:GAL4-VP16) line to temporo-spatially control the expression of effectors including fluorescent reporters or nitroreductase based on the GAL4/UAS system, which expresses GAL4-VP16 chimeric transcription factors in most ciliated tissues during embryonic development. To analyze the function of IFT46-expressing ciliated cells during zebrafish development, we generated the Tg(IFT46:GAL4-VP16;UAS;nfsb-mCherry) line, a ciliated cell-specific injury model induced by nitroreductase (NTR)/metrodinazole (MTZ). Conditionally, controlled ablation of ciliated cells in transgenic animals exhibited ciliopathy-like phenotypes including cystic kidneys and pericardial and periorbital edema. Altogether, we established a zebrafish NTR/MTZ-mediated ciliated cell injury model that recapitulates ciliopathy-like phenotypes and may be a vertebrate animal model to further investigate the etiology and therapeutic approaches to human ciliopathies.