Morpholino-mediated Knockdown Research Articles

N-cadherin facilitates trigeminal sensory neuron outgrowth and target tissue innervation.

Our findings are significant because they demonstrate how neurons derived from two distinct cell populations, neural crest and placode cells, coordinate the outgrowth of their axons in time and space to generate the trigeminal ganglion using the cell adhesion molecule N-cadherin. Notably, our results provide evidence for the existence of subpopulations of neurons within the trigeminal ganglion that differentially require N-cadherin to facilitate axon outgrowth, and hint at the possibility that trigeminal pioneer neurons are derived from placode cells while followers arise from both placode and neural crest cells. These studies provide new insight into trigeminal gangliogenesis that will likely be translatable to other cranial ganglia and vertebrate species.

Fbrsl1 is required for heart development in Xenopus laevis and de novo variants in FBRSL1 can cause human heart defects.

De novo truncating variants in fibrosin-like 1 (FBRSL1), a member of the AUTS2 gene family, cause a disability syndrome, including organ malformations such as heart defects. Here, we use Xenopus laevis to investigate whether Fbrsl1 plays a role in heart development. Xenopus laevis fbrsl1 is expressed in tissues relevant for heart development, and morpholino-mediated knockdown of Fbrsl1 results in severely hypoplastic hearts. Our data suggest that Fbrsl1 is required for the development of the first heart field, which contributes to the ventricle and the atria, but not for the second heart field, which gives rise to the outflow tract. The morphant heart phenotype could be rescued using a human N-terminal FBRSL1 isoform that contains an alternative exon, but lacks the AUTS2 domain. N-terminal isoforms carrying patient variants failed to rescue. Interestingly, a long human FBRSL1 isoform, harboring the AUTS2 domain, also did not rescue the morphant heart defects. Thus, our data suggest that different FBRSL1 isoforms may have distinct functions and that only the short N-terminal isoform, appears to be critical for heart development.

Differentiation and functioning of the lateral line organ in zebrafish require Smpx activity

The small muscle protein, X-linked (SMPX) gene encodes a cytoskeleton-associated protein, highly expressed in the inner ear hair cells (HCs), possibly regulating auditory function. In the last decade, several mutations in SMPX have been associated with X-chromosomal progressive non syndromic hearing loss in humans and, in line with this, Smpx-deficient animal models, namely zebrafish and mouse, showed significant impairment of inner ear HCs development, maintenance, and functioning. In this work, we uncovered smpx expression in the neuromast mechanosensory HCs of both Anterior and Posterior Lateral Line (ALL and PLL, respectively) of zebrafish larvae and focused our attention on the PLL. Smpx was subcellularly localized throughout the cytoplasm of the HCs, as well as in their primary cilium. Loss-of-function experiments, via both morpholino-mediated gene knockdown and CRISPR/Cas9 F0 gene knockout, revealed that the lack of Smpx led to fewer properly differentiated and functional neuromasts, as well as to a smaller PLL primordium (PLLp), the latter also Smpx-positive. In addition, the kinocilia of Smpx-deficient neuromast HCs appeared structurally and numerically altered. Such phenotypes were associated with a significant reduction in the mechanotransduction activity of the neuromast HCs, in line with their positivity for Smpx. In summary, this work highlights the importance of Smpx in lateral line development and, specifically, in proper HCs differentiation and/or maintenance, and in the mechanotransduction process carried out by the neuromast HCs. Because lateral line HCs are both functionally and structurally analogous to the cochlear HCs, the neuromasts might represent an invaluable—and easily accessible—tool to dissect the role of Smpx in HCs development/functioning and shed light on the underlying mechanisms involved in hearing loss.

The transcription of the main gene associated with Treacher–Collins syndrome (TCOF1) is regulated by G-quadruplexes and cellular nucleic acid binding protein (CNBP)

Treacle ribosome biogenesis factor 1 (TCOF1) is responsible for about 80% of mandibular dysostosis (MD) cases. We have formerly identified a correlation between TCOF1 and CNBP (CCHC-type zinc finger nucleic acid binding protein) expression in human mesenchymal cells. Given the established role of CNBP in gene regulation during rostral development, we explored the potential for CNBP to modulate TCOF1 transcription. Computational analysis for CNBP binding sites (CNBP-BSs) in the TCOF1 promoter revealed several putative binding sites, two of which (Hs791 and Hs2160) overlap with putative G-quadruplex (G4) sequences (PQSs). We validated the folding of these PQSs measuring circular dichroism and fluorescence of appropriate synthetic oligonucleotides. In vitro studies confirmed binding of purified CNBP to the target PQSs (both folded as G4 and unfolded) with Kd values in the nM range. ChIP assays conducted in HeLa cells chromatin detected the CNBP binding to TCOF1 promoter. Transient transfections of HEK293 cells revealed that Hs2160 cloned upstream SV40 promoter increased transcription of downstream firefly luciferase reporter gene. We also detected a CNBP-BS and PQS (Dr2393) in the zebrafish TCOF1 orthologue promoter (nolc1). Disrupting this G4 in zebrafish embryos by microinjecting DNA antisense oligonucleotides complementary to Dr2393 reduced the transcription of nolc1 and recapitulated the craniofacial anomalies characteristic of Treacher Collins Syndrome. Both cnbp overexpression and Morpholino-mediated knockdown in zebrafish induced nolc1 transcription. These results suggest that CNBP modulates the transcriptional expression of TCOF1 through a mechanism involving G-quadruplex folding/unfolding, and that this regulation is active in vertebrates as distantly related as bony fish and humans. These findings may have implications for understanding and treating MD.

Apela promotes blood vessel regeneration and remodeling in zebrafish

In contrast to adult mammals, zebrafish display a high capacity to heal injuries and repair damage to various organs. One of the earliest responses to injury in adult zebrafish is revascularization, followed by tissue morphogenesis. Tissue vascularization entails the formation of a blood vessel plexus that remodels into arteries and veins. The mechanisms that coordinate these processes during vessel regeneration are poorly understood. Hence, investigating and identifying the factors that promote revascularization and vessel remodeling have great therapeutic potential. Here, we revealed that fin vessel remodeling critically depends on Apela peptide. We found that Apela selectively accumulated in newly formed zebrafish fin tissue and vessels. The temporal expression of Apela, Apln, and their receptor Aplnr is different during the regenerative process. While morpholino-mediated knockdown of Apela (Mo-Apela) prevented vessel remodeling, exogenous Apela peptide mediated plexus repression and the development of arteries in regenerated fins. In contrast, Apela enhanced subintestinal venous plexus formation (SIVP). The use of sunitinib completely inhibited vascular plexus formation in zebrafish, which was not prevented by exogenous application. Furthermore, Apela regulates the expression of vessel remolding-related genes including VWF, IGFPB3, ESM1, VEGFR2, Apln, and Aplnr, thereby linking Apela to the vascular plexus factor network as generated by the STRING online database. Together, our findings reveal a new role for Apela in vessel regeneration and remodeling in fin zebrafish and provide a framework for further understanding the cellular and molecular mechanisms involved in vessel regeneration.

Identification of End-Binding 1 Protein as Novel α-4 Giardin-Binding Partners in Giardia lamblia Trophozoites.

Giardia lamblia (syn. G. intestinalis, G. duodenalis) is a primitive opportunistic protozoon, and one of the earliest differentiated eukaryotes. Despite its primitive nature, G. lamblia has a sophisticated cytoskeleton system, which is closely related to its proliferation and pathogenicity. Meanwhile, α giardin is a G. lamblia-specific cytoskeleton protein, which belongs to the annexin superfamily. Interestingly, G. lamblia has 21 annexin-like α giardins, i.e., more than higher eukaryotes. The functional differences among α giardin members are not fully understood. We took α-4 giardin, a member of α giardin family, as a research object. A morpholino-mediated knockdown experiment was performed to identify the effect of α-4 giardin on G. lamblia trophozoites biological traits. A yeast two-hybrid cDNA library of G. lamblia strain C2 trophozoites was screened for interaction partners of α-4 giardin. Co-immunoprecipitation and fluorescent colocalization confirmed the relationship between G. lamblia EB1 (gEB1) and α-4 giardin. α-4 Giardin could inhibit the proliferation and adhesion of G. lamblia trophozoites. In addition, it interacted with G. lamblia EB1 (gEB1). α-4 Giardin was involved in proliferation and adhesion in G. lamblia trophozoites, and EB1, a crucial roles in mitosis, was an interacting partner of α-4 giardin.

Developmental adcyap1b loss leads to hemorrhage, disrupted hemostasis, and a blood coagulation cascade in zebrafish

BackgroundPituitary adenylate cyclase–activating polypeptide is a neuropeptide with diverse roles in biological processes. Its involvement in the blood coagulation cascade is unclear. ObjectivesThis study unraveled adcyap1b’s role in blood coagulation using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 in zebrafish. Effects were validated via adcyap1b knockdown. Gene expression changes in adcyap1b mutants were explored, linking them to clotting disorders. An analysis of proca gene splicing illuminated its role in adcyap1b-related anticoagulation deficiencies. MethodsZebrafish were genetically modified using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 to induce adcyap1b knockout. Morpholino-mediated gene knockdown was employed for validation. Expression levels of coagulation factors, anticoagulant proteins, and fibrinolytic system genes were assessed in adcyap1b mutant zebrafish. Alternative splicing of proca gene was analyzed. ResultsAdcyap1b mutant zebrafish exhibited severe hemorrhage, clotting disorders, and disrupted blood coagulation. Morpholino-mediated knockdown replicated observed phenotypes. Downregulation in transcripts related to coagulation factors V and IX, anticoagulation protein C, and plasminogen was observed. Abnormal alternative splicing of the proca gene was identified, providing a mechanistic explanation for anticoagulation system deficiencies. ConclusionAdcyap1b plays a crucial role in maintaining zebrafish blood coagulation and hemostasis. Its influence extends to the regulation of procoagulant and anticoagulant pathways, with abnormal alternative splicing contributing to observed deficiencies. These findings unveil a novel aspect of adcyap1b function, offering potential insights into similar processes in mammalian systems.

Leukotriene B4 receptor 2 governs macrophage migration during tissue inflammation

Chronic inflammation is the underlying cause of many diseases, including type 1 diabetes, obesity, and non-alcoholic fatty liver disease. Macrophages are continuously recruited to tissues during chronic inflammation where they exacerbate or resolve the pro-inflammatory environment. Although leukotriene B4 receptor 2 (BLT2) has been characterized as a low affinity receptor to several key eicosanoids and chemoattractants, its precise roles in the setting of inflammation and macrophage function remain incompletely understood. Here we used zebrafish and mouse models to probe the role of BLT2 in macrophage function during inflammation. We detected BLT2 expression in bone marrow derived and peritoneal macrophages of mouse models. Transcriptomic analysis of Ltb4r2-/- and WT macrophages suggested a role for BLT2 in macrophage migration, and studies invitro confirmed that whereas BLT2 does not mediate macrophage polarization, it is required for chemotactic function, possibly mediated by downstream genes Ccl5 and Lgals3. Using a zebrafish model of tailfin injury, we demonstrated that antisense morpholino-mediated knockdown of blt2a or chemical inhibition of BLT2 signaling impairs macrophage migration. We further replicated these findings in zebrafish models of islet injury and liver inflammation. Moreover, we established the applicability of our zebrafish findings to mammals by showing that macrophages of Ltb4r2-/- mice have defective migration during lipopolysaccharide stimulation invivo. Collectively, our results demonstrate that BLT2 mediates macrophage migration during inflammation, which implicates it as a potential therapeutic target for inflammatory pathologies.

The Crucial Role of CTCF in Mitotic Progression during Early Development of Sea Urchin.

CCCTC-binding factor (CTCF), an insulator protein with 11 zinc fingers, is enriched at the boundaries of topologically associated domains (TADs) in eukaryotic genomes. In this study, we isolated and analyzed the cDNAs encoding HpCTCF, the CTCF homolog in the sea urchin Hemicentrotus pulcherrimus, to investigate its expression patterns and functions during early development of sea urchin. HpCTCF contains nine zinc fingers corresponding to fingers 2-10 of the vertebrate CTCF. The expression pattern analysis revealed that HpCTCF mRNA was detected at all developmental stages and in the entire embryo. Upon expressing the HpCTCF-GFP fusion protein in early embryos, we observed its uniform distribution within interphase nuclei. However, during mitosis, it disappeared from the chromosomes and subsequently reassembled on the chromosome during telophase. Moreover, the morpholino-mediated knockdown of HpCTCF resulted in mitotic arrest during the morula-to-blastula stage. Most of the arrested chromosomes were not phospholylated at serine 10 of histone H3, indicating that mitosis was arrested at the telophase by HpCTCF depletion. Furthermore, impaired sister chromatid segregation was observed using time-lapse imaging of HpCTCF-knockdown embryos. Thus, HpCTCF is essential for mitotic progression during the early development of sea urchins, especially during the telophase-to-interphase transition. However, the normal development of pluteus larvae in CRISPR-mediated HpCTCF-knockout embryos suggests that disruption of zygotic HpCTCF expression has little effect on embryonic and larval development. This article is protected by copyright. All rights reserved.

Plexina4 and cell survival in the developing zebrafish hindbrain.

Growth factors are important in the developing and mature nervous system to support the survival of neurons. Developmental signaling molecules are known for their roles in controlling neurogenesis and neural circuit formation. Whether or not these molecules also have roles in cell survival in the developing nervous system is poorly understood. Plexins are a family of transmembrane receptors that bind Semaphorin ligands and are known to function in the guidance of developing axons and blood vessels. In embryonic zebrafish, plexina4 is expressed widely in the brain, becoming largely restricted to the hindbrain as neurogenesis and differentiation proceed. Apoptosis is increased in the embryonic hindbrain of a plexina4ca307/ca307 CRISPR mutant. Based on the literature, we tested the secreted heat shock protein, Clusterin, as a candidate ligand to mediate cell survival through Plexina4. clusterin is expressed by the floor plate of the embryonic zebrafish hindbrain, in proximity to plexina4-expressing hindbrain cells. Morpholino-mediated knockdown of Clusterin increases cell apoptosis in the hindbrain, with additional cell death observed in epistasis experiments where Clusterin is knocked down in a plexina4 mutant background. Our data suggest that Plexina4 promotes cell survival in the developing zebrafish hindbrain, likely through a pathway independent of Clusterin.

Functional Differentiation of Cyclins and Cyclin-Dependent Kinases in Giardia lamblia.

Cyclin-dependent kinases (CDKs) are serine/threonine kinases that control the eukaryotic cell cycle. Limited information is available on Giardia lamblia CDKs (GlCDKs), GlCDK1 and GlCDK2. After treatment with the CDK inhibitor flavopiridol-HCl (FH), division of Giardia trophozoites was transiently arrested at the G1/S phase and finally at the G2/M phase. The percentage of cells arrested during prophase or cytokinesis increased, whereas DNA synthesis was not affected by FH treatment. Morpholino-mediated depletion of GlCDK1 caused arrest at the G2/M phase, while GlCDK2 depletion resulted in an increase in the number of cells arrested at the G1/S phase and cells defective in mitosis and cytokinesis. Coimmunoprecipitation experiments with GlCDKs and the nine putative G. lamblia cyclins (Glcyclins) identified Glcyclins 3977/14488/17505 and 22394/6584 as cognate partners of GlCDK1 and GlCDK2, respectively. Morpholino-based knockdown of Glcyclin 3977 or 22394/6584 arrested cells in the G2/M phase or G1/S phase, respectively. Interestingly, GlCDK1- and Glcyclin 3977-depleted Giardia showed significant flagellar extension. Altogether, our results suggest that GlCDK1/Glcyclin 3977 plays an important role in the later stages of cell cycle control and in flagellar biogenesis. In contrast, GlCDK2 along with Glcyclin 22394 and 6584 functions from the early stages of the Giardia cell cycle. IMPORTANCE Giardia lamblia CDKs (GlCDKs) and their cognate cyclins have not yet been studied. In this study, the functional roles of GlCDK1 and GlCDK2 were distinguished using morpholino-mediated knockdown and coimmunoprecipitation. GlCDK1 with Glcyclin 3977 plays a role in flagellum formation as well as cell cycle control of G. lamblia, whereas GlCDK2 with Glcyclin 22394/6584 is involved in cell cycle control.

CCN6 influences transcription and controls mitochondrial mass and muscle organization.

Mutations in Cellular Communication Network Factor 6 (CCN6) are linked to the debilitating musculoskeletal disease Progressive Pseudo Rheumatoid Dysplasia (PPRD), which disrupts mobility. Yet, much remains unknown about CCN6 function at the molecular level. In this study, we revealed a new function of CCN6 in transcriptional regulation. We demonstrated that CCN6 localizes to chromatin and associates with RNA Polymerase II in human chondrocyte lines. Using zebrafish as a model organism we validated the nuclear presence of CCN6 and its association with RNA Polymerase II in different developmental stages from 10 hpf embryo to adult fish muscle. In concurrence with these findings, we confirmed the requirement of CCN6 in the transcription of several genes encoding mitochondrial electron transport complex proteins in the zebrafish, both in the embryonic stages and in the adult muscle. Reduction in the expression of these genes upon morpholino-mediated knockdown of CCN6 protein expression led to reduced mitochondrial mass, which correlated with defective myotome organization during zebrafish muscle development. Overall, this study suggests that the developmental musculoskeletal abnormalities linked with PPRD could be contributed at least partly by impaired expression of genes encoding mitochondrial electron transport complexes due to defects in CCN6 associated transcriptional regulation.

Effects of Morpholino-mediated knockdown of the Angiotensin-converting enzyme 2 (ACE-2) on dopamine Neurogenesis and the Hypothalamic Pituitary-adrenal (HPA) Axis in Larval Zebrafish (Danio rerio)

Effects of Morpholino-mediated knockdown of the Angiotensin-converting enzyme 2 (ACE-2) on dopamine Neurogenesis and the Hypothalamic Pituitary-adrenal (HPA) Axis in Larval Zebrafish (Danio rerio)

Identification of the genes encoding candidate septate junction components expressed during early development of the sea urchin, Strongylocentrotus purpuratus, and evidence of a role for Mesh in the formation of the gut barrier

Septate junctions (SJs) evolved as cell-cell junctions that regulate the paracellular barrier and integrity of epithelia in invertebrates. Multiple morphological variants of SJs exist specific to different epithelia and/or phyla but the biological significance of varied SJ morphology is unclear because the knowledge of the SJ associated proteins and their functions in non-insect invertebrates remains largely unknown. Here we report cell-specific expression of nine candidate SJ genes in the early life stages of the sea urchin Strongylocentrotus purpuratus. By use of in situ RNA hybridization and single cell RNA-seq we found that the expression of selected genes encoding putatively SJ associated transmembrane and cytoplasmic scaffold molecules was dynamically regulated during epithelial development in the embryos and larvae with different epithelia expressing different cohorts of SJ genes. We focused a functional analysis on SpMesh, a homolog of the Drosophila smooth SJ component Mesh, which was highly enriched in the endodermal epithelium of the mid- and hindgut. Functional perturbation of SpMesh by both CRISPR/Cas9 mutagenesis and vivo morpholino-mediated knockdown shows that loss of SpMesh does not disrupt the formation of the gut epithelium during gastrulation. However, loss of SpMesh resulted in a severely reduced gut-paracellular barrier as quantitated by increased permeability to 3–5 ​kDa FITC-dextran. Together, these studies provide a first look at the molecular SJ physiology during the development of a marine organism and suggest a shared role for Mesh-homologous proteins in forming an intestinal barrier in invertebrates. Results have implications for consideration of the traits underlying species-specific sensitivity of marine larvae to climate driven ocean change.

Non-additive mixture effects of benzo[a]pyrene and pesticides in vitro and in vivo: Role of AhR signaling

Polycyclic aromatic hydrocarbons (PAHs) and pesticides are two major groups of environmental contaminants which humans are simultaneously exposed to. However, potential mixture interactions of these groups of chemicals are not well-studied. In this study, the effects of binary mixtures of the PAH benzo[a]pyrene (B[a]P) and the commonly used pesticides chlorpyrifos, paraquat and tebuconazole on human liver HepG2 cells were investigated. The results showed that binary mixtures of B[a]P and paraquat or tebuconazole mainly caused additive effects on cell viability and cytochrome P4501a1 (CYP1A1) expression compared to single compound exposures. In contrast, the binary mixture with chlorpyrifos interacted antagonistically on cell viability and ROS production, whereas synergistic effects were observed for induction of CYP1A1 expression. B[a]P and chlorpyrifos also inhibited the activity of recombinant human CYP1A1 enzyme. To verify the synergistic in vitro results, zebrafish (Danio rerio) embryos were exposed to binary mixtures of B[a]P and chlorpyrifos. The mixtures caused synergistic induction of CYP1A expression, as well as synergistic developmental toxicity on multiple endpoints including non-inflated swim bladder, yolk-sac and pericardial edema, and spinal deformation. The effects were reduced upon morpholino-mediated knockdown of the aryl hydrocarbon receptor (AhR), indicating an AhR-dependence of the synergistic toxicity. Altogether, these data suggest that the combination of AhR activation and CYP1A1 inhibition is responsible for the underlying non-additive interaction between B[a]P and chlorpyrifos in vitro and in vivo.

Biallelic mutations of TTC12 and TTC21B were identified in Chinese patients with multisystem ciliopathy syndromes

BackgroundAbnormalities in cilia ultrastructure and function lead to a range of human phenotypes termed ciliopathies. Many tetratricopeptide repeat domain (TTC) family members have been reported to play critical roles in cilium organization and function.ResultsHere, we describe five unrelated family trios with multisystem ciliopathy syndromes, including situs abnormality, complex congenital heart disease, nephronophthisis or neonatal cholestasis. Through whole-exome sequencing and Sanger sequencing confirmation, we identified compound heterozygous mutations of TTC12 and TTC21B in six affected individuals of Chinese origin. These nonsynonymous mutations affected highly conserved residues and were consistently predicted to be pathogenic. Furthermore, ex vivo cDNA amplification demonstrated that homozygous c.1464 + 2 T > C of TTC12 would cause a whole exon 16 skipping. Both mRNA and protein levels of TTC12 were significantly downregulated in the cells derived from the patient carrying TTC12 mutation c.1464 + 2 T > C by real-time qPCR and immunofluorescence assays when compared with two healthy controls. Transmission electron microscopy analysis further identified ultrastructural defects of the inner dynein arms in this patient. Finally, the effect of TTC12 deficiency on cardiac LR patterning was recapitulated by employing a morpholino-mediated knockdown of ttc12 in zebrafish.ConclusionsTo the best of our knowledge, this is the first study reporting the association between TTC12 variants and ciliopathies in a Chinese population. In addition to nephronophthisis and laterality defects, our findings demonstrated that TTC21B should also be considered a candidate gene for biliary ciliopathy, such as TTC26, which further expands the phenotypic spectrum of TTC21B deficiency in humans.

Characterization of two novel ammonia transporters, Hiat1a and Hiat1b, in the teleost model system Danio rerio.

Ammonia excretion in fish excretory epithelia is a complex interplay of multiple membrane transport proteins and mechanisms. Using the model system of zebrafish (Danio rerio) larvae, here we identified three paralogues of a novel ammonia transporter, hippocampus-abundant transcript 1 (DrHiat1), also found in most vertebrates. When functionally expressed in Xenopus laevis oocytes, DrHiat1a and DrHiat1b promoted methylamine uptake in a competitive manner with ammonia. In situ hybridization experiments showed that both transporters were expressed as early as the 4-cell stage in zebrafish embryos and could be identified in most tissues 4days post-fertilization. Larvae experiencing morpholino-mediated knockdown of DrHiat1b exhibited significantly lower whole-body ammonia excretion rates compared with control larvae. Markedly decreased site-specific total ammonia excretion of up to 85% was observed in both the pharyngeal region (site of developing gills) and the yolk sac (region shown to have the highest NH4+ flux). This study is the first to identify DrHiat1b/DrHIAT1 in particular as an important contributor to ammonia excretion in larval zebrafish. Being evolutionarily conserved, these proteins are likely involved in multiple other general ammonia-handling mechanisms, making them worthy candidates for future studies on nitrogen regulation in fishes and across the animal kingdom.

Ifi30 Is Required for Sprouting Angiogenesis During Caudal Vein Plexus Formation in Zebrafish.

Interferon-gamma-inducible protein 30 (IFI30) is a critical enzyme that mainly exists in immune cells and functions in reducing protein disulfide bonds in endocytosis-mediated protein degradation. Regardless of this, it is also found to be expressed in vascular system. However, the functions of IFI30 in vascular development remains unknown. Vascular network formation is a tightly controlled process coordinating a series of cell behaviors, including endothelial cell (EC) sprouting, proliferation, and anastomosis. In this work, we analyzed the function of zebrafish Ifi30, orthologous to the human IFI30, in vascular development during embryogenesis. The ifi30 gene was found to be highly expressed in the caudal vein plexus (CVP) region of zebrafish embryos. Morpholino-mediated Ifi30 knockdown in zebrafish resulted in incomplete CVP formation with reduced loop numbers, area, and width. Further analyses implied that Ifi30 deficiency impaired cell behaviors of both ECs and macrophages, including cell proliferation and migration. Here, we demonstrate a novel role of IFI30, which was originally identified as a lysosomal thiol reductase involved in immune responses, in CVP development during embryogenesis. Our results suggest that Ifi30 is required for sprouting angiogenesis during CVP formation, which may offer an insight into the function of human IFI30 in angiogenesis under physiological or pathological conditions.

Elucidating the Role of N‐cadherin during Trigeminal Gangliogenesis

Cadherin‐based adhesion is critical to the formation of multicellular tissues, including the cranial sensory ganglia of the peripheral nervous system. Initial assembly of the cranial trigeminal ganglion, which houses the cell bodies for the trigeminal nerve, relies on the coalescence of N‐cadherin (N‐cad)‐expressing placode cell‐derived neurons and Cadherin‐7‐expressing neural crest cells. The trigeminal ganglion has a role that is primarily sensory, transmitting information on pain, touch, and temperature from different facial regions back to the central nervous system. Proper survival and differentiation of trigeminal neurons relies, in part, upon binding of neurotrophic and other growth factors secreted from target tissues to their cognate receptors found on peripheral axons. These complexes are then trafficked to neuronal cell bodies, resulting in the activation of downstream signaling pathways. Prior studies have shown that N‐cad internalization and trafficking are required for proper neuronal outgrowth. Moreover, N‐cad knockdown in chick trigeminal placode cells leads to early defects in trigeminal ganglion assembly by increasing placodal neuron dispersal. Later functions for N‐cad in chick trigeminal gangliogenesis, however, remain obscure. We hypothesize that N‐cad controls trigeminal ganglion development through the regulation of multiple signaling pathways. To address this, we performed morpholino‐mediated knockdown of N‐cad in chick trigeminal placode cells followed by immunohistochemistry and biochemistry to evaluate trigeminal gangliogenesis. Our preliminary data show that N‐cad depletion has a sustained negative impact on the trigeminal ganglion through cell and non‐cell autonomous effects, resulting in decreased ganglion size, aberrant nerve outgrowth and branching, and reduced levels of Cadherin‐7, neurotrophins and their receptors, and Smad family members. Future studies will aim to elucidate the mechanisms by which N‐cad controls trigeminal ganglion development via these molecules and pathways, including experiments designed to examine protein trafficking in trigeminal neurons. Taken together, our findings reveal potential adhesion‐dependent and ‐independent functions for N‐cad in the trigeminal ganglion, which will aid in the understanding of its tissue‐specific roles. This knowledge will shed light on the etiology of disorders characterized by abnormal neural crest cell and placode cell development and those affecting the peripheral nervous system.

The CHARGE syndrome ortholog CHD-7 regulates TGF-β pathways in Caenorhabditis elegans

CHARGE syndrome is a complex developmental disorder caused by mutations in the chromodomain helicase DNA-binding protein-7 (CHD7) and characterized by retarded growth and malformations in the heart and nervous system. Despite the public health relevance of this disorder, relevant cellular pathways and targets of CHD7 that relate to disease pathology are still poorly understood. Here we report that chd-7, the nematode ortholog of Chd7, is required for dauer morphogenesis, lifespan determination, stress response, and body size determination. Consistent with our discoveries, we found chd-7 to be allelic to scd-3, a previously identified dauer suppressor from the DAF-7/ tumor growth factor-β (TGF-β) pathway. Epistatic analysis places CHD-7 at the level of the DAF-3/DAF-5 complex, but we found that CHD-7 also directly impacts the expression of multiple components of this pathway. Transcriptomic analysis revealed that chd-7 mutants fail to repress daf-9 for execution of the dauer program. In addition, CHD-7 regulates the DBL-1/BMP pathway components and shares roles in male tail development and cuticle synthesis. To explore a potential conserved function for chd-7 in vertebrates, we used Xenopus laevis embryos, an established model to study craniofacial development. Morpholino-mediated knockdown of Chd7 led to a reduction in col2a1 messenger RNA (mRNA) levels, a collagen whose expression depends on TGF-β signaling. Both embryonic lethality and craniofacial defects in Chd7-depleted tadpoles were partially rescued by overexpression of col2a1 mRNA. We suggest that Chd7 has conserved roles in regulation of the TGF-β signaling pathway and pathogenic Chd7 could lead to a defective extracellular matrix deposition.