Mutant Zebrafish Research Articles

Prox1a promotes liver growth and differentiation by repressing cdx1b expression and intestinal fate transition in zebrafish

The liver is a key endoderm-derived multifunctional organ within the digestive system. Prospero homeobox 1 (Prox1) is an essential transcription factor for liver development, but its specific function is not well understood. Here, we show that hepatic development, including the formation of intrahepatic biliary and vascular networks, is severely disrupted in prox1a mutant zebrafish. We find that Prox1a is essential for liver growth and proper differentiation but not required for early hepatic cell fate specification. Intriguingly, prox1a depletion leads to ectopic initiation of a Cdx1b-mediated intestinal program and the formation of intestinal lumen-like structures within the liver. Morpholino knockdown of cdx1b alleviates liver defects in the prox1a mutant zebrafish. Finally, chromatin immunoprecipitation analysis reveals that Prox1a binds directly to the promoter region of cdx1b, thereby repressing its expression. Overall, our findings indicate that Prox1a is required to promote and protect hepatic development by repression of Cdx1b-mediated intestinal cell fate in zebrafish.

The people behind the papers - Joey Ghersi and Stefania Nicoli.

Akt is believed to be important for vascular development, but complex tissue-specific loss-of-function studies have been difficult in mice because of embryonic lethality. In a new study, Wenping Zhou, Joey Ghersi, Stefania Nicoli and colleagues report the phenotype of a complete Akt mutant zebrafish and its role in arterial specification via Notch signaling. To learn more about their work, we spoke with corresponding authors Joey Ghersi, Assistant Professor at Centre Hospitalier Universitaire (CHU) Sainte-Justine, Canada, and Stefania Nicoli, Associate Professor Tenure at Yale University, USA.

Genetic and Environmental Factors Co-Contributing to Behavioral Abnormalities in adnp/adnp2 Mutant Zebrafish.

Human mutations of ADNP and ADNP2 are known to be associated with neural developmental disorders (NDDs), including autism spectrum disorders (ASDs) and schizophrenia (SZ). However, the underlying mechanisms remain elusive. In this study, using CRISPR/Cas9 gene editing technology, we generated adnp and adnp2 mutant zebrafish models, which exhibited developmental delays, brain deficits, and core behavioral features of NDDs. RNA sequencing analysis of adnpa-/-; adnpb-/- and adnp2a-/-; adnp2b-/- larval brains revealed altered gene expression profiles affecting synaptic transmission, autophagy, apoptosis, microtubule dynamics, hormone signaling, and circadian rhythm regulation. Validation using whole-mount in situ hybridization (WISH) and real-time quantitative PCR (qRT-PCR) corroborated these findings, supporting the RNA-seq results. Additionally, loss of adnp and adnp2 resulted in significant downregulation of pan-neuronal HuC and neuronal fiber network α-Tubulin signals. Importantly, prolonged low-dose exposure to environmental endocrine disruptors (EEDs) aggravated behavioral abnormalities in adnp and adnp2 mutants. This comprehensive approach enhances our understanding of the complex interplay between genetic mutations and environmental factors in NDDs. Our findings provide novel insights and experimental foundations into the roles of adnp and adnp2 in neurodevelopment and behavioral regulation, offering a framework for future preclinical drug screening aimed at elucidating the pathogenesis of NDDs and related conditions.

Photoreceptor regeneration occurs normally in microglia-deficient irf8 mutant zebrafish following acute retinal damage

Microglia are resident immune cells in the central nervous system, including the retina that surveil the environment for damage and infection. Following retinal damage, microglia undergo morphological changes, migrate to the site of damage, and express and secrete pro-inflammatory signals. In the zebrafish retina, inflammation induces the reprogramming and proliferation of Müller glia and the regeneration of neurons following damage or injury. Immunosuppression or pharmacological ablation of microglia reduce or abolish Müller glia proliferation. We evaluated the retinal architecture and retinal regeneration in adult zebrafish irf8 mutants, which have significantly depleted numbers of microglia. We show that irf8 mutants have normal retinal structure at 3 months post fertilization (mpf) and 6 mpf but fewer cone photoreceptors by 10 mpf. Surprisingly, light-induced photoreceptor ablation induced Müller glia proliferation in irf8 mutants and cone and rod photoreceptor regeneration. Light-damaged retinas from both wild-type and irf8 mutants show upregulated expression of mmp-9, il8, and tnfβ pro-inflammatory cytokines. Our data demonstrate that adult zebrafish irf8 mutants can regenerate normally following acute retinal injury. These findings suggest that microglia may not be essential for retinal regeneration in zebrafish and that other mechanisms can compensate for the reduction in microglia numbers.

The Accordion Zebrafish tq206 Mutant in the Assessment of a Novel Pharmaceutical Approach to Brody Myopathy.

Brody disease (BD) is an "ultra-rare" human genetic disorder of skeletal muscle function due to defects in the atp2a1 gene causing deficiency of the SERCA protein, isoform1. The main clinical signs are exercise-induced stiffness and delayed muscular relaxation after physical exercises, even mild ones. No mouse model nor specific therapies exist for Brody myopathy, which is therefore considered an orphan disease. Bovine congenital pseudomyotonia (PMT) is a muscular disorder characterized by an impairment of muscle relaxation and is the only mammalian model of human BD. The pathogenetic mechanism underlying bovine PMT has been recently clarified. These findings prompted us to purpose a potential pharmacological approach addressing a specific population of BD patients who exhibit reduced expression but still exhibit activity of the SERCA1 pump. Preclinical research involving in vivo studies is essential and necessary before clinical trials can be pursued and SERCA protein shows a high degree of conservation among species. So far, the only animal models available to study BD in vivo are a group of zebrafish mutant lines known as accordion zebrafish (acc). In this paper, we focused on a comprehensive characterization of the "acctq206" zebrafish variant. Our aim was to use this mutant line as an experimental animal model for testing the novel therapeutic approach for BD.

Functional analysis of ESRP1/2 gene variants and CTNND1 isoforms in orofacial cleft pathogenesis

Orofacial cleft (OFC) is a common human congenital anomaly. Epithelial-specific RNA splicing regulators ESRP1 and ESRP2 regulate craniofacial morphogenesis and their disruption result in OFC in zebrafish, mouse and humans. Using esrp1/2 mutant zebrafish and murine Py2T cell line models, we functionally tested the pathogenicity of human ESRP1/2 gene variants. We found that many variants predicted by in silico methods to be pathogenic were functionally benign. Esrp1 also regulates the alternative splicing of Ctnnd1 and these genes are co-expressed in the embryonic and oral epithelium. In fact, over-expression of ctnnd1 is sufficient to rescue morphogenesis of epithelial-derived structures in esrp1/2 zebrafish mutants. Additionally, we identified 13 CTNND1 variants from genome sequencing of OFC cohorts, confirming CTNND1 as a key gene in human OFC. This work highlights the importance of functional assessment of human gene variants and demonstrates the critical requirement of Esrp-Ctnnd1 acting in the embryonic epithelium to regulate palatogenesis.

Voltage-gated calcium channels generate blastema Ca 2+ fluxes restraining zebrafish fin regenerative outgrowth.

Adult zebrafish fins regenerate to their original size regardless of damage extent, providing a tractable model of organ size and scale control. Gain-of-function of voltage-gated K + channels expressed in fibroblast-lineage blastema cells promotes excessive fin outgrowth, leading to a long-finned phenotype. Similarly, inhibition of the Ca 2+ -dependent phosphatase calcineurin during regeneration causes dramatic fin overgrowth. However, Ca 2+ fluxes and their potential origins from dynamic membrane voltages have not been explored or linked to fin size restoration. We used fibroblast-lineage GCaMP imaging of regenerating adult fins to identify dynamic and heterogeneous Ca 2+ transients in distal blastema cells. Membrane depolarization of isolated regenerating fin fibroblasts triggered Ca 2+ spikes dependent on voltage-gated Ca 2+ channel activity. Single cell transcriptomics identified the voltage-gated Ca 2+ channels cacna1c (L-type channel), cacna1ba (N-type), and cacna1g (T-type) as candidate mediators of fibroblast-lineage Ca 2+ signaling. Small molecule inhibition revealed L- and/or N-type voltage-gated Ca 2+ channels act during regenerative outgrowth to restore fins to their original scale. Strikingly, cacna1g homozygous mutant zebrafish regenerated extraordinarily long fins due to prolonged outgrowth. The regenerated fins far exceeded their original length but with otherwise normal ray skeletons. Therefore, cacna1g mutants uniquely provide a genetic loss-of-function long-finned model that decouples developmental and regenerative fin outgrowth. Live GCaMP imaging of regenerating fins showed T-type Cacna1g channels enable Ca 2+ dynamics in distal fibroblast-lineage blastemal mesenchyme during the outgrowth phase. We conclude "bioelectricity" for fin size control likely entirely reflects voltage-modulated Ca 2+ dynamics in fibroblast-lineage blastemal cells that specifically and steadily decelerates outgrowth at a rate tuned to restore the original fin size.

Activation of Nrf2 at Critical Windows of Development Alters Tissue-Specific Protein S-Glutathionylation in the Zebrafish (Danio rerio) Embryo.

Activation of Nrf2-the master regulator of antioxidative response-at different stages of embryonic development has been shown to result in changes in gene expression, but the tissue-specific and downstream effects of Nrf2 activation during development remain unclear. This work seeks to elucidate the tissue-specific Nrf2 cellular localization and the downstream changes in protein S-glutathionylation during critical windows of zebrafish (Danio rerio) development. Wild-type and mutant zebrafish embryos with a loss-of-function mutation in Nrf2a were treated with two canonical activators, sulforaphane (SFN; 40 µM) or tert-butylhydroquinone (tBHQ; 1 µM), for 6 h at either pharyngula, hatching, or the protruding-mouth stage. Nrf2a protein and S-glutathionylation were visualized in situ using immunohistochemistry. At the hatching stage, Nrf2a protein levels were decreased with SFN, but not tBHQ, exposure. Exposure to both activators, however, decreased downstream S-glutathionylation. Stage- and tissue-specific differences in Nrf2a protein and S-glutathionylation were identified in the pancreatic islet and liver. Protein S-glutathionylation in Nrf2a mutant fish was increased in the liver by both activators, but not the islets, indicating a tissue-specific and Nrf2a-dependent dysregulation. This work demonstrates that critical windows of exposure and Nrf2a activity may influence redox homeostasis and highlights the importance of considering tissue-specific outcomes and sensitivity in developmental redox biology.

Lowered GnT-I Activity Decreases Complex-Type N-Glycan Amounts and Results in an Aberrant Primary Motor Neuron Structure in the Spinal Cord.

The attachment of sugar to proteins and lipids is a basic modification needed for organismal survival, and perturbations in glycosylation cause severe developmental and neurological difficulties. Here, we investigated the neurological consequences of N-glycan populations in the spinal cord of Wt AB and mgat1b mutant zebrafish. Mutant fish have reduced N-acetylglucosaminyltransferase-I (GnT-I) activity as mgat1a remains intact. GnT-I converts oligomannose N-glycans to hybrid N-glycans, which is needed for complex N-glycan production. MALDI-TOF MS profiles identified N-glycans in the spinal cord for the first time and revealed reduced amounts of complex N-glycans in mutant fish, supporting a lesion in mgat1b. Further lectin blotting showed that oligomannose N-glycans were more prevalent in the spinal cord, skeletal muscle, heart, swim bladder, skin, and testis in mutant fish relative to WT AB, supporting lowered GnT- I activity in a global manner. Developmental delays were noted in hatching and in the swim bladder. Microscopic images of caudal primary (CaP) motor neurons of the spinal cord transiently expressing EGFP in mutant fish were abnormal with significant reductions in collateral branches. Further motor coordination skills were impaired in mutant fish. We conclude that identifying the neurological consequences of aberrant N-glycan processing will enhance our understanding of the role of complex N-glycans in development and nervous system health.

Deficiency of leap2 promotes somatic growth in zebrafish: Involvement of the growth hormone system

PurposeLiver-expressed antimicrobial peptide-2 (LEAP2) is identified as an endogenous antagonist and inverse agonist of the growth hormone secretagogue receptor type 1a (GHSR1a), its effect on the GHSR1a is contrary to the role of GHRELIN. Growth hormone (GH) is a crucial hormone for early development. Previous studies report that LEAP2 dose-dependently attenuates ghrelin-induced GH secretion, and Leap2-knockout mice exhibit increased plasma GH levels after GHRELIN administration. Clinical data revealed a possible correlation between LEAP2 and height development. However, the role of LEAP2 in early development remains unclear. This study aimed to investigate the role of LEAP2 in early development using leap2 mutant zebrafish larvae as a model. MethodWe analyzed the conservation of LEAP2 peptide across multiple species and generated leap2 mutants in zebrafish by CRISPR-Cas9, dynamically observed and measured the growth and development of zebrafish larvae from fertilization to 5 day post fertilization (dpf). In situ hybridization, transcriptome sequencing, quantitative real-time PCR and Western blot were used to detect the expression levels of GH and its signaling in early stage of embryonic development. ResultOur data demonstrate that zebrafish with a knockout of the leap2 gene display a significant increase in hatching rate, body length, and the distance between their eyes, all without visible developmental defects in the early stages of development. In addition, both RNA and protein analyses revealed a significant increase in GH expression in leap2 mutant. ConclusionIn general, this study demonstrates that LEAP2 regulates the expression of GH during early development, particularly influencing body length.

A lmod1a mutation causes megacystis microcolon intestinal hypoperistalsis in a CRISPR/Cas9-modified zebrafish model.

Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is defined as a congenital visceral myopathy with genetic mutations. However, the etiology and pathophysiology are not fully understood. We aimed to generate a gene leiomodin-1a (lmod1a) modification technique to establish a zebrafish model of MMIHS. We targeted lmod1a in zebrafish using CRISPR/Cas9. After confirming the genotype, we measured the expression levels of the target gene and protein associated with MMIHS. A gut transit assay and spatiotemporal mapping were conducted to analyze the intestinal function. Genetic confirmation showed a 5-base-pair deletion in exon 1 of lmod1a, which caused a premature stop codon. We observed significant mRNA downregulation of lmod1a, myh11, myod1, and acta2 and the protein expression of Lmod1 and Acta2 in the mutant group. A functional analysis of the lmod1a mutant zebrafish showed that its intestinal peristalsis was fewer, slower, and shorter in comparison to the wild type. This study showed that targeted deletion of lmod1a in zebrafish resulted in depletion of MMIHS-related genes and proteins, resulting in intestinal hypoperistalsis. This model may have the potential to be utilized in future therapeutic approaches, such as drug discovery screening and gene repair therapy for MMIHS.

Gata6 functions in zebrafish endoderm to regulate late differentiating arterial pole cardiogenesis.

Mutations in GATA6 are associated with congenital heart disease, most notably conotruncal structural defects. However, how GATA6 regulates cardiac morphology during embryogenesis is undefined. We used knockout and conditional mutant zebrafish alleles to investigate the spatiotemporal role of gata6 during cardiogenesis. Loss of gata6 specifically impacts atrioventricular valve formation and recruitment of epicardium, with a prominent loss of arterial pole cardiac cells, including those of the ventricle and outflow tract. However, there are no obvious defects in cardiac progenitor cell specification, proliferation or death. Conditional loss of gata6 starting at 24 h is sufficient to disrupt the addition of late differentiating cardiomyocytes at the arterial pole, with decreased expression levels of anterior secondary heart field (SHF) markers spry4 and mef2cb. Conditional loss of gata6 in the endoderm is sufficient to phenocopy the straight knockout, resulting in a significant loss of ventricular and outflow tract tissue. Exposure to a Dusp6 inhibitor largely rescues the loss of ventricular cells in gata6-/- larvae. Thus, gata6 functions in endoderm are mediated by FGF signaling to regulate the addition of anterior SHF progenitor derivatives during heart formation.

A deleterious variant of INTS1 leads to disrupted sleep-wake cycles.

Sleep disturbances are common among children with neurodevelopmental disorders. Here, we report a syndrome characterized by prenatal microcephaly, intellectual disability and severe disruption of sleep-wake cycles in a consanguineous family. Exome sequencing revealed homozygous variants (c.5224G>A and c.6506G>T) leading to the missense mutations E1742K and G2169V in integrator complex subunit 1 (INTS1), the core subunit of the Integrator complex. Conservation and structural analyses suggest that G2169V has a minor impact on the structure and function of the complex, while E1742K significantly alters a negatively charged conserved patch on the surface of the protein. The severe sleep-wake cycles disruption in human carriers highlights a new aspect of Integrator complex impairment. To further study INTS1 pathogenicity, we generated Ints1-deficient zebrafish lines. Mutant zebrafish larvae displayed abnormal circadian rhythms of locomotor activity and sleep, as is the case with the affected humans. Furthermore, Ints1-deficent larvae exhibited elevated levels of dopamine β-hydroxylase (dbh) mRNA in the locus coeruleus, a wakefulness-inducing brainstem center. Altogether, these findings suggest a significant, likely indirect, effect of INTS1 and the Integrator complex on maintaining circadian rhythms of locomotor activity and sleep homeostasis across vertebrates.

A zebrafish gephyrinb mutant distinguishes synaptic and enzymatic functions of Gephyrin

Gephyrin is thought to play a critical role in clustering glycine receptors at synapses within the central nervous system (CNS). The main in vivo evidence for this comes from Gephyrin (Gphn)-null mice, where glycine receptors are depleted from synaptic regions. However, these mice die at birth, possibly due to impaired molybdenum cofactor (MoCo) synthesis, an essential role Gephyrin assumes throughout an animal. This complicates the interpretation of synaptic phenotypes in Gphn-null mice and raises the question whether the synaptic and enzymatic functions of Gephyrin can be investigated separately. Here, we generated a gephyrinb zebrafish mutant, vo84, that almost entirely lacks Gephyrin staining in the spinal cord. gephyrinbvo84 mutants exhibit normal gross morphology at both larval and adult stages. In contrast to Gphn-null mice, gephyrinbvo84 mutants exhibit normal motor activity and MoCo-dependent enzyme activity. Instead, gephyrinbvo84 mutants display impaired rheotaxis and increased mortality in late development. To investigate what may mediate these defects in gephyrinbvo84 mutants, we examined the cell density of neurons and myelin in the spinal cord and found no obvious changes. Surprisingly, in gephyrinbvo84 mutants, glycine receptors are still present in the synaptic regions. However, their abundance is reduced, potentially contributing to the observed defects. These findings challenge the notion that Gephyrin is absolutely required to cluster glycine receptors at synapses and reveals a new role of Gephyrin in regulating glycine receptor abundance and rheotaxis. They also establish a powerful new model for studying the mechanisms underlying synaptic, rather than enzymatic, functions of Gephyrin.

A syngeneic spontaneous zebrafish model of tp53-deficient, EGFRvIII, and PI3KCAH1047R-driven glioblastoma reveals inhibitory roles for inflammation during tumor initiation and relapse in vivo.

High-throughput vertebrate animal model systems for the study of patient-specific biology and new therapeutic approaches for aggressive brain tumors are currently lacking, and new approaches are urgently needed. Therefore, to build a patient-relevant in vivo model of human glioblastoma, we expressed common oncogenic variants including activated human EGFRvIII and PI3KCAH1047R under the control of the radial glial-specific promoter her4.1 in syngeneic tp53 loss-of-function mutant zebrafish. Robust tumor formation was observed prior to 45 days of life, and tumors had a gene expression signature similar to human glioblastoma of the mesenchymal subtype, with a strong inflammatory component. Within early stage tumor lesions, and in an in vivo and endogenous tumor microenvironment, we visualized infiltration of phagocytic cells, as well as internalization of tumor cells by mpeg1.1:EGFP+ microglia/macrophages, suggesting negative regulatory pressure by pro-inflammatory cell types on tumor growth at early stages of glioblastoma initiation. Furthermore, CRISPR/Cas9-mediated gene targeting of master inflammatory transcription factors irf7 or irf8 led to increased tumor formation in the primary context, while suppression of phagocyte activity led to enhanced tumor cell engraftment following transplantation into otherwise immune-competent zebrafish hosts. Altogether, we developed a genetically relevant model of aggressive human glioblastoma and harnessed the unique advantages of zebrafish including live imaging, high-throughput genetic and chemical manipulations to highlight important tumor-suppressive roles for the innate immune system on glioblastoma initiation, with important future opportunities for therapeutic discovery and optimizations.

Functional knockout of the Oatp1d1 membrane transporter affects toxicity of diclofenac in zebrafish embryos

Organic anion transporting polypeptides (OATPs) facilitate the cellular uptake of a large number of compounds. Zebrafish Oatp1d1 matches the functional capabilities of human OATP orthologs, particularly in hormone and drug transport. It is highly expressed in the liver and later stages of embryonic development, indicating its critical role in zebrafish physiology and development. Data from previous in vitro analyses have shown a high affinity of zebrafish Oatp1d1 for pharmaceuticals and xenobiotics, providing the basis for further in vivo studies on its defence and developmental functions. Using CRISPR-Cas9 technology, we have generated an Oatp1d1 zebrafish mutant that has highly reduced Oatp1d1 expression in embryos and adult tissues compared to wild type (WT). The absence of Oatp1d1 was confirmed using custom-made antibodies. To evaluate its ecotoxicological relevance, mutant and WT embryos were exposed to increasing concentrations of diclofenac, an NSAID known for its wide and frequent use, environmental pseudo-persistence and ecological implications. WT embryos showed developmental delays and malformations such as spinal curvature, cardiac edema and blood pooling at higher diclofenac concentrations, whereas the Oatp1d1 mutant embryos showed marked resilience, with milder developmental defects and delayed toxic effects. These observations suggest that the absence of Oatp1d1 impedes the efficient entry of diclofenac into hepatocytes, thereby slowing its biotransformation into potentially more toxic metabolites. In addition, the changes in transcript expression of other uptake transporters revealed a highly probable and complex network of compensatory mechanisms. Therefore, the results of this study point to the importance of Oatp1d1-mediated transport of diclofenac, as demonstrated for the first time in vivo using an Oatp1 deficient zebrafish line. Finally, our data indicates that the compensatory role of other transporters with overlapping substrate preferences needs to be considered for a reliable understanding of the physiological and/or defensive role(s) of membrane transporters.

Highly sensitive response to the toxicity of environmental chemicals in transparent casper zebrafish

The response sensitivity to toxic substances is the most concerned performance of animal model in chemical risk assessment. Casper (mitfaw2/w2;mpv17a9/a9), a transparent zebrafish mutant, is a useful in vivo model for toxicological assessment. However, the ability of casper to respond to the toxicity of exogenous chemicals is unknown. In this study, zebrafish embryos were exposed to five environmental chemicals, chlorpyrifos, lindane, α-endosulfan, bisphenol A, tetrabromobisphenol A (TBBPA), and an antiepileptic drug valproic acid. The half-lethal concentration (LC50) values of these chemicals in casper embryos were 62–87 % of that in the wild-type. After TBBPA exposure, the occurrence of developmental defects in the posterior blood island of casper embryos was increased by 67–77 % in relative to the wild-type, and the half-maximal effective concentration (EC50) in casper was 73 % of that in the wild-type. Moreover, the casper genetic background significantly increased the hyperlocomotion caused by chlorpyrifos and lindane exposure compared with the wild-type. These results demonstrated that casper had greater susceptibility to toxicity than wild-type zebrafish in acute toxicity, developmental toxicity and neurobehavioral toxicity assessments. Our data will inform future toxicological studies in casper and accelerate the development of efficient approaches and strategies for toxicity assessment via the use of casper.

Epidermal growth factor-like domain 7 drives brain lymphatic endothelial cell development through integrin αvβ3

In zebrafish, brain lymphatic endothelial cells (BLECs) are essential for meningeal angiogenesis and cerebrovascular regeneration. Although epidermal growth factor-like domain 7 (Egfl7) has been reported to act as a pro-angiogenic factor, its roles in lymphangiogenesis remain unclear. Here, we show that Egfl7 is expressed in both blood and lymphatic endothelial cells. We generate an egfl7cq180 mutant with a 13-bp-deletion in exon 3 leading to reduced expression of Egfl7. The egfl7cq180 mutant zebrafish exhibit defective formation of BLEC bilateral loop-like structures, although trunk and facial lymphatic development remains unaffected. Moreover, while the egfl7cq180 mutant displays normal BLEC lineage specification, the migration and proliferation of these cells are impaired. Additionally, we identify integrin αvβ3 as the receptor for Egfl7. αvβ3 is expressed in the CVP and sprouting BLECs, and blocking this integrin inhibits the formation of BLEC bilateral loop-like structures. Thus, this study identifies a role for Egfl7 in BLEC development that is mediated through the integrin αvβ3.

Electrical Synapses Mediate Embryonic Hyperactivity in a Zebrafish Model of Fragile X Syndrome.

Although hyperactivity is associated with a wide variety of neurodevelopmental disorders, the early embryonic origins of locomotion have hindered investigation of pathogenesis of these debilitating behaviors. The earliest motor output in vertebrate animals is generated by clusters of early-born motor neurons (MNs) that occupy distinct regions of the spinal cord, innervating stereotyped muscle groups. Gap junction electrical synapses drive early spontaneous behavior in zebrafish, prior to the emergence of chemical neurotransmitter networks. We use a genetic model of hyperactivity to gain critical insight into the consequences of errors in motor circuit formation and function, finding that Fragile X syndrome model mutant zebrafish are hyperexcitable from the earliest phases of spontaneous behavior, show altered sensitivity to blockade of electrical gap junctions, and have increased expression of the gap junction protein Connexin 34/35. We further show that this hyperexcitable behavior can be rescued by pharmacological inhibition of electrical synapses. We also use functional imaging to examine MN and interneuron (IN) activity in early embryogenesis, finding genetic disruption of electrical gap junctions uncouples activity between mnx1 + MNs and INs. Taken together, our work highlights the importance of electrical synapses in motor development and suggests that the origins of hyperactivity in neurodevelopmental disorders may be established during the initial formation of locomotive circuits.

Functional analysis of ESRP1/2 gene variants and CTNND1 isoforms in orofacial cleft pathogenesis.

Orofacial cleft (OFC) is a common human congenital anomaly. Epithelial-specific RNA splicing regulators ESRP1 and ESRP2 regulate craniofacial morphogenesis and their disruption result in OFC in zebrafish, mouse and humans. Using esrp1/2 mutant zebrafish and murine Py2T cell line models, we functionally tested the pathogenicity of human ESRP1/2 gene variants. We found that many variants predicted by in silico methods to be pathogenic were functionally benign. Esrp1 also regulates the alternative splicing of Ctnnd1 and these genes are co-expressed in the embryonic and oral epithelium. In fact, over-expression of ctnnd1 is sufficient to rescue morphogenesis of epithelial-derived structures in esrp1/2 zebrafish mutants. Additionally, we identified 13 CTNND1 variants from genome sequencing of OFC cohorts, confirming CTNND1 as a key gene in human OFC. This work highlights the importance of functional assessment of human gene variants and demonstrates the critical requirement of Esrp-Ctnnd1 acting in the embryonic epithelium to regulate palatogenesis.