Knockout Zebrafish Research Articles

Biallelic loss-of-function variants in GON4L cause microcephaly and brain structure abnormalities

We identified two homozygous truncating variants in GON4L [NM_001282860.2:c.62_63del, p.(Gln21Argfs*12) and c.5517+1G>A] in two unrelated families who presented prenatal-onset growth impairment, microcephaly, characteristic face, situs inversus, and developmental delay. The frameshift variant is predicted to invoke nonsense-mediated mRNA decay of all five known GON4L isoforms resulting in the complete loss of GON4L function. The splice site variant located at a region specific to the longer isoforms; therefore, defects of long GON4L isoforms may explain the phenotypes observed in the three patients. Knockdown of Gon4l in rat PC12 cells suppressed neurite outgrowth in vitro. gon4lb knockdown and knockout zebrafish successfully recapitulated the patients’ phenotypes including craniofacial abnormalities. We also observed situs inversus in gon4lb-knockout zebrafish embryo. To our knowledge, the relationship between craniofacial abnormalities or situs inversus and gon4lb has not been reported before. Thus, our data provide evidence that GON4L is involved in craniofacial and left-right patterning during development.

Social isolation intensifies adgrl3.1-related externalizing and internalizing behaviors in zebrafish

Externalizing disorders (EDs) are characterized by outward-directed behaviors such as aggression and hyperactivity. They are influenced by gene-environment interactions, yet our understanding of the genetic predispositions and environmental contexts that give rise to them is incomplete. Additionally, people with EDs often exhibit comorbid internalizing symptoms, which can complicate the clinical presentation and treatment strategies. Following on from our previous studies, we examined genes x environment interaction as a risk factor for EDs by looking at internalizing and externalizing behaviors after social isolation. Specifically, we subjected adgrl3.1 knockout zebrafish — characterized by hyperactivity and impulsivity — to a 2-week social isolation protocol. We subsequently assessed the impact on anxiety-like behavior, abnormal repetitive behaviors, working memory, and social interactions. Genotype-specific additive effects emerged, with socially isolated adgrl3.1 knockout fish exhibiting intensified comorbid phenotypes, including increased anxiety, abnormal repetitive behaviors, reduced working memory, and altered shoaling, when compared to WT fish. The findings demonstrate that genetic predispositions interact with environmental stressors, such as social isolation, to exacerbate both externalizing and internalizing symptoms. This underlines the necessity for comprehensive diagnostic and intervention strategies.

SRPK3 Is Essential for Cognitive and Ocular Development in Humans and Zebrafish, Explaining X-Linked Intellectual Disability.

Intellectual disability is often the outcome of neurodevelopmental disorders and is characterized by significant impairments in intellectual and adaptive functioning. X-linked intellectual disability (XLID) is a subset of these disorders caused by genetic defects on the X chromosome, affecting about 2 out of 1,000 males. In syndromic form, it leads to a broad range of cognitive, behavioral, ocular, and physical disabilities. Employing exome or genome sequencing, here we identified 4 missense variants (c.475C > G; p.H159D, c.1373C > A; p.T458N, and c.1585G > A; p.E529K, c.953C > T; p.S318L) and a putative truncating variant (c.1413_1414del; p.Y471*) in the SRPK3 gene in 9 XLID patients from 5 unrelated families. To validate SRPK3 as a novel XLID gene, we established a knockout (KO) model of the SRPK3 orthologue in zebrafish. The 8 patients ascertained postnatally shared common clinical features including intellectual disability, agenesis of the corpus callosum, abnormal eye movement, and ataxia. A ninth case, ascertained prenatally, had a complex structural brain phenotype. Together, these data indicate a pathological role of SRPK3 in neurodevelopmental disorders. In post-fertilization day 5 larvae (free swimming stage), KO zebrafish exhibited severe deficits in eye movement and swim bladder inflation, mimicking uncontrolled ocular movement and physical clumsiness observed in human patients. In adult KO zebrafish, cerebellar agenesis and behavioral abnormalities were observed, recapitulating human phenotypes of cerebellar atrophy and intellectual disability. Overall, these results suggest a crucial role of SRPK3 in the pathogenesis of syndromic X-linked intellectual disability and provide new insights into brain development, cognitive and ocular dysfunction in both humans and zebrafish. ANN NEUROL 2024;96:914-931.

Planar cell polarity zebrafish models of congenital scoliosis reveal underlying defects in notochord morphogenesis.

Congenital scoliosis (CS) is a type of vertebral malformation for which the etiology remains elusive. The notochord is pivotal for vertebrae development, but its role in CS is still understudied. Here, we generated a zebrafish knockout of ptk7a, a planar cell polarity (PCP) gene that is essential for convergence and extension (C&E) of the notochord, and detected congenital scoliosis-like vertebral malformations (CVMs). Maternal zygotic ptk7a mutants displayed severe C&E defects of the notochord. Excessive apoptosis occurred in the malformed notochord, causing a significantly reduced number of vacuolated cells, and compromising the mechanical properties of the notochord. The latter manifested as a less-stiff extracellular matrix along with a significant reduction in the number of the caveolae and severely loosened intercellular junctions in the vacuolated region. These defects led to focal kinks, abnormal mineralization, and CVMs exclusively at the anterior spine. Loss of function of another PCP gene, vangl2, also revealed excessive apoptosis in the notochord associated with CVMs. This study suggests a new model for CS pathogenesis that is associated with defects in notochord C&E and highlights an essential role of PCP signaling in vertebrae development.

Membrane progesterone receptor γ (paqr5b) is essential for the formation of neurons in the zebrafish olfactory rosette

Paqr5b is a gene encoding membrane progesterone receptor γ (mPRγ), which is one of five mPR subtypes. Paqr5b belongs to the progestin and adipoQ receptor (PAQR) family, which consists of 11 genes. To elucidate the physiological functions of the mPR subtypes, we established gene knockout (KO) zebrafish strains by genetically editing seven paqr genes and analyzed their phenotypes. The null-mutant strain of paqr5b (paqr5b−/−) that we established in this study showed low fecundity, reduced chorion elevation and a high percentage of abnormal embryos. Embryos showed curvature of the spine and an abnormal head morphology. Individuals with abnormal head morphology continued to develop a phenotype of markedly abnormal palatine bone. The length of the brain of paqr5b−/− zebrafish was short, and the position of the cerebellum moved to the front and overlapped with that of the midbrain. Micro-CT scans revealed that the olfactory rosettes (ORs) were so shrunken that they were difficult to identify and connected with the olfactory bulbs (OBs) by thread-like structures. Immunohistochemical staining of OR with an anti-Paqr5b antibody revealed that Paqr5b was extensively expressed in neurons in the OR in wild-type zebrafish, whereas signals were not detected in paqr5b−/− zebrafish. In histological sections, the neurons disappeared, and the lamellar layer of the OR became thinner. These results indicate that Paqr5b is required for the formation of neurons in the OR. This is the first report demonstrating a distinct role for the mPR gene.

Unraveling the socio-cognitive consequences of KCC2 disruption in zebrafish: implications for neurodevelopmental disorders and therapeutic interventions.

During postnatal brain development, maintaining a delicate balance between excitation and inhibition (E/I) is essential for the precise formation of neuronal circuits. The K+/cl- cotransporter 2 (KCC2) is instrumental in this process, and its dysregulation is implicated in various neurological disorders. This study utilized zebrafish (Danio rerio) to investigate the socio-cognitive consequences of KCC2 disruption. Through CRISPR-Cas9 technology, biallelic kcc2a knockout zebrafish larvae were generated, revealing behavioral abnormalities, including impaired social interactions and memory deficits. Molecular analyses unveiled alterations in key genes associated with the GABAergic and glutamatergic systems, potentially contributing to E/I imbalance. Additionally, KCC2 disruption influenced the expression of oxytocin and BDNF, crucial regulators of social behaviors, synaptic plasticity, and memory formation. The study also explored the therapeutic potential of KCC2 modulation using pharmaceuticals, showing the rescuing effects of CLP-290 and LIT-001 on social abnormalities. However, the selective impact of LIT-001 on social behaviors, not memory, highlights the complexity of neurobehavioral modulation. In summary, this study sheds light on the pivotal role of KCC2 in shaping socio-cognitive functions and suggests potential therapeutic avenues for KCC2-related neurological disorders.

Lactic acid-fermented by-product of Shochu distillery reduces anxiety behavior in Neuropeptide Y knockout zebrafish by the regulation of isotoin neuron

The development of techniques to reduce stress in the aquaculture industry is essential for the good growth, low mortality, and high flesh quality of farmed fish. Shochu is a traditional Japanese alcoholic beverage, and the disposal of large amounts of Shochu lees waste has been a problem. As Shochu lees contain low-molecular-weight compounds, which are thought to have health benefits, the lactic acid-fermented by-product of Shochu distillery (FBPSD) was tested to estimate its anti-stress effects in neuropeptide Y-knockout zebrafish (NPY-KO), which exhibit high anxiety and low sociability, as a farmed fish model.In the Black-White preference test, NPY-KO preferred the black area over the white area due to fear, whereas NPY-KO fed the FBPSD showed a higher preference for the white area with more swimming distance than the control group. In the 3-chambers test, NPY-KO showed low sociability with the unfamiliar zebrafish group, whereas FBPSD-fed NPY-KO showed high sociability with the group. The mRNA level of isotocin, the homologue of human oxytocin, was significantly increased in the brains of FBPSD-fed zebrafish. The oxytocin receptor antagonists L-368,899 attenuated the effects of FBPSD in the 3-chambers test. These results suggest that FBPSD attenuates anxiety in NPY-KO zebrafish by activating isotocin neurons.

CRISPR/Cas9-Mediated fech Knockout Zebrafish: Unraveling the Pathogenesis of Erythropoietic Protoporphyria and Facilitating Drug Screening

Erythropoietic protoporphyria (EPP1) results in painful photosensitivity and severe liver damage in humans due to the accumulation of fluorescent protoporphyrin IX (PPIX). While zebrafish (Danio rerio) models for porphyria exist, the utility of ferrochelatase (fech) knockout zebrafish, which exhibit EPP, for therapeutic screening and biological studies remains unexplored. This study investigated the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated fech-knockout zebrafish larvae as a model of EPP1 for drug screening. CRISPR/Cas9 was employed to generate fech-knockout zebrafish larvae exhibiting morphological defects without lethality prior to 9 days post-fertilization (dpf). To assess the suitability of this model for drug screening, ursodeoxycholic acid (UDCA), a common treatment for cholestatic liver disease, was employed. This treatment significantly reduced PPIX fluorescence and enhanced bile-secretion-related gene expression (abcb11a and abcc2), indicating the release of PPIX. Acridine orange staining and quantitative reverse transcription polymerase chain reaction analysis of the bax/bcl2 ratio revealed apoptosis in fech−/− larvae, and this was reduced by UDCA treatment, indicating suppression of the intrinsic apoptosis pathway. Neutral red and Sudan black staining revealed increased macrophage and neutrophil production, potentially in response to PPIX-induced cell damage. UDCA treatment effectively reduced macrophage and neutrophil production, suggesting its potential to alleviate cell damage and liver injury in EPP1. In conclusion, CRISPR/Cas9-mediated fech−/− zebrafish larvae represent a promising model for screening drugs against EPP1.

EZH2 specifically regulates ISL1 during embryonic urinary tract formation

Isl1 has been described as an embryonic master control gene expressed in the pericloacal mesenchyme. Deletion of Isl1 from the genital mesenchyme in mice leads to an ectopic urethral opening and epispadias-like phenotype. Using genome wide association methods, we identified ISL1 as the key susceptibility gene for classic bladder exstrophy (CBE), comprising epispadias and exstrophy of the urinary bladder. The most significant marker (rs6874700) identified in our recent GWAS meta-analysis achieved a p value of 1.48 × 10− 24 within the ISL1 region. In silico analysis of rs6874700 and all other genome-wide significant markers in Linkage Disequilibrium (LD) with rs6874700 (D’ = 1.0; R2 > 0.90) revealed marker rs2303751 (p value 8.12 × 10− 20) as the marker with the highest regulatory effect predicted. Here, we describe a novel 1.2 kb intragenic promoter residing between 6.2 and 7.4 kb downstream of the ISL1 transcription starting site, which is located in the reverse DNA strand and harbors a binding site for EZH2 at the exact region of marker rs2303751. We show, that EZH2 silencing in HEK cells reduces ISL1 expression. We show that ezh2−/− knockout (KO) zebrafish larvae display tissues specificity of ISL1 regulation with reduced expression of Isl1 in the pronephric region of zebrafish larvae. In addition, a shorter and malformed nephric duct is observed in ezh2−/− ko zebrafish Tg(wt1ß:eGFP) reporter lines. Our study shows, that Ezh2 is a key regulator of Isl1 during urinary tract formation and suggests tissue specific ISL1 dysregulation as an underlying mechanism for CBE formation.

RFC2 may contribute to the pathogenicity of Williams syndrome revealed in a zebrafish model

Williams syndrome (WS) is a rare multisystemic disorder caused by recurrent microdeletions on 7q11.23, characterized by intellectual disability, distinctive craniofacial and dental features, and cardiovascular problems. Previous studies have explored the roles of individual genes within these microdeletions in contributing to WS phenotypes. Here, we report five patients with WS with 1.4 Mb–1.5 Mb microdeletions that include RFC2, as well as one patient with a 167 kb microdeletion involving RFC2 and six patients with intragenic variants within RFC2. To investigate the potential involvement of RFC2 in WS pathogenicity, we generate a rfc2 knockout (KO) zebrafish using CRISPR-Cas9 technology. Additionally, we generate a KO zebrafish of its paralog gene, rfc5, to better understand the functions of these RFC genes in development and disease. Both rfc2 and rfc5 KO zebrafish exhibit similar phenotypes reminiscent of WS, including small head and brain, jaw and dental defects, and vascular problems. RNA-seq analysis reveals that genes associated with neural cell survival and differentiation are specifically affected in rfc2 KO zebrafish. In addition, heterozygous rfc2 KO adult zebrafish demonstrate an anxiety-like behavior with increased social cohesion. These results suggest that RFC2 may contribute to the pathogenicity of Williams syndrome, as evidenced by the zebrafish model.

Zebrafish reveal new roles for Fam83f in hatching and the DNA damage-mediated autophagic response.

The FAM83 (Family with sequence similarity 83) family is highly conserved in vertebrates, but little is known of the functions of these proteins beyond their association with oncogenesis. Of the family, FAM83F is of particular interest because it is the only membrane-targeted FAM83 protein. When overexpressed, FAM83F activates the canonical Wnt signalling pathway and binds to and stabilizes p53; it therefore interacts with two pathways often dysregulated in disease. Insights into gene function can often be gained by studying the roles they play during development, and here we report the generation of fam83f knock-out (KO) zebrafish, which we have used to study the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the hatching gland of developing zebrafish embryos, and that fam83f KO embryos hatch earlier than their wild-type (WT) counterparts, despite developing at a comparable rate. We also demonstrate that fam83f KO embryos are more sensitive to ionizing radiation than WT embryos-an unexpected finding, bearing in mind the previously reported ability of FAM83F to stabilize p53. Transcriptomic analysis shows that loss of fam83f leads to downregulation of phosphatidylinositol-3-phosphate (PI(3)P) binding proteins and impairment of cellular degradation pathways, particularly autophagy, a crucial component of the DNA damage response. Finally, we show that Fam83f protein is itself targeted to the lysosome when overexpressed in HEK293T cells, and that this localization is dependent upon a C' terminal signal sequence. The zebrafish lines we have generated suggest that Fam83f plays an important role in autophagic/lysosomal processes, resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo.

Knockout, Knockdown, and the Schrödinger Paradox: Genetic Immunity to Phenotypic Recapitulation in Zebrafish.

Previous research has highlighted significant phenotypic discrepancies between knockout and knockdown approaches in zebrafish, raising concerns about the reliability of these methods. However, our study suggests that these differences are not as pronounced as was once believed. By carefully examining the roles of maternal and zygotic gene contributions, we demonstrate that these factors significantly influence phenotypic outcomes, often accounting for the observed discrepancies. Our findings emphasize that morpholinos, despite their potential off-target effects, can be effective tools when used with rigorous controls. We introduce the concept of graded maternal contribution, which explains how the uneven distribution of maternal mRNA and proteins during gametogenesis impacts phenotypic variability. Our research categorizes genes into three types-susceptible, immune, and "Schrödinger" (conditional)-based on their phenotypic expression and interaction with genetic compensation mechanisms. This distinction provides new insights into the paradoxical outcomes observed in genetic studies. Ultimately, our work underscores the importance of considering both maternal and zygotic contributions, alongside rigorous experimental controls, to accurately interpret gene function and the mechanisms underlying disease. This study advocates for the continued use of morpholinos in conjunction with advanced genetic tools like CRISPR/Cas9, stressing the need for a meticulous experimental design to optimize the utility of zebrafish in genetic research and therapeutic development.

Tcap deficiency impedes striated muscle function and heart regeneration with elevated ROS and autophagy

Telethonin/titin-cap (TCAP) encodes a Z-disc protein that plays important roles in sarcomere/T-tubule interactions, stretch-sensing and signaling. Mutations in TCAP are associated with muscular dystrophy and cardiomyopathy; however, the complete etiology and its roles in myocardial infarction and regeneration are not fully understood. Here, we generated tcap gene knockout zebrafish with CRISPR/Cas9 technology and observed muscular dystrophy-like phenotypes and abnormal mitochondria in skeletal muscles. The stretch-sensing ability was inhibited in tcap‐/‐ mutants. Moreover, Tcap deficiency led to alterations in cardiac morphology and function as well as increases in reactive oxygen species (ROS) and mitophagy. In addition, the cardiac regeneration and cardiomyocyte proliferation ability of tcap‐/‐ mutants were impaired, but these impairments could be rescued by supplementation with ROS scavengers or autophagy inhibitors. Overall, our study demonstrates the essential roles of Tcap in striated muscle function and heart regeneration. Additionally, elevations in ROS and autophagy may account for the phenotypes resulting from Tcap deficiency and could serve as novel therapeutic targets for muscular dystrophy and cardiomyopathy.

ABCA7-dependent induction of neuropeptide Y is required for synaptic resilience in Alzheimer’s disease through BDNF/NGFR signaling

Genetic variants in ABCA7, an Alzheimer's disease (AD)-associated gene, elevate AD risk, yet its functional relevance to the etiology is unclear. We generated a CRISPR-Cas9-mediated abca7 knockout zebrafish to explore ABCA7's role in AD. Single-cell transcriptomics in heterozygous abca7+/- knockout combined with Aβ42 toxicity revealed that ABCA7 is crucial for neuropeptide Y (NPY), brain-derived neurotrophic factor (BDNF), and nerve growth factor receptor (NGFR) expressions, which are crucial for synaptic integrity, astroglial proliferation, and microglial prevalence. Impaired NPY induction decreased BDNF and synaptic density, which are rescuable with ectopic NPY. In induced pluripotent stem cell-derived human neurons exposed to Aβ42, ABCA7-/- suppresses NPY. Clinical data showed reduced NPY in AD correlated with elevated Braak stages, genetic variants in NPY associated with AD, and epigenetic changes in NPY, NGFR, and BDNF promoters linked to ABCA7 variants. Therefore, ABCA7-dependent NPY signaling via BDNF-NGFR maintains synaptic integrity, implicating its impairment in increased AD risk through reduced brain resilience.

The metalloproteinase PAPP-A is required for IGF-dependent chondrocyte differentiation and organization

Insulin-like growth factor (IGF) signaling is required for proper growth and skeletal development in vertebrates. Consequently, its dysregulation may lead to abnormalities of growth or skeletal structures. IGF is involved in the regulation of cell proliferation and differentiation of chondrocytes. However, the availability of bioactive IGF may be controlled by antagonizing IGF binding proteins (IGFBPs) in the circulation and tissues. As the metalloproteinase PAPP-A specifically cleaves members of the IGFBP family, we hypothesized that PAPP-A activity liberates bioactive IGF in cartilage. In PAPP-A knockout mice, the femur length was reduced and the mice showed a disorganized columnar organization of growth plate chondrocytes. Similarly, zebrafish lacking pappaa showed reduced length of Meckel’s cartilage and disorganized chondrocytes, reminiscent of the mouse knockout phenotype. Expression of chondrocyte differentiation markers (sox9a, ihha, and col10a1) was markedly affected in Meckel’s cartilage of pappaa knockout zebrafish, indicating that differentiation of chondrocytes was compromised. Additionally, the zebrafish pappaa knockout phenotype was mimicked by pharmacological inhibition of IGF signaling, and it could be rescued by treatment with exogenous recombinant IGF-I. In conclusion, our data suggests that IGF activity in the growing cartilage, and hence IGF signaling in chondrocytes, requires the presence of PAPP-A. The absence of PAPP-A causes aberrant chondrocyte organization and compromised growth in both mice and zebrafish.

Effects of two chd2-knockout strains on the morphology and behavior in zebrafish.

The chromodomain helicase DNA binding domain 2 (CHD2) gene is an ATPase and a member of the SNF2-like family of helicase-related enzymes. CHD2 plays critical roles in human brain development and function, and homozygous mutation of Chd2 in mice results in perinatal lethality. To further elucidate the effects of chd2, we used CRISPR/Cas9 to create two chd2-knockout strains (fdu901, 11,979-11982delGGGT, and fdu902, 27350delG) in zebrafish. We found that the deformity and mortality rates of fdu901 and fdu902 were higher than those of the wild type. Developmental delay was more obvious and embryo mortality was higher in fdu901 than in fdu902. However, the embryo deformity rate in fdu902 was higher than that in fdu901. Although there were no significant differences in behavior between the two knockout zebrafish and wild-type zebrafish at 7days post fertilization (dpf), fdu901 and fdu902 zebrafish showed different alterations. The excitability of fdu902 was higher than that of fdu901. Overall, our data demonstrate that two homozygous chd2 knockout mutations were survivable and could be stably inherited and that fdu901 and fdu902 zebrafish differed in behavior and morphology. These two models might be good tools for understanding the functions of the different domains of chd2.

Lysosomal membrane protein TMEM106B modulates hematopoietic stem and progenitor cell proliferation and differentiation by regulating LAMP2A stability.

Hematopoietic stem and progenitor cells (HSPCs) are successfully employed for hematological transplantations, and impaired HSPC function causes hematological diseases and aging. HSPCs maintain the lifelong homeostasis of blood and immune cells through continuous self-renewal and maintenance of the multilineage differentiation potential. TMEM106B is a transmembrane protein localized on lysosomal membranes and associated with neurodegenerative and cardiovascular diseases; however, its roles in HSPCs and hematopoiesis are unknown. Here, we established tmem106bb-/- knockout (KO) zebrafish and showed that tmem106bb KO reduced the proliferation of HSPCs during definitive hematopoiesis. The differentiation potential of HSPCs to lymphoid lineage was reduced, whereas the myeloid and erythroid differentiation potentials of HPSCs were increased in tmem106bb-/- zebrafish. Similar results were obtained with morpholino knockdown of tmem106bb. Mechanistically, TMEM106B interacted with LAMP2A, the lysosomal associated membrane protein 2A, impaired LAMP2A-Cathepsin A interaction, and enhanced LAMP2A stability; tmem106bb KO or TMEM106B knockdown caused LAMP2A degradation and impairment of chaperone-mediated autophagy (CMA). Knockdown of lamp2a caused similar phenotypes to that in tmem106bb-/- zebrafish, and overexpression of lamp2a rescued the impaired phenotypes of HSPCs in tmem106bb-/- embryos. These results uncover a novel molecular mechanism for the maintenance of HSPC proliferation and differentiation through stabilizing LAMP2A via TMEM106B-LAMP2A interaction.

HSP60 chaperone deficiency disrupts the mitochondrial matrix proteome and dysregulates cholesterol synthesis

ObjectiveMitochondrial proteostasis is critical for cellular function. The molecular chaperone HSP60 is essential for cell function and dysregulation of HSP60 expression has been implicated in cancer and diabetes. The few reported patients carrying HSP60 gene variants show neurodevelopmental delay and brain hypomyelination. Hsp60 interacts with more than 260 mitochondrial proteins but the mitochondrial proteins and functions affected by HSP60 deficiency are poorly characterized. MethodsWe studied two model systems for HSP60 deficiency: (1) engineered HEK cells carrying an inducible dominant negative HSP60 mutant protein, (2) zebrafish HSP60 knockout larvae. Both systems were analyzed by RNASeq, proteomics, and targeted metabolomics, and several functional assays relevant for the respective model. In addition, skin fibroblasts from patients with disease-associated HSP60 variants were analyzed by proteomics. ResultsWe show that HSP60 deficiency leads to a differentially downregulated mitochondrial matrix proteome, transcriptional activation of stress responses, and dysregulated cholesterol biosynthesis. This leads to lipid accumulation in zebrafish knockout larvae. ConclusionsOur data provide a compendium of the effects of HSP60 deficiency on the mitochondrial matrix proteome. We show that HSP60 is a master regulator and modulator of mitochondrial functions and metabolic pathways. HSP60 dysfunction also affects cellular metabolism and disrupts the integrated stress response. The effect on cholesterol synthesis explains the effect of HSP60 dysfunction on myelination observed in patients carrying genetic variants of HSP60.

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.

Loss of FMRP affects ovarian development and behaviour through multiple pathways in a zebrafish model of fragile X syndrome.

Fragile X syndrome (FXS) is an inherited neurodevelopmental disorder and the leading genetic cause of autism spectrum disorders. FXS is caused by loss of function mutations in Fragile X mental retardation protein (FMRP), an RNA binding protein that is known to regulate translation of its target mRNAs, predominantly in the brain and gonads. The molecular mechanisms connecting FMRP function to neurodevelopmental phenotypes are well understood. However, neither the full extent of reproductive phenotypes, nor the underlying molecular mechanisms have been as yet determined. Here, we developed new fmr1 knockout zebrafish lines and show that they mimic key aspects of FXS neuronal phenotypes across both larval and adult stages. Results from the fmr1 knockout females also showed that altered gene expression in the brain, via the neuroendocrine pathway contribute to distinct abnormal phenotypes during ovarian development and oocyte maturation. We identified at least three mechanisms underpinning these defects, including altered neuroendocrine signaling in sexually mature females resulting in accelerated ovarian development, altered expression of germ cell and meiosis promoting genes at various stages during oocyte maturation, and finally a strong mitochondrial impairment in late stage oocytes from knockout females. Our findings have implications beyond FXS in the study of reproductive function and female infertility. Dissection of the translation control pathways during ovarian development using models like the knockout lines reported here may reveal novel approaches and targets for fertility treatments.