Homozygous Mutant Zebrafish Research Articles

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.

A screen of pharmacologically active compounds to identify modulators of the Adgrg6/Gpr126 signalling pathway in zebrafish embryos.

Adhesion G protein-coupled receptors (GPCRs) are an underrepresented class of GPCRs in drug discovery. We previously developed an in vivo drug screening pipeline to identify compounds with agonist activity for Adgrg6 (Gpr126), an adhesion GPCR required for myelination of the peripheral nervous system in vertebrates. The screening assay tests for rescue of an ear defect found in adgrg6tb233c-/- hypomorphic homozygous mutant zebrafish, using the expression of versican b (vcanb) mRNA as an easily identifiable phenotype. In the current study, we used the same assay to screen a commercially available library of 1280 diverse bioactive compounds (Sigma LOPAC). Comparison with published hits from two partially overlapping compound collections (Spectrum, Tocris) confirms that the screening assay is robust and reproducible. Using a modified counter screen for myelin basic protein (mbp) gene expression, we have identified 17 LOPAC compounds that can rescue both inner ear and myelination defects in adgrg6tb233c-/- hypomorphic mutants, three of which (ebastine, S-methylisothiourea hemisulfate, and thapsigargin) are new hits. A further 25 LOPAC hit compounds were effective at rescuing the otic vcanb expression but not mbp. Together, these and previously identified hits provide a wealth of starting material for the development of novel and specific pharmacological modulators of Adgrg6 receptor activity.

Pleiotropic effects of a high confidence Autism Spectrum Disorder gene, arid1b, on zebrafish sleep

Sleep fulfills critical functions in neurodevelopment, such as promoting synaptic plasticity, neuronal wiring, and brain connectivity which are critical phenomena in Autism Spectrum Disorder (ASD) pathophysiology. Sleep disturbance, specifically insomnia, accompanies ASD and is associated with more severe core symptoms (e.g., social impairment). It is possible that focusing on identifying effective ways to treat sleep problems can help alleviate other ASD-related symptoms. A body of evidence indicates shared mechanisms and neurobiological substrates between sleep and ASD and investigation of these may inform therapeutic effects of improving sleep at both behavioral and molecular levels. In this study, we tested if sleep and social behavior were different in a zebrafish model with the arid1b gene mutated compared to controls. This gene was selected for study as expert curations conducted for the Simons Foundation for Autism Research Institute (SFARI) Gene database define it is as a ‘high confidence’ ASD gene (i.e., clearly implicated) encoding a chromatin remodeling protein. Homozygous arid1b mutants displayed increased arousability and light sleep compared to their heterozygous and wild type counterparts, based on testing a mechano-acoustic stimulus presenting different vibration frequencies of increasing intensity to detect sleep depth. In addition, decreased social preference was observed in arid1b heterozygous and homozygous mutant zebrafish. The behavioral phenotypes reported in our study are in line with findings from mouse models and human studies and demonstrate the utility of zebrafish as a vertebrate model system with high throughput phenotyping in the investigation of changes in sleep in models relevant to ASD. Furthermore, we demonstrate the importance of including assessments of arousal threshold when studying sleep using in vivo models.

Characterization of a mutant samhd1 zebrafish model implicates dysregulation of cholesterol biosynthesis in Aicardi-Goutières syndrome.

Aicardi-Goutières syndrome (AGS1-9) is a genetically determined encephalopathy that falls under the type I interferonopathy disease class, characterized by excessive type I interferon (IFN-I) activity, coupled with upregulation of IFN-stimulated genes (ISGs), which can be explained by the vital role these proteins play in self-non-self-discrimination. To date, few mouse models fully replicate the vast clinical phenotypes observed in AGS patients. Therefore, we investigated the use of zebrafish as an alternative species for generating a clinically relevant model of AGS. Using CRISPR-cas9 technology, we generated a stable mutant zebrafish line recapitulating AGS5, which arises from recessive mutations in SAMHD1. The resulting homozygous mutant zebrafish larvae possess a number of neurological phenotypes, exemplified by variable, but increased expression of several ISGs in the head region, a significant increase in brain cell death, microcephaly and locomotion deficits. A link between IFN-I signaling and cholesterol biosynthesis has been highlighted by others, but not previously implicated in the type I interferonopathies. Through assessment of neurovascular integrity and qPCR analysis we identified a significant dysregulation of cholesterol biosynthesis in the zebrafish model. Furthermore, dysregulation of cholesterol biosynthesis gene expression was also observed through RNA sequencing analysis of AGS patient whole blood. From this novel finding, we hypothesize that cholesterol dysregulation may play a role in AGS disease pathophysiology. Further experimentation will lend critical insight into the molecular pathophysiology of AGS and the potential links involving aberrant type I IFN signaling and cholesterol dysregulation.

Loss of pex5 sensitizes zebrafish to fasting due to deregulated mitochondria, mTOR, and autophagy

Animal models have been utilized to understand the pathogenesis of Zellweger spectrum disorders (ZSDs); however, the link between clinical manifestations and molecular pathways has not yet been clearly established. We generated peroxin 5 homozygous mutant zebrafish (pex5−/−) to gain insight into the molecular pathogenesis of peroxisome dysfunction. pex5−/− display hallmarks of ZSD in humans and die within one month after birth. Fasting rapidly depletes lipids and glycogen in pex5−/− livers and expedites their mortality. Mechanistically, deregulated mitochondria and mechanistic target of rapamycin (mTOR) signaling act together to induce metabolic alterations that deplete hepatic nutrients and accumulate damaged mitochondria. Accordingly, chemical interventions blocking either the mitochondrial function or mTOR complex 1 (mTORC1) or a combination of both improve the metabolic imbalance shown in the fasted pex5−/− livers and extend the survival of animals. In addition, the suppression of oxidative stress by N-acetyl L-cysteine (NAC) treatment rescued the apoptotic cell death and early mortality observed in pex5−/−. Furthermore, an autophagy activator effectively ameliorated the early mortality of fasted pex5−/−. These results suggest that fasting may be detrimental to patients with peroxisome dysfunction, and that modulating the mitochondria, mTORC1, autophagy activities, or oxidative stress may provide a therapeutic option to alleviate the symptoms of peroxisomal diseases associated with metabolic dysfunction.

Lrpap1 deficiency leads to myopia through TGF-β-induced apoptosis in zebrafish

BackgroundFrameshift mutations in LRPAP1 are responsible for autosomal recessive high myopia in human beings but its underlying mechanism remains elusive. This study aims to investigate the effect of LRPAP1 defect on ocular refractive development and its involved mechanism.MethodsA lrpap1 mutant zebrafish line with homozygous frameshift mutation was generated by CRISPR/Cas9 technology and confirmed by Sanger sequencing. The ocular refractive phenotype was analyzed by calculating the relative refractive error (RRE) with vivo photography and histological analysis at different development stages, together with examining ocular structure change via transmission electron microscopy. Further, RNA sequencing and bioinformatics analysis were performed. The potentially involved signaling pathway as well as the interacted protein were investigated in vivo.ResultsThe lrpap1 homozygous mutant zebrafish line showed myopic phenotype. Specifically, the mutant lines showed larger eye axial length-to-body length in one-month old individuals and a myopic shift with an RRE that changed after two months. Collagen fibers became thinning and disordered in the sclera. Further, RNA sequencing and bioinformatics analysis indicated that apoptosis signaling was activated in mutant line; this was further confirmed by acridine orange and TUNEL staining. Moreover, the expression of TGF-β protein was elevated in the mutant lines. Finally, the treatment of wild-type embryos with a TGF-β agonist aggravated the degree of eyeball apoptosis; conversely, the use of a TGF-β inhibitor mitigated apoptosis in mutant embryos.ConclusionThe study provides functional evidence of a link between lrpap1 and myopia, suggesting that lrpap1 deficiency could lead to myopia through TGF-β-induced apoptosis signaling.7w_R-rNe6ADVminp521PHYVideo abstract

Selective Targeting of Splicing Factor Mutant Hematopoietic Stem and Progenitor Cells Via STAT3 Inhibition

Myelodysplastic syndrome (MDS) is a bone marrow failure disorder driven by dysfunction of hematopoietic stem and progenitor cells (HSPCs). Patient sequencing studies over the last decade have revealed that mutations in splicing machinery predominate in MDS, thus selective targeting of these cells is therapeutically attractive. STAT3 inhibition has been explored previously as a means to eradicate HSPCs in MDS. While efficacy was demonstrated in a subset of samples, the underlying mechanism for this selectivity remains unknown. We examined RNAseq of MDS CD34+ HSPCs with splicing factor mutations versus wildtype, finding alternative splicing and differential expression of STAT3 pathway components. Functionally, we explored if STAT3 signaling represents a novel vulnerability in SF3B1 mutant HSPCs using a multi-model approach of in vivo zebrafish and mouse systems, and in vitro assays of CRISPR-engineered human leukemia K562 cells and primary MDS samples. Utilizing the small molecule STAT3 inhibitor STATTIC, we found that human cells carrying MDS-associated SF3B1 point mutations had heightened sensitivity to STAT3 inhibition compared to wildtype controls. To evaluate the activity of STAT3 inhibition in vivo, we utilized an Mx1-cre conditional knock-in mouse model of mutant SF3B1 (Sf3b1+/K700E). We demonstrated that in vivo STATTIC treatment selectively depleted Sf3b1 mutant cells over wildtype in vivo. RNAseq of sf3b1 homozygous mutantzebrafish cells revealed conserved dysregulation of STAT3 pathway splicing and target expression. Diminishing Stat3 (via morpholino knockdown, stable mutants, or STATTIC treatment) decreased HSPCs in sf3b1 heterozygotes but not wildtype embryos, demonstrating synthetic lethality between Sf3b1 and Stat3. Our data indicate that SF3B1 heterozygosity, regardless of the type of mutation, confers a heightened sensitivity to STAT3 inhibition in zebrafish, mouse, and human HSPCs. Critically, our data indicate that SF3B1-mutant cells can be selectively killed in vivo while sparing wildtype cells. We sought to rescue HSPCs in sf3b1 homozygous mutant zebrafish, however overexpression of ligands Osm and Il6 or wildtype Stat3 was insufficient. Instead, overexpression of constitutively-active Stat3 partially restored HSPCs, indicating that functional Stat3 signaling downstream of Sf3b1 is critical for HSPC formation. To investigate the specificity of the synthetic lethality for SF3B1, we assessed the STAT3 synthetic lethal interaction with other mutated splicing factors in MDS. Similar to SF3B1, we demonstrated STAT3 synthetic lethality with U2AF1 and SRSF2 heterozygosity in zebrafish and human cells. RNA-sequencing analysis of STATTIC-treated K562 cells revealed an exacerbation of splicing alterations upon STAT3 inhibition that was more pronounced in SF3B1+/K666N cells compared to wildtype. Even more strikingly, we demonstrated that constitutive activation of STAT3 could partially reverse defective splicing in zebrafish sf3b1 homozygous mutant cells. Mechanistically, these data strongly support coordinated splicing dysfunction as the underlying cause for STAT3-SF3B1 synthetic lethality. Together, we demonstrated a conserved and selective synthetic lethal interaction between STAT3 function and splicing factor defects that represents a novel liability for mutant HSPCs with important implications for MDS treatment. DisclosuresShastri: Guidepoint: Consultancy; Kymera Therapeutics: Research Funding; Onclive: Honoraria; GLC: Consultancy. Verma: Curis: Research Funding; BMS: Research Funding; Stelexis: Consultancy, Current equity holder in publicly-traded company; Eli Lilly: Research Funding; Medpacto: Research Funding; Incyte: Research Funding; GSK: Research Funding; Novartis: Consultancy; Acceleron: Consultancy; Celgene: Consultancy; Stelexis: Current equity holder in publicly-traded company; Throws Exception: Current equity holder in publicly-traded company.

Long noncoding RNAs implicated in embryonic development in Ybx1 knockout zebrafish.

Y-box-binding protein 1 (Ybx1, YB-1), also known as Y-box transcription factor, is involved in a variety of biological processes (BPs) and pathways, including embryogenesis, reproduction and development in vertebrates. Several noncoding RNAs regulate Ybx1 signaling. However, the role of long noncoding RNAs (lncRNAs) in embryogenesis remains incompletely understood. Here, we investigated the possible involvement of lncRNAs in Ybx1-mediated regulation of vertebrate development by performing systematic transcriptome analysis of RNA sequencing data derived from ybx1 homozygous mutant zebrafish on day 5 (day5_ybx1-/- ) and wild-type zebrafish on days 5 and 6 (day5_ybx1+/+ and day6_ybx1+/+ ). We identified several lncRNAs affected by ybx1 disruption that may target reduction-oxidation-related genes, such as duox (NADPH oxidase) and noxo1a (NADPH oxidase organizer). Knockdown of three selected lncRNAs led to morphological deformation of larvae, implying an involvement of these lncRNAs in zebrafish embryo development. In summary, our study provides new insights into the lncRNA-mediated mechanisms underlying development in Ybx1-deficient zebrafish larvae.

Zebrafish stm is involved in the development of otoliths and of the fertilization envelope.

SummaryUsing an in vivo assay, we selected 11 genes that were highly upregulated during the induction of ovulation in zebrafish using microarray analysis and RNA sequencing. The starmaker gene (stm) was one of these genes. Although stm has been previously reported to be involved in otolith formation during the early development of zebrafish, we detected its expression in eggs and showed that stm was related to fertilization by establishing an stm gene knockout strain using the CRISPR/Cas9 system. Further phenotypic analysis of stm knockout fish was conducted in this study. With a higher nonfertilization rate, the stm mutant strain showed an extremely low survival rate. Otoliths of stm homozygous mutant zebrafish showed abnormal morphology in embryos and adult fish. However, fish did not show any abnormalities in swimming behaviour in either embryos or adults. Stm proteins were detected on the chorion of ovulated eggs before spawning. Fibre-supported knob-like structures on the fertilization envelope (FE) also showed abnormal structures in stm mutants. The Stm protein is necessary for otolith formation, and a lack of Stm causes abnormal otolith formation. The partial defect of otolith formation does not cause defects in swimming behaviour. The Stm protein is expressed in the chorion and is responsible for the formation of fibre-supported knob-like structures on the FE. It was suggested that a lack of Stm caused a lower fertilization rate due to inadequate formation of the FE.Lay summaryIn zebrafish, the protein Starmaker (Stm) was identified as having a role in ovulation. Stm is also known to be required for the formation of ear stones (otoliths) which are needed to keep the body in balance. Zebrafish lacking Stm were produced by genome editing. As expected, Stm-deficient fish formed abnormal otoliths. To investigate the role of Stm in ovulation, fertilization and early development, we tried mating of Stm mutants and observed their juveniles. Although no problem found in ovulation, we found low fertilization rate and abnormal structure of knob-like structure (small pit) on the egg membrane. Survival rate of embryos with abnormal egg membrane was extremely low. It was demonstrated that Stm protein is necessary to form the functional egg membrane to protect embryos from the outside environment.

Pax2a is expressed in oocytes and is responsible for early development and oogenesis in zebrafish

Eleven genes, including pax2a, were selected as candidate ovulation-inducing genes on the basis of microarray analysis and RNA sequencing in our previous study. The purpose of this study was to investigate the role of the pax2a gene in the ovulation-inducing process. F2 pax2a homozygous mutant zebrafish possessing a deletion of 6 nucleotides were established in this study. However, the deletion included the start codon (ATG) of the pax2a gene, and the Pax2a protein was still detected, which indicated that the deletion caused a shift in the start codon to the next ATG, resulting in a 12-amino acid deletion. F2 pax2a homozygous mutant zebrafish showed ovulation. However, the embryos showed an abnormal oval shape at the epiboly stage that resulted in yolk and tail formation abnormalities and heart edema. The surviving F3 homozygous mutants did not develop ovaries. Pax2a was detected in oocytes and eggs but not after the Prim-22 stage. It is suggested that pax2a is expressed as a maternal gene in oocytes and is necessary for oogenesis and early development.

ZEBRAFISH AS MODELS OF TYROSINE KINASE‐DRIVEN CANCER

The protein kinase gene family encodes proteins involved in several essential functions, with cell growth and migration being essential outcomes. Mutations in kinases underlie a broad range of diseases, primarily inflammation and cancer. Tyrosine kinase inhibitors (TKIs) in particular, are successfully used as a targeted therapy for cancer and inflammation‐driven disorders. Current unmet needs include addressing the adverse effects of TKIs, the emergence of resistance, and the necessity of newer TKIs. Experiments using accessible animal models such as zebrafish can help address these needs. Zebrafish studies can help in efficient characterization of toxicological and pharmacological effects of inhibition using known or potential TKIs, via the combination of phenotypic and molecular analysis. The recent application of CRISPR‐Cas9 further enables the development of genetically engineered cancer models in zebrafish. Following our previous work showing conservation of zebrafish tyrosine kinases compared to humans, we are now studying the effects of chemical or genetic tyrosine kinase modulation in zebrafish.To identify the phenotypic endpoints of tyrosine kinase inhibition in zebrafish, we selected a few drugs approved by the USFDA for treating cancer and inflammation‐related disorders in humans. We evaluated phenotypic effects including adverse effects to identify NOELs and detectable and consistent phenotypic endpoints of such compounds in zebrafish. These effects provide a basis to evaluate novel TKI compounds. Further, tyrosine kinase genes involved in inflammation and cancer were selected for targeted genetic modification in zebrafish using CRISPR Cas9. Our initial data demonstrate the development of MET (HGFR) homozygous mutant zebrafish and the phenotypes associated with this mutant. Detailed characterization of this mutant including molecular analysis is under way. The characteristics of this mutant provide the basis to identify MET‐specific inhibitors. Development of zebrafish models carrying oncogenic TK mutants, and detailed characterization of the effects of TKIs in zebrafish are ongoing.In summary, our work is focussed on identifying TKI treatment‐related phenotypes in zebrafish, and generation of TK‐crispant zebrafish as cancer models.Support or Funding InformationDepartment of Biotechnology and the Indian Council of Medical Research, Govt. of India

Modeling ZNF408-Associated FEVR in Zebrafish Results in Abnormal Retinal Vasculature.

PurposeFamilial exudative vitreoretinopathy (FEVR) is an inherited retinal disease in which the retinal vasculature is affected. Patients with FEVR typically lack or have abnormal vasculature in the peripheral retina, the outcome of which can range from mild visual impairment to complete blindness. A missense mutation (p.His455Tyr) in ZNF408 was identified in an autosomal dominant FEVR family. Little, however, is known about the molecular role of ZNF408 and how its defect leads to the clinical features of FEVR.MethodsUsing CRISPR/Cas9 technology, two homozygous mutant zebrafish models with truncated znf408 were generated, as well as one heterozygous and one homozygous missense znf408 model in which the human p.His455Tyr mutation is mimicked.ResultsIntriguingly, all three znf408-mutant zebrafish strains demonstrated progressive retinal vascular pathology, initially characterized by a deficient hyaloid vessel development at 5 days postfertilization (dpf) leading to vascular insufficiency in the retina. The generation of stable mutant lines allowed long-term follow up studies, which showed ectopic retinal vascular hyper-sprouting at 90 dpf and extensive vascular leakage at 180 dpf.ConclusionsTogether, our data demonstrate an important role for znf408 in the development and maintenance of the vascular system within the retina.

Targeted deletion of the zebrafish actin-bundling protein L-plastin (lcp1).

Regulation of the cytoskeleton is essential for cell migration in health and disease. Lymphocyte cytosolic protein 1 (lcp1, also called L-plastin) is a hematopoietic-specific actin-bundling protein that is highly conserved in zebrafish, mice and humans. In addition, L-plastin expression is documented as both a genetic marker and a cellular mechanism contributing to the invasiveness of tumors and transformed cell lines. Despite L-plastin’s role in both immunity and cancer, in zebrafish there are no direct studies of its function, and no mutant, knockout or reporter lines available. Using CRISPR-Cas9 genome editing, we generated null alleles of zebrafish lcp1 and examined the phenotypes of these fish throughout the life cycle. Our editing strategy used gRNA to target the second exon of lcp1, producing F0 mosaic fish that were outcrossed to wild types to confirm germline transmission. F1 heterozygotes were then sequenced to identify three unique null alleles, here called ‘Charlie’, ‘Foxtrot’ and ‘Lima’. In silico, each allele truncates the endogenous protein to less than 5% normal size and removes both essential actin-binding domains (ABD1 and ABD2). Although none of the null lines express detectable LCP1 protein, homozygous mutant zebrafish (-/-) can develop and reproduce normally, a finding consistent with that of the L-plastin null mouse (LPL -/-). However, such mice do have a profound immune defect when challenged by lung bacteria. Interestingly, we observed reduced long-term survival of zebrafish lcp1 -/- homozygotes (~30% below the expected numbers) in all three of our knockout lines, with greatest mortality corresponding to the period (4–6 weeks post-fertilization) when the innate immune system is functional, but the adaptive immune system is not yet mature. This suggests that null zebrafish may have reduced capacity to combat opportunistic infections, which are more easily transmissible in the aquatic environment. Overall, our novel mutant lines establish a sound genetic model and an enhanced platform for further studies of L-plastin gene function in hematopoiesis and cancer.

Combining genotypic and phenotypic analyses on single mutant zebrafish larvae

Zebrafish is a powerful animal model used to study vertebrate embryogenesis, organ development and diseases (Gut et al., 2017) [1]. The usefulness of the model was established as a result of various large forward genetic screens identifying mutants in almost every organ or cell type (Driever et al., 1996; Haffter et al., 1996) [2,3]. More recently, the advent of genome editing methodologies, including TALENs (Sander et al., 2011) [4] and the CRISPR/Cas9 technology (Hwang et al., 2013) [5], led to an increase in the production of zebrafish mutants. A number of these mutations are homozygous lethal at the embryonic or larval stages preventing the generation of homozygous mutant zebrafish lines. Here, we present a method allowing both genotyping and phenotype analyses of mutant zebrafish larvae from heterozygous zebrafish incrosses. The procedure is based on the genotyping of the larval tail after transection, whereas phenotypic studies are performed on the anterior part of the zebrafish larvae.•The method includes (i) a protocol for genotyping, (ii) protocols for paraffin embedding and histological analyses, (iii) protocols for protein and histone extraction and characterization by Western blot, (iv) protocols for RNA extraction and characterization by RT-PCR, and (v) protocols to study caudal spinal cord regeneration.•The technique is optimized in order to be applied on single zebrafish embryos and larvae.

Genetic Disruption of 21-Hydroxylase in Zebrafish Causes Interrenal Hyperplasia.

Congenital adrenal hyperplasia is a group of common inherited disorders leading to glucocorticoid deficiency. Most cases are caused by 21-hydroxylase deficiency (21OHD). The systemic consequences of imbalanced steroid hormone biosynthesis due to severe 21OHD remains poorly understood. Therefore, we developed a zebrafish model for 21OHD, which focuses on the impairment of glucocorticoid biosynthesis. A single 21-hydroxylase gene (cyp21a2) is annotated in the zebrafish genome based on sequence homology. Our in silico analysis of the 21-hydroxylase (Cyp21a2) protein sequence suggests a sufficient degree of similarity for the usage of zebrafish cyp21a2 to model aspects of human 21OHD in vivo. We determined the spatiotemporal expression patterns of cyp21a2 by whole-mount in situ hybridization and reverse transcription polymerase chain reaction throughout early development. Early cyp21a2 expression is restricted to the interrenal gland (zebrafish adrenal counterpart) and the brain. To further explore the in vivo consequences of 21OHD we created several cyp21a2 null-allele zebrafish lines by using a transcription activator–like effector nuclease genomic engineering strategy. Homozygous mutant zebrafish larvae showed an upregulation of the hypothalamic–pituitary–interrenal (HPI) axis and interrenal hyperplasia. Furthermore, Cyp21a2-deficient larvae had a typical steroid profile, with reduced concentrations of cortisol and increased concentrations of 17-hydroxyprogesterone and 21-deoxycortisol. Affected larvae showed an upregulation of the HPI axis and interrenal hyperplasia. Downregulation of the glucocorticoid-responsive genes pck1 and fkbp5 indicated systemic glucocorticoid deficiency. Our work demonstrates the crucial role of Cyp21a2 in glucocorticoid biosynthesis in zebrafish larvae and establishes an in vivo model allowing studies of systemic consequences of altered steroid hormone synthesis.

The Roles of RNA Polymerase I and III Subunits Polr1c and Polr1d in Craniofacial Development and in Zebrafish Models of Treacher Collins Syndrome.

Ribosome biogenesis is a global process required for growth and proliferation of all cells, yet perturbation of ribosome biogenesis during human development often leads to tissue-specific defects termed ribosomopathies. Transcription of the ribosomal RNAs (rRNAs) by RNA polymerases (Pol) I and III, is considered a rate limiting step of ribosome biogenesis and mutations in the genes coding for RNA Pol I and III subunits, POLR1C and POLR1D cause Treacher Collins syndrome, a rare congenital craniofacial disorder. Our understanding of the functions of individual RNA polymerase subunits, however, remains poor. We discovered that polr1c and polr1d are dynamically expressed during zebrafish embryonic development, particularly in craniofacial tissues. Consistent with this pattern of activity, polr1c and polr1d homozygous mutant zebrafish exhibit cartilage hypoplasia and cranioskeletal anomalies characteristic of humans with Treacher Collins syndrome. Mechanistically, we discovered that polr1c and polr1d loss-of-function results in deficient ribosome biogenesis, Tp53-dependent neuroepithelial cell death and a deficiency of migrating neural crest cells, which are the primary progenitors of the craniofacial skeleton. More importantly, we show that genetic inhibition of tp53 can suppress neuroepithelial cell death and ameliorate the skeletal anomalies in polr1c and polr1d mutants, providing a potential avenue to prevent the pathogenesis of Treacher Collins syndrome. Our work therefore has uncovered tissue-specific roles for polr1c and polr1d in rRNA transcription, ribosome biogenesis, and neural crest and craniofacial development during embryogenesis. Furthermore, we have established polr1c and polr1d mutant zebrafish as models of Treacher Collins syndrome together with a unifying mechanism underlying its pathogenesis and possible prevention.

Normal and Malignant Muscle Cell Transplantation into Immune Compromised Adult Zebrafish

Zebrafish have become a powerful tool for assessing development, regeneration, and cancer. More recently, allograft cell transplantation protocols have been developed that permit engraftment of normal and malignant cells into irradiated, syngeneic, and immune compromised adult zebrafish. These models when coupled with optimized cell transplantation protocols allow for the rapid assessment of stem cell function, regeneration following injury, and cancer. Here, we present a method for cell transplantation of zebrafish adult skeletal muscle and embryonal rhabdomyosarcoma (ERMS), a pediatric sarcoma that shares features with embryonic muscle, into immune compromised adult rag2E450fs homozygous mutant zebrafish. Importantly, these animals lack T cells and have reduced B cell function, facilitating engraftment of a wide range of tissues from unrelated donor animals. Our optimized protocols show that fluorescently labeled muscle cell preparations from α-actin-RFP transgenic zebrafish engraft robustly when implanted into the dorsal musculature of rag2 homozygous mutant fish. We also demonstrate engraftment of fluorescent-transgenic ERMS where fluorescence is confined to cells based on differentiation status. Specifically, ERMS were created in AB-strain myf5-GFP; mylpfa-mCherry double transgenic animals and tumors injected into the peritoneum of adult immune compromised fish. The utility of these protocols extends to engraftment of a wide range of normal and malignant donor cells that can be implanted into dorsal musculature or peritoneum of adult zebrafish.

Normal and malignant muscle cell transplantation into immune compromised adult zebrafish.

Zebrafish have become a powerful tool for assessing development, regeneration, and cancer. More recently, allograft cell transplantation protocols have been developed that permit engraftment of normal and malignant cells into irradiated, syngeneic, and immune compromised adult zebrafish. These models when coupled with optimized cell transplantation protocols allow for the rapid assessment of stem cell function, regeneration following injury, and cancer. Here, we present a method for cell transplantation of zebrafish adult skeletal muscle and embryonal rhabdomyosarcoma (ERMS), a pediatric sarcoma that shares features with embryonic muscle, into immune compromised adult rag2E450fs homozygous mutant zebrafish. Importantly, these animals lack T cells and have reduced B cell function, facilitating engraftment of a wide range of tissues from unrelated donor animals. Our optimized protocols show that fluorescently labeled muscle cell preparations from α-actin-RFP transgenic zebrafish engraft robustly when implanted into the dorsal musculature of rag2 homozygous mutant fish. We also demonstrate engraftment of fluorescent-transgenic ERMS where fluorescence is confined to cells based on differentiation status. Specifically, ERMS were created in AB-strain myf5-GFP; mylpfa-mCherry double transgenic animals and tumors injected into the peritoneum of adult immune compromised fish. The utility of these protocols extends to engraftment of a wide range of normal and malignant donor cells that can be implanted into dorsal musculature or peritoneum of adult zebrafish.

The Translation Initiation Factor eIF3i Up-regulates Vascular Endothelial Growth Factor A, Accelerates Cell Proliferation, and Promotes Angiogenesis in Embryonic Development and Tumorigenesis

Vascular endothelial growth factor A (VEGFA) is a critical proangiogenic factor that is activated by hypoxia at both the transcriptional and post-transcriptional levels. In hypoxia conditions, stabilized hypoxia-inducible factor 1α (HIF1A) is the key regulator for transcriptional activation of VEGFA. However, the post-transcriptional control of VEGFA expression remains poorly understood. Here, we report that the eukaryotic translation initiation factor 3i (eIF3i) is required for VEGFA protein expression in both normal embryonic and tumorigenic angiogenesis. eIF3i is dynamically expressed in the early stages of zebrafish embryogenesis and in human hepatocellular carcinoma tissues. eIF3i homozygous mutant zebrafish embryos show severe angiogenesis defects and human hepatocellular cancer cells with depletion of eIF3i to induce less angiogenesis in tumor models. Under hypoxia, the HIF1A protein can interact with its binding sequence in the eIF3i promoter and activate eIF3i transcription. The expression of VEGFA, which should rise in hypoxia, is significantly inhibited by eIF3i siRNA treatment. Moreover, eIF3i knockdown did not cause a general translation repression but specifically reduced the translation efficiency of the VEGFA mRNAs. Taken together, our results suggest that eIF3i is induced by HIF1A under hypoxia and controls normal and tumorigenic angiogenesis through regulating VEGFA protein translation.

Loss of function tp53 mutations do not accelerate the onset of myc‐induced T‐cell acute lymphoblastic leukaemia in the zebrafish

The TP53 tumour suppressor is activated in response to distinct stimuli, including an ARF-dependent response to oncogene stress and an ATM/ATR-dependent response to DNA damage. In human T-cell acute lymphoblastic leukaemia (T-ALL), TP53-dependent tumour suppression is typically disabled via biallelic ARF deletions. In murine models, loss of Arf (Cdkn2a) or Tp53 markedly accelerates the onset of Myc-induced lymphoblastic malignancies. In zebrafish, no ARF ortholog has been identified, but the sequence of ARF is very poorly conserved evolutionarily, making it difficult to exclude the presence of a zebrafish ARF ortholog without functional studies. Here we show that tp53 mutations have no significant influence on the onset of myc-induced T-ALL in zebrafish, consistent with the lack of additional effects of Tp53 loss on lymphomagenesis in Arf-deficient mice. By contrast, irradiation leads to complete T-ALL regression in tp53 wild-type but not homozygous mutant zebrafish, indicating that the tp53-dependent DNA damage response is intact. We conclude that tp53 inactivation has no impact on the onset of myc-induced T-ALL in the zebrafish, consistent with the lack of a functional ARF ortholog linking myc-induced oncogene stress to tp53-dependent tumour suppression. Thus, the zebrafish model is well suited to the study of ARF-independent pathways in T-ALL pathobiology.