Zebrafish Injectable Plastic for Identification Tagging (ZIP IT) for larvae to adults using a fluorescent Visible Implant Elastomer.

Distinguishing individuals or small groups is essential for many experiments. The regenerative properties of zebrafish make traditional marking methods for rodent models (e.g., tattoos, tissue clipping) ineffective. Fluorescent Visible Implant Elastomer (VIE) can permanently mark juvenile and adult zebrafish but to date no marking technique has been described for larval stage zebrafish. This protocol, Zebrafish Injectable Plastic for Identification Tagging (ZIP IT), utilizes VIE in zebrafish as early as 2 days post fertilization (dpf) using standard microinjection methods and direct injection using an insulin syringe at one month. Larval zebrafish between 2 and 7 dpf were injected in the dorsal musculature. At one month, retention and visibility of the VIE was observed in 72% of the injected fish with no effect on growth; however, a variable change in mortality was observed, generally higher than the uninjected fish. This demonstrates that VIE can be used in very early stages of fish development, providing the first procedure to track individuals or groups within a larger population. Subcutaneous injection of juvenile zebrafish starting at one month has greater than 99%-mark retention and visibility with very low mortality. The combination of larvae and juvenile VIE injections also provide a powerful tool to track and gather data from marked fish throughout their lifespan.•We present a method for tagging individuals or groups of zebrafish at most life stages (larvae or juvenile/adult) with Visible Implant Elastomer (VIE).•The larvae and juvenile injection procedures can be combined so that an individual fish can be tagged for its entire lifetime. Larvae injections become significantly less visible after one month; however, the fish can be reinjected using the juvenile procedure, thus allowing continuity of the visible mark.•This protocol was empirically built on the "Evaluation of VIE tags in Zebrafish (Danio rerio)" by Hohn and Petrie-Hanson (2013) and expanded to include larvae and a small batch elastomer mixing technique.

Transcriptomic profiling of tissue environments critical for post-embryonic patterning and morphogenesis of zebrafish skin.

Pigment patterns and skin appendages are prominent features of vertebrate skin. In zebrafish, regularly patterned pigment stripes and an array of calcified scales form simultaneously in the skin during post-embryonic development. Understanding the mechanisms that regulate stripe patterning and scale morphogenesis may lead to the discovery of fundamental mechanisms that govern the development of animal form. To learn about cell types and signaling interactions that govern skin patterning and morphogenesis, we generated and analyzed single-cell transcriptomes of skin from wild-type fish as well as fish having genetic or transgenically induced defects in squamation or pigmentation. These data reveal a previously undescribed population of epidermal cells that express transcripts encoding enamel matrix proteins, suggest hormonal control of epithelial-mesenchymal signaling, clarify the signaling network that governs scale papillae development, and identify a critical role for the hypodermis in supporting pigment cell development. Additionally, these comprehensive single-cell transcriptomic data representing skin phenotypes of biomedical relevance should provide a useful resource for accelerating the discovery of mechanisms that govern skin development and homeostasis.

Fluorescent PCR-based Screening Methods for Precise Knock-in of Small DNA Fragments and Point Mutations in Zebrafish.

Generation of zebrafish (Danio rerio) models with targeted insertion of epitope tags and point mutations is highly desirable for functional genomics and disease modeling studies. Currently, CRISPR/Cas9-mediated knock-in is the method of choice for insertion of exogeneous sequences by providing a repair template for homology-directed repair (HDR). A major hurdle in generating knock-in models is the labor and cost involved in screening of injected fish to identify the precise knock-in events due to low efficiency of the HDR pathway in zebrafish. Thus, we developed fluorescent PCR-based high-throughput screening methods for precise knock-in of epitope tags and point mutations in zebrafish. Here, we provide a step-by-step guide that describes selection of an active sgRNA near the intended knock-in site, design of single-stranded oligonucleotide (ssODN) templates for HDR, quick validation of somatic knock-in using injected embryos, and screening for germline transmission of precise knock-in events to establish stable lines. Our screening method relies on the size-based separation of all fragments in an amplicon by fluorescent PCR and capillary electrophoresis, thus providing a robust and cost-effective strategy. Although we present the use of this protocol for insertion of epitope tags and point mutations, it can be used for insertion of any small DNA fragments (e.g., LoxP sites, in-frame codons). Furthermore, the screening strategy described here can be used to screen for precise knock-in of small DNA sequences in any model system, as PCR amplification of the target region is its only requirement. Key features This protocol expands the use of fluorescent PCR and CRISPR-STAT for screening of precise knock-in of small insertions and point mutations in zebrafish. Allows validation of selected sgRNA and HDR template within two weeks by somatic knock-in screening. Allows robust screening of point mutations by combining restriction digest with CRISPR-STAT. Graphical overview Overview of the three-phase knock-in pipeline in zebrafish (created with BioRender.com).

A novel de novo TP63 mutation in whole-exome sequencing of a Syrian family with Oral cleft and ectrodactyly.

BACKGROUND: Oral clefts and ectrodactyly are common, heterogeneous birth defects. We performed whole-exome sequencing (WES) analysis in a Syrian family. The proband presented with both orofacial clefting and ectrodactyly but not ectodermal dysplasia as typically seen in ectrodactyly, ectodermal dysplasia, and cleft lip/palate syndrome-3. A paternal uncle with only an oral cleft was deceased and unavailable for analysis. METHODS: Variant annotation, Mendelian inconsistencies, and novel variants in known cleft genes were examined. Candidate variants were validated using Sanger sequencing, and pathogenicity assessed by knocking out the tp63 gene in zebrafish to evaluate its role during zebrafish development. RESULTS: Twenty-eight candidate de novo events were identified, one of which is in a known oral cleft and ectrodactyly gene, TP63 (c.956G > T, p.Arg319Leu), and confirmed by Sanger sequencing. CONCLUSION: TP63 mutations are associated with multiple autosomal dominant orofacial clefting and limb malformation disorders. The p.Arg319Leu mutation seen in this patient is de novo but also novel. Two known mutations in the same codon (c.956G > A, p.(Arg319His; rs121908839, c.955C > T), p.Arg319Cys) cause ectrodactyly, providing evidence that mutating this codon is deleterious. While this TP63 mutation is the best candidate for the patient's clinical presentation, whether it is responsible for the entire phenotype is unclear. Generation and characterization of tp63 knockout zebrafish showed necrosis and rupture of the head at 3 days post-fertilization (dpf). The embryonic phenotype could not be rescued by injection of zebrafish or human messenger RNA (mRNA). Further functional analysis is needed to determine what proportion of the phenotype is due to this mutation.

A RUNX1-FPDMM rhesus macaque model reproduces the human phenotype and predicts challenges to curative gene therapies.

Germ line loss-of-function heterozygous mutations in the RUNX1 gene cause familial platelet disorder with associated myeloid malignancies (FPDMM) characterized by thrombocytopenia and a life-long risk of hematological malignancies. Although gene therapies are being considered as promising therapeutic options, current preclinical models do not recapitulate the human phenotype and are unable to elucidate the relative fitness of mutation-corrected and RUNX1-heterozygous mutant hematopoietic stem and progenitor cells (HSPCs) in vivo long term. We generated a rhesus macaque with an FPDMM competitive repopulation model using CRISPR/Cas9 nonhomologous end joining editing in the RUNX1 gene and the AAVS1 safe-harbor control locus. We transplanted mixed populations of edited autologous HSPCs and tracked mutated allele frequencies in blood cells. In both animals, RUNX1-edited cells expanded over time compared with AAVS1-edited cells. Platelet counts remained below the normal range in the long term. Bone marrows developed megakaryocytic dysplasia similar to human FPDMM, and CD34+ HSPCs showed impaired in vitro megakaryocytic differentiation, with a striking defect in polyploidization. In conclusion, the lack of a competitive advantage for wildtype or control-edited HSPCs over RUNX1 heterozygous-mutated HSPCs long term in our preclinical model suggests that gene correction approaches for FPDMM will be challenging, particularly to reverse myelodysplastic syndrome/ acute myeloid leukemia predisposition and thrombopoietic defects.

A robust pipeline for efficient knock-in of point mutations and epitope tags in zebrafish using fluorescent PCR based screening.

BACKGROUND: Genome editing using CRISPR/Cas9 has become a powerful tool in zebrafish to generate targeted gene knockouts models. However, its use for targeted knock-in remains challenging due to inefficient homology directed repair (HDR) pathway in zebrafish, highlighting the need for efficient and cost-effective screening methods.  RESULTS: Here, we present our fluorescent PCR and capillary electrophoresis based screening approach for knock-in using a single-stranded oligodeoxynucleotide donor (ssODN) as a repair template for the targeted insertion of epitope tags, or single nucleotide changes to recapitulate pathogenic human alleles. For the insertion of epitope tags, we took advantage of the expected change in size of the PCR product. For point mutations, we combined fluorescent PCR with restriction fragment length polymorphism (RFLP) analysis to distinguish the fish with the knock-in allele. As a proof-of-principle, we present our data on the generation of fish lines with insertion of a FLAG tag at the tcnba locus, an HA tag at the gata2b locus, and a point mutation observed in Gaucher disease patients in the gba gene. Despite the low number of germline transmitting founders (1-5%), combining our screening methods with prioritization of founder fish by fin biopsies allowed us to establish stable knock-in lines by screening 12 or less fish per gene. CONCLUSIONS: We have established a robust pipeline for the generation of zebrafish models with precise integration of small DNA sequences and point mutations at the desired sites in the genome. Our screening method is very efficient and easy to implement as it is PCR-based and only requires access to a capillary sequencer.

Zrsr2 Is Essential for the Embryonic Development and Splicing of Minor Introns in RNA and Protein Processing Genes in Zebrafish.

ZRSR2 (zinc finger CCCH-type, RNA binding motif and serine/arginine rich 2) is an essential splicing factor involved in 3' splice-site recognition as a component of both the major and minor spliceosomes that mediate the splicing of U2-type (major) and U12-type (minor) introns, respectively. Studies of ZRSR2-depleted cell lines and ZRSR2-mutated patient samples revealed its essential role in the U12-dependent minor spliceosome. However, the role of ZRSR2 during embryonic development is not clear, as its function is compensated for by Zrsr1 in mice. Here, we utilized the zebrafish model to investigate the role of zrsr2 during embryonic development. Using CRISPR/Cas9 technology, we generated a zrsr2-knockout zebrafish line, termed zrsr2hg129/hg129 (p.Trp167Argfs*9) and examined embryo development in the homozygous mutant embryos. zrsr2hg129/hg129 embryos displayed multiple developmental defects starting at 4 days post fertilization (dpf) and died after 8 dpf, suggesting that proper Zrsr2 function is required during embryonic development. The global transcriptome analysis of 3 dpf zrsr2hg129/hg129 embryos revealed that the loss of Zrsr2 results in the downregulation of essential metabolic pathways and the aberrant retention of minor introns in about one-third of all minor intron-containing genes in zebrafish. Overall, our study has demonstrated that the role of Zrsr2 as a component of the minor spliceosome is conserved and critical for proper embryonic development in zebrafish.

A Comprehensive Review of Indel Detection Methods for Identification of Zebrafish Knockout Mutants Generated by Genome-Editing Nucleases.

The use of zebrafish in functional genomics and disease modeling has become popular due to the ease of targeted mutagenesis with genome editing nucleases, i.e., zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9). These nucleases, specifically CRISPR/Cas9, are routinely used to generate gene knockout mutants by causing a double stranded break at the desired site in the target gene and selecting for frameshift insertions or deletions (indels) caused by the errors during the repair process. Thus, a variety of methods have been developed to identify fish with indels during the process of mutant generation and phenotypic analysis. These methods range from PCR and gel-based low-throughput methods to high-throughput methods requiring specific reagents and/or equipment. Here, we provide a comprehensive review of currently used indel detection methods in zebrafish. By discussing the molecular basis for each method as well as their pros and cons, we hope that this review will serve as a comprehensive resource for zebrafish researchers, allowing them to choose the most appropriate method depending upon their budget, access to required equipment and the throughput needs of the projects.

Pancytopenia and thrombosis defects in zebrafish mutants of Fanconi anemia genes.

Progressive pancytopenia is a common feature observed in DNA crosslink repair deficiency disorder, Fanconi anemia (FA). However, this phenotype has not been recapitulated in single FA gene knockout animal models. In this study, we analyzed hematological characteristics in zebrafish null mutants for two FA genes, fanca and fanco. In adult mutants, we demonstrate age-associated reduction in blood cell counts for all lineages, resembling progressive pancytopenia in FA patients. In larval mutants, we demonstrate vascular injury-induced thrombosis defects, particularly upon treatment with crosslinking agent diepoxybutane (DEB), indicating DNA damage induced inefficiency of thrombocytes.

Redundant mechanisms driven independently by RUNX1 and GATA2 for hematopoietic development.

RUNX1 is essential for the generation of hematopoietic stem cells (HSCs). Runx1-null mouse embryos lack definitive hematopoiesis and die in mid-gestation. However, although zebrafish embryos with a runx1 W84X mutation have defects in early definitive hematopoiesis, some runx1W84X/W84X embryos can develop to fertile adults with blood cells of multilineages, raising the possibility that HSCs can emerge without RUNX1. Here, using 3 new zebrafish runx1-/- lines, we uncovered the compensatory mechanism for runx1-independent hematopoiesis. We show that, in the absence of a functional runx1, a cd41-green fluorescent protein (GFP)+ population of hematopoietic precursors still emerge from the hemogenic endothelium and can colonize the hematopoietic tissues of the mutant embryos. Single-cell RNA sequencing of the cd41-GFP+ cells identified a set of runx1-/--specific signature genes during hematopoiesis. Significantly, gata2b, which normally acts upstream of runx1 for the generation of HSCs, was increased in the cd41-GFP+ cells in runx1-/- embryos. Interestingly, genetic inactivation of both gata2b and its paralog gata2a did not affect hematopoiesis. However, knocking out runx1 and any 3 of the 4 alleles of gata2a and gata2b abolished definitive hematopoiesis. Gata2 expression was also upregulated in hematopoietic cells in Runx1-/- mice, suggesting the compensatory mechanism is conserved. Our findings indicate that RUNX1 and GATA2 serve redundant roles for HSC production, acting as each other's safeguard.

Large-scale generation and phenotypic characterization of zebrafish CRISPR mutants of DNA repair genes.

A systematic knowledge of the roles of DNA repair genes at the level of the organism has been limited due to the lack of appropriate experimental approaches using animal model systems. Zebrafish has become a powerful vertebrate genetic model system with availability due to the ease of genome editing and large-scale phenotype screening. Here, we generated zebrafish mutants for 32 DNA repair and replication genes through multiplexed CRISPR/Cas9-mediated mutagenesis. Large-scale phenotypic characterization of our mutant collection revealed that three genes (atad5a, ddb1, pcna) are essential for proper embryonic development and hematopoiesis; seven genes (apex1, atrip, ino80, mre11a, shfm1, telo2, wrn) are required for growth and development during juvenile stage and six genes (blm, brca2, fanci, rad51, rad54l, rtel1) play critical roles in sex development. Furthermore, mutation in six genes (atad5a, brca2, polk, rad51, shfm1, xrcc1) displayed hypersensitivity to DNA damage agents. Our zebrafish mutant collection provides a unique resource for understanding of the roles of DNA repair genes at the organismal level.

Compound heterozygous KCTD7 variants in progressive myoclonus epilepsy.

KCTD7 is a member of the potassium channel tetramerization domain-containing protein family and has been associated with progressive myoclonic epilepsy (PME), characterized by myoclonus, epilepsy, and neurological deterioration. Here we report four affected individuals from two unrelated families in which we identified KCTD7 compound heterozygous single nucleotide variants through exome sequencing. RNAseq was used to detect a non-annotated splicing junction created by a synonymous variant in the second family. Whole-cell patch-clamp analysis of neuroblastoma cells overexpressing the patients' variant alleles demonstrated aberrant potassium regulation. While all four patients experienced many of the common clinical features of PME, they also showed variable phenotypes not previously reported, including dysautonomia, brain pathology findings including a significantly reduced thalamus, and the lack of myoclonic seizures. To gain further insight into the pathogenesis of the disorder, zinc finger nucleases were used to generate kctd7 knockout zebrafish. Kctd7 homozygous mutants showed global dysregulation of gene expression and increased transcription of c-fos, which has previously been correlated with seizure activity in animal models. Together these findings expand the known phenotypic spectrum of KCTD7-associated PME, report a new animal model for future studies, and contribute valuable insights into the disease.

Clinical and genomic analysis of a large Chinese family with familial cortical myoclonic tremor with epilepsy and SAMD12 intronic repeat expansion.

Objective: Our goal was to perform detailed clinical and genomic analysis of a large multigenerational Chinese family with 21 individuals showing symptoms of Familial Cortical Myoclonic Tremor with Epilepsy (FCMTE) that we have followed for over 20 years. Methods: Patients were subjected to clinical evaluation, routine EEG, and structural magnetic resonance imaging. Whole exome sequencing, repeat-primed PCR, long-range PCR, and PacBio sequencing were performed to characterize the disease-causing mutation in this family. Results: All evaluated patients manifested adult-onset seizures and presented with progressive myoclonic postural tremors starting after the third or fourth decade of life. Seizures typically diminished markedly in frequency with implementation of antiseizure medications but did not completely cease. The electroencephalogram of affected individuals showed generalized or multifocal spikes and slow wave complexes. An expansion of TTTTA motifs with addition of TTTCA motifs in intron 4 of SAMD12 was identified to segregate with the disease phenotype in this family. Furthermore, we found that the mutant allele is unstable and can undergo both contraction and expansion by changes in the number of repeat motifs each time it is passed to the next generation. The size of mutant allele varied from 5 to 5.5 kb with 549-603 copies of TTTTA and 287-343 copies of TTTCA repeat motifs in this family. Significance: Our study provides a detailed description of clinical progression of FCMTE symptoms and its management with antiseizure medications. Our method of repeat analysis by PacBio sequencing of long-range PCR products does not require high-quality DNA and hence can be easily applied to other families to elucidate any correlation between the repeat size and phenotypic variables, such as, age of onset, and severity of symptoms.

Rare hypomorphic human variation in the heptahelical domain of SMO contributes to holoprosencephaly phenotypes.

Holoprosencephaly (HPE) is the most common congenital anomaly affecting the forebrain and face in humans and occurs as frequently as 1:250 conceptions or 1:10,000 livebirths. Sonic Hedgehog signaling molecule is one of the best characterized HPE genes that plays crucial roles in numerous developmental processes including midline neural patterning and craniofacial development. The Frizzled class G-protein coupled receptor Smoothened (SMO), whose signaling activity is tightly regulated, is the sole obligate transducer of Hedgehog-related signals. However, except for previous reports of somatic oncogenic driver mutations in human cancers (or mosaic tumors in rare syndromes), any potential disease-related role of SMO genetic variation in humans is largely unknown. To our knowledge, ours is the first report of a human hypomorphic variant revealed by functional testing of seven distinct nonsynonymous SMO variants derived from HPE molecular and clinical data. Here we describe several zebrafish bioassays developed and guided by a systems biology analysis. This analysis strategy, and detection of hypomorphic variation in human SMO, demonstrates the necessity of integrating the genomic variant findings in HPE probands with other components of the Hedgehog gene regulatory network in overall medical interpretations.

Functional analysis of Sonic Hedgehog variants associated with holoprosencephaly in humans using a CRISPR/Cas9 zebrafish model.

Genetic variation in the highly conserved Sonic Hedgehog (SHH) gene is one of the most common genetic causes for the malformations of the brain and face in humans described as the holoprosencephaly clinical spectrum. However, only a minor fraction of known SHH variants have been experimentally proven to lead to abnormal function. Employing a phenotypic rescue assay with synthetic human messenger RNA variant constructs in shha-/- knockout zebrafish, we evaluated 104 clinically reported in-frame and missense SHH variants. Our data helped us to classify them into loss of function variants (31), hypomorphic variants (33), and nonpathogenic variants (40). We discuss the strengths and weaknesses of currently accepted predictors of variant deleteriousness and the American College of Medical Genetics and Genomics guidelines for variant interpretation in the context of this functional model; furthermore, we demonstrate the robustness of model systems such as zebrafish as a rapid method to resolve variants of uncertain significance.

BE4max and AncBE4max Are Efficient in Germline Conversion of C:G to T:A Base Pairs in Zebrafish.

The ease of use and robustness of genome editing by CRISPR/Cas9 has led to successful use of gene knockout zebrafish for disease modeling. However, it still remains a challenge to precisely edit the zebrafish genome to create single-nucleotide substitutions, which account for ~60% of human disease-causing mutations. Recently developed base editing nucleases provide an excellent alternate to CRISPR/Cas9-mediated homology dependent repair for generation of zebrafish with point mutations. A new set of cytosine base editors, termed BE4max and AncBE4max, demonstrated improved base editing efficiency in mammalian cells but have not been evaluated in zebrafish. Therefore, we undertook this study to evaluate their efficiency in converting C:G to T:A base pairs in zebrafish by somatic and germline analysis using highly active sgRNAs to twist and ntl genes. Our data demonstrated that these improved BE4max set of plasmids provide desired base substitutions at similar efficiency and without any indels compared to the previously reported BE3 and Target-AID plasmids in zebrafish. Our data also showed that AncBE4max produces fewer incorrect and bystander edits, suggesting that it can be further improved by codon optimization of its components for use in zebrafish.

Generation of Novel Genetic Models to Dissect Resistance to Thyroid Hormone Receptor α in Zebrafish.

Background: Patients with mutations of the thyroid hormone receptor alpha (THRA) gene show resistance to thyroid hormone alpha (RTHα). No amendable mouse models are currently available to elucidate deleterious effects of TRα1 mutants during early development. Zebrafish with transient suppressed expression by morpholino knockdown and ectopic expression of TRα1 mutants in the embryos have been reported. However, zebrafish with germline transmittable mutations have not been reported. The stable expression of thra mutants from embryos to adulthood facilitated the study of molecular actions of TRα1 mutants during development. Methods: In contrast to human and mice, the thra gene is duplicated in zebrafish, thraa, and thrab. Using CRISPR/Cas9-mediated targeted mutagenesis, we created dominant negative mutations in the two duplicated thra genes. We comprehensively analyzed the molecular and phenotypic characteristics of mutant fish during development. Results: Adult and juvenile homozygous thrab 1-bp ins (m/m) mutants exhibited severe growth retardation, but adult homozygous thraa 8-bp ins (m/m) mutants had very mild growth impairment. Expression of the growth hormone (gh1) and insulin-like growth factor 1 was markedly suppressed in homozygous thrab 1-bp ins (m/m) mutants. Decreased messenger RNA and protein levels of triiodothyronine-regulated keratin genes and inhibited keratinocyte proliferation resulted in hypoplasia of the epidermis in adult and juvenile homozygous thrab 1-bp ins (m/m) mutants, but not homozygous thraa 8-bp ins (m/m) mutants. RNA-seq analysis showed that homozygous thrab 1-bp ins (m/m) mutation had global impact on the functions of the adult pituitary. However, no morphological defects nor any changes in the expression of gh1 and keratin genes were observed in the embryos and early larvae. Thus, mutations of either the thraa or thrab gene did not affect initiation of embryogenesis. But the mutation of the thrab gene, but not the thraa gene, is detrimental in postlarval growth and skin development. Conclusions: The thra duplicated genes are essential to control temporal coordination in postlarval growth and development in a tissue-specific manner. We uncovered novel functions of the duplicated thra genes in zebrafish in development. These mutant zebrafish could be used as a model for further analysis of TRα1 mutant actions and for rapid screening of therapeutics for RTHα.

Splicing factor DHX15 affects tp53 and mdm2 expression via alternate splicing and promoter usage.

DHX15, a DEAH box containing RNA helicase, is a splicing factor required for the last step of splicing. Recent studies identified a recurrent mutational hotspot, R222G, in DHX15 in ∼ 6% of acute myeloid leukemia (AML) patients that carry the fusion protein RUNX1-RUNX1T1 produced by t (8;21) (q22;q22). Studies using yeast mutants showed that substitution of G for the residue equivalent to R222 leads to loss of its helicase function, suggesting that it is a loss-of-function mutation. To elucidate the role of DHX15 during development, we established the first vertebrate knockout model with CRISPR/Cas9 in zebrafish. Our data showed that dhx15 expression is enriched in the brain, eyes, pectoral fin primordia, liver and intestinal bulb during embryonic development. Dhx15 deficiency leads to pleiotropic morphological phenotypes in homozygous mutant embryos starting at 3 days post fertilization (dpf) that result in lethality by 7 dpf, revealing an essential role during embryonic development. RNA-seq analysis suggested important roles of Dhx15 in chromatin and nucleosome assembly and regulation of the Mdm2-p53 pathway. Interestingly, exons corresponding to the alternate transcriptional start sites for tp53 and mdm2 were preferentially expressed in the mutant embryos, leading to significant upregulation of their alternate isoforms, Δ113p53 (orthologous to Δ133p53 isoform in human) and mdm2-P2 (isoform using distal promoter P2), respectively. We speculate that these alterations in the Mdm2-p53 pathway contribute to the development of AML in patients with t(8;21) and somatically mutated DHX15.

A model for reticular dysgenesis shows impaired sensory organ development and hair cell regeneration linked to cellular stress.

Mutations in the gene AK2 are responsible for reticular dysgenesis (RD), a rare and severe form of primary immunodeficiency in children. RD patients have a severely shortened life expectancy and without treatment die, generally from sepsis soon after birth. The only available therapeutic option for RD is hematopoietic stem cell transplantation (HSCT). To gain insight into the pathophysiology of RD, we previously created zebrafish models for Ak2 deficiencies. One of the clinical features of RD is hearing loss, but its pathophysiology and causes have not been determined. In adult mammals, sensory hair cells of the inner ear do not regenerate; however, their regeneration has been observed in several non-mammalian vertebrates, including zebrafish. Therefore, we used our RD zebrafish models to determine whether Ak2 deficiency affects sensory organ development and/or hair cell regeneration. Our studies indicated that Ak2 is required for the correct development, survival and regeneration of sensory hair cells. Interestingly, Ak2 deficiency induces the expression of several oxidative stress markers and it triggers an increased level of cell death in the hair cells. Finally, we show that glutathione treatment can partially rescue hair cell development in the sensory organs in our RD models, pointing to the potential use of antioxidants as a therapeutic treatment supplementing HSCT to prevent or ameliorate sensorineural hearing deficits in RD patients.

Multiplexed CRISPR/Cas9-mediated knockout of 19 Fanconi anemia pathway genes in zebrafish revealed their roles in growth, sexual development and fertility.

Fanconi Anemia (FA) is a genomic instability syndrome resulting in aplastic anemia, developmental abnormalities, and predisposition to hematological and other solid organ malignancies. Mutations in genes that encode proteins of the FA pathway fail to orchestrate the repair of DNA damage caused by DNA interstrand crosslinks. Zebrafish harbor homologs for nearly all known FA genes. We used multiplexed CRISPR/Cas9-mediated mutagenesis to generate loss-of-function mutants for 17 FA genes: fanca, fancb, fancc, fancd1/brca2, fancd2, fance, fancf, fancg, fanci, fancj/brip1, fancl, fancm, fancn/palb2, fanco/rad51c, fancp/slx4, fancq/ercc4, fanct/ube2t, and two genes encoding FA-associated proteins: faap100 and faap24. We selected two indel mutations predicted to cause premature truncations for all but two of the genes, and a total of 36 mutant lines were generated for 19 genes. Generating two independent mutant lines for each gene was important to validate their phenotypic consequences. RT-PCR from homozygous mutant fish confirmed the presence of transcripts with indels in all genes. Interestingly, 4 of the indel mutations led to aberrant splicing, which may produce a different protein than predicted from the genomic sequence. Analysis of RNA is thus critical in proper evaluation of the consequences of the mutations introduced in zebrafish genome. We used fluorescent reporter assay, and western blots to confirm loss-of-function for several mutants. Additionally, we developed a DEB treatment assay by evaluating morphological changes in embryos and confirmed that homozygous mutants from all the FA genes that could be tested (11/17), displayed hypersensitivity and thus were indeed null alleles. Our multiplexing strategy helped us to evaluate 11 multiple gene knockout combinations without additional breeding. Homozygous zebrafish for all 19 single and 11 multi-gene knockouts were adult viable, indicating FA genes in zebrafish are generally not essential for early development. None of the mutant fish displayed gross developmental abnormalities except for fancp-/- fish, which were significantly smaller in length than their wildtype clutch mates. Complete female-to-male sex reversal was observed in knockouts for 12/17 FA genes, while partial sex reversal was seen for the other five gene knockouts. All adult females were fertile, and among the adult males, all were fertile except for the fancd1 mutants and one of the fancj mutants. We report here generation and characterization of zebrafish knockout mutants for 17 FA disease-causing genes, providing an integral resource for understanding the pathophysiology associated with the disrupted FA pathway.