Mutant Zebrafish Research Articles

Bmp4 in Zebrafish Enhances Antiviral Innate Immunity through p38 MAPK (Mitogen-Activated Protein Kinases) Pathway.

Bone morphogenetic proteins (BMPs) are a group of structurally and functionally related signaling molecules that comprise a subfamily, belonging to the TGF-β superfamily. Most BMPs play roles in the regulation of embryonic development, stem cell differentiation, tumor growth and some cardiovascular and cerebrovascular diseases. Although evidence is emerging for the antiviral immunity of a few BMPs, more BMPs are needed to determine whether this function is universal. Here, we identified the zebrafish bmp4 ortholog, whose expression is up-regulated through challenge with grass carp reovirus (GCRV) or its mimic poly(I:C). The overexpression of bmp4 in epithelioma papulosum cyprini (EPC) cells significantly decreased the viral titer of GCRV-infected cells. Moreover, compared to wild-type zebrafish, viral load and mortality were significantly increased in both larvae and adults of bmp4-/- mutant zebrafish infected with GCRV virus. We further demonstrated that Bmp4 promotes the phosphorylation of Tbk1 and Irf3 through the p38 MAPK pathway, thereby inducing the production of type I IFNs in response to virus infection. These data suggest that Bmp4 plays an important role in the host defense against virus infection. Our study expands the understanding of BMP protein functions and opens up new targets for the control of viral infection.

Osteomodulin downregulation is associated with osteoarthritis development

Abnormal subchondral bone remodeling leading to sclerosis is a main feature of osteoarthritis (OA), and osteomodulin (OMD), a proteoglycan involved in extracellular matrix mineralization, is associated with the sclerotic phenotype. However, the functions of OMD remain poorly understood, specifically in vivo. We used Omd knockout and overexpressing male mice and mutant zebrafish to study its roles in bone and cartilage metabolism and in the development of OA. The expression of Omd is deeply correlated with bone and cartilage microarchitectures affecting the bone volume and the onset of subchondral bone sclerosis and spontaneous cartilage lesions. Mechanistically, OMD binds to RANKL and inhibits osteoclastogenesis, thus controlling the balance of bone remodeling. In conclusion, OMD is a key factor in subchondral bone sclerosis associated with OA. It participates in bone and cartilage homeostasis by acting on the regulation of osteoclastogenesis. Targeting OMD may be a promising new and personalized approach for OA.

Ligament injury in adult zebrafish triggers ECM remodeling and cell dedifferentiation for scar-free regeneration

After traumatic injury, healing of mammalian ligaments is typically associated with fibrotic scarring as opposed to scar-free regeneration. In contrast, here we show that the ligament supporting the jaw joint of adult zebrafish is capable of rapid and complete scar-free healing. Following surgical transection of the jaw joint ligament, we observe breakdown of ligament tissue adjacent to the cut sites, expansion of mesenchymal tissue within the wound site, and then remodeling of extracellular matrix (ECM) to a normal ligament morphology. Lineage tracing of mature ligamentocytes following transection shows that they dedifferentiate, undergo cell cycle re-entry, and contribute to the regenerated ligament. Single-cell RNA sequencing of the regenerating ligament reveals dynamic expression of ECM genes in neural-crest-derived mesenchymal cells, as well as diverse immune cells expressing the endopeptidase-encoding gene legumain. Analysis of legumain mutant zebrafish shows a requirement for early ECM remodeling and efficient ligament regeneration. Our study establishes a new model of adult scar-free ligament regeneration and highlights roles of immune-mesenchyme cross-talk in ECM remodeling that initiates regeneration.

Depletion of Igfbp7 alleviates zebrafish NAFLD progression through inhibiting hepatic ferroptosis

AimsThe global increased expression of Insulin-like growth factor binding protein 7 (IGFBP7) has been detected in non-alcoholic fatty liver disease (NAFLD) patients, however, its roles in NAFLD and the mechanism remain largely unclear. The goal of this study is to investigate the effect and mechanism of Igfbp7 using a zebrafish NAFLD model. Main methodsThe igfbp7−/− null zebrafish mutant and the Igfbp7 liver overexpressed (LOE) transgenic zebrafish based on Gal4/UAS system were generated by CRISPR/Cas9 and Tol2 transgenic technique, respectively. The zebrafish NAFLD models in wildtypes, igfbp7−/− mutants and Igfbp7 LOE fishes have been established by high-fat diet feeding. The Igfbp7 dynamic expression and its effects on NAFLD progression have been detected and analyzed in both human NAFLD patients and zebrafish models. And the potential mechanism has been investigated through transcriptome analysis and subsequent detection and verification. Key findingsHigh Igfbp7 levels in NASH and fibrosis stages have been detected in liver tissues of both human NAFLD patients and zebrafish models. Depletion of Igfbp7 significantly alleviated liver steatosis, inflammation, and fibrosis, whereas liver specific Igfbp7 overexpression dramatically exacerbated liver fibrosis in zebrafish NAFLD model. The hepatic iron deposition, lipid peroxidation products, and ferroptosis-related index were also significantly reduced at the NASH stage in the absence of Igfbp7. Igfbp7 promotes NAFLD progression through regulating ferroptosis, and Ncoa4-mediated ferritinophagy may be the pathway of Igfbp7-regulated ferroptosis. SignificanceIgfbp7 is confirmed as an important regulator in NAFLD progression. Depleting Igfbp7 effectively alleviates zebrafish NAFLD progression by inhibiting hepatic ferroptosis, suggesting a novel potential target for NAFLD treatment.

The exploration of neuroinflammatory mechanism by which CRHR2 deficiency induced anxiety disorder

Inflammation stimulates the hypothalamic-pituitary adrenal (HPA) axis and triggers glial neuroinflammatory phenotypes, which reduces monoamine neurotransmitters by activating indoleamine 2,3-dioxygenase enzyme. These changes can induce psychiatric diseases, including anxiety. Corticotropin releasing hormone receptor 2 (CRHR2) in the HPA axis is involved in the etiology of anxiety. Omega(n)-3 polyunsaturated fatty acids (PUFAs) can attenuate anxiety through anti-inflammation and HPA axis modulation. However, the underlying molecular mechanism by CRHR2 modulates anxiety and its correlation with neuroinflammation remain unclear. Here, we first constructed a crhr2 zebrafish mutant line, and evaluated anxiety-like behaviors, gene expression associated with the HPA axis, neuroinflammatory response, neurotransmitters, and PUFAs profile in crhr2+/+ and crhr2−/− zebrafish. The crhr2 deficiency decreased cortisol levels and up-regulated crhr1 and down-regulated crhb, crhbp, ucn3l and proopiomelanocortin a (pomc a) in zebrafish. Interestingly, a significant increase in the neuroinflammatory markers, translocator protein (TSPO) and the activation of microglia M1 phenotype (CD11b) were found in crhr2−/− zebrafish. As a consequence, the expression of granulocyte-macrophage colony-stimulating factor, pro-inflammatory cytokines vascular endothelial growth factor, and astrocyte A1 phenotype c3 were up-regulated. While microglia anti-inflammatory phenotype (CD206), central anti-inflammatory cytokine interleukin-4, arginase-1, and transforming growth factor-β were downregulated. In parallel, crhr2-deficient zebrafish showed an upregulation of vdac1 protein, a TSPO ligand, and its downstream caspase-3. Furthermore, 5-HT/5-HIAA ratio was decreased and n-3 PUFAs deficiency was identified in crhr2−/− zebrafish. In conclusion, anxiety-like behavior displayed by crhr2-deficient zebrafish may be caused by the HPA axis dysfunction and enhanced neuroinflammation, which resulted in n-3 PUFAs and monoamine neurotransmitter reductions.

Novel Development of Magnetic Resonance Imaging to Quantify the Structural Anatomic Growth of Diverse Organs in Adult and Mutant Zebrafish.

Zebrafish (Danio rerio) is a widely used vertebrate animal for modeling genetic diseases by targeted editing strategies followed by gross phenotypic and biomarker characterization. While larval transparency permits microscopic detection of anatomical defects, histological adult screening for organ-level defects remains invasive, tedious, inefficient, and subject to technical artifact. Here, we describe a noninvasive magnetic resonance imaging (MRI) approach to systematically screen adult zebrafish for anatomical growth defects. An anatomical atlas of wild-type (WT) zebrafish at 5-31 months post-fertilization was created by ex vivo MRI with a 9.4 T magnet. Volumetric growth over time was measured of animals and major organs, including the brain, spinal cord, heart, eyes, optic nerve, ear, liver, kidneys, and swim bladder. Subsequently, surf1-/-, fbxl4-/-, and opa1+/- mitochondrial disease mutant adult zebrafish were quantitatively studied to compare organ volumes with age-matched WT zebrafish. Results demonstrated that MRI enabled noninvasive, high-resolution, rapid screening of mutant adult zebrafish for overall and organ-specific growth abnormalities. Detailed volumetric analyses of three mitochondrial disease mutants delineated specific organ differences, including significantly increased brain growth in surf1-/- and opa1+/-, and marginally significant decreased heart and spinal cord volumes in surf1-/- mutants. This is interesting as we know neurological involvement can be seen in SURF1-/- patients with ataxia, dystonia, and lesions in basal ganglia, as well as in OPA1+/- patients with spasticity, ataxia, and hyperreflexia indicative of neuropathology. Similarly, cardiomyopathy is a known sequelae of cardiac pathology in patients with SURF1-/--related disease. Future studies will define MRI signaling patterns of organ dysfunction to further delineate specific pathology.

Cooperative contributions of the klf1 and klf17 genes in zebrafish primitive erythropoiesis

Krüppel-like transcription factors (Klfs), which are characterized by the three conserved C-terminal zinc fingers, are involved in various biological processes, such as haematopoiesis and angiogenesis. However, how the Klf family of transcription factors cooperate in organogenesis remains elusive. During zebrafish embryogenesis, both klf1 and klf17 are expressed in the intermediate cell mass (ICM), where primitive erythroid cells are produced. Using CRISPR–Cas9 genome editing technology, we established klf1-klf17 double mutant zebrafish to investigate the functionally interactive roles of the klf1 and klf17 genes. The klf1-klf17 mutant exhibited a diminished number of circulating primitive erythroid cells at 2 days postfertilization (dpf), while klf1 or klf17 single mutants and wild-type embryos produced comparable numbers of primitive erythroid cells. Circulating erythroid cells from the klf1-klf17 mutant possessed larger nuclei at 2 dpf than wild-type cells, suggesting the impairment of primitive erythroid cell maturation. The expression of the erythroid cell maturation markers band3 and mitoferrin, but not the haematopoietic progenitor markers c-myb and scl, was decreased in the klf1-klf17 mutant at 1 dpf. Thus, these results illustrate the cooperative function of klf1 and klf17 in the maturation processes of zebrafish primitive erythroid cells.

The RNA-binding protein Adad1 is necessary for germ cell maintenance and meiosis in zebrafish.

The double stranded RNA binding protein Adad1 (adenosine deaminase domain containing 1) is a member of the adenosine deaminase acting on RNAs (Adar) protein family with germ cell-specific expression. In mice, Adad1 is necessary for sperm differentiation, however its function outside of mammals has not been investigated. Here, through an N-ethyl-N-nitrosourea (ENU) based forward genetic screen, we identified an adad1 mutant zebrafish line that develops as sterile males. Further histological examination revealed complete lack of germ cells in adult mutant fish, however germ cells populated the gonad, proliferated, and entered meiosis in larval and juvenile fish. Although meiosis was initiated in adad1 mutant testes, the spermatocytes failed to progress beyond the zygotene stage. Thus, Adad1 is essential for meiosis and germline maintenance in zebrafish. We tested if spermatogonial stem cells were affected using nanos2 RNA FISH and a label retaining cell (LRC) assay, and found that the mutant testes had fewer LRCs and nanos2-expressing cells compared to wild-type siblings, suggesting that failure to maintain the spermatogonial stem cells resulted in germ cell loss by adulthood. To identify potential molecular processes regulated by Adad1, we sequenced bulk mRNA from mutants and wild-type testes and found mis-regulation of genes involved in RNA stability and modification, pointing to a potential broader role in post-transcriptional regulation. Our findings suggest that the RNA regulatory protein Adad1 is required for fertility through regulation of spermatogonial stem cell maintenance in zebrafish.

The zebrafish mutant dreammist implicates sodium homeostasis in sleep regulation.

Sleep is a nearly universal feature of animal behaviour, yet many of the molecular, genetic, and neuronal substrates that orchestrate sleep/wake transitions lie undiscovered. Employing a viral insertion sleep screen in larval zebrafish, we identified a novel gene, dreammist (dmist), whose loss results in behavioural hyperactivity and reduced sleep at night. The neuronally expressed dmist gene is conserved across vertebrates and encodes a small single-pass transmembrane protein that is structurally similar to the Na+,K+-ATPase regulator, FXYD1/Phospholemman. Disruption of either fxyd1 or atp1a3a, a Na+,K+-ATPase alpha-3 subunit associated with several heritable movement disorders in humans, led to decreased night-time sleep. Since atpa1a3a and dmist mutants have elevated intracellular Na+ levels and non-additive effects on sleep amount at night, we propose that Dmist-dependent enhancement of Na+ pump function modulates neuronal excitability to maintain normal sleep behaviour.

The zebrafish mutant dreammist implicates sodium homeostasis in sleep regulation

Sleep is a nearly universal feature of animal behaviour, yet many of the molecular, genetic, and neuronal substrates that orchestrate sleep/wake transitions lie undiscovered. Employing a viral insertion sleep screen in larval zebrafish, we identified a novel gene, dreammist (dmist), whose loss results in behavioural hyperactivity and reduced sleep at night. The neuronally expressed dmist gene is conserved across vertebrates and encodes a small single-pass transmembrane protein that is structurally similar to the Na+,K+-ATPase regulator, FXYD1/Phospholemman. Disruption of either fxyd1 or atp1a3a, a Na+,K+-ATPase alpha-3 subunit associated with several heritable movement disorders in humans, led to decreased night-time sleep. Since atpa1a3a and dmist mutants have elevated intracellular Na+ levels and non-additive effects on sleep amount at night, we propose that Dmist-dependent enhancement of Na+ pump function modulates neuronal excitability to maintain normal sleep behaviour.

Cfdp1 Is Essential for Cardiac Development and Function.

Cardiovascular diseases (CVDs) are the prevalent cause of mortality worldwide. A combination of environmental and genetic effectors modulates the risk of developing them. Thus, it is vital to identify candidate genes and elucidate their role in the manifestation of the disease. Large-scale human studies have revealed the implication of Craniofacial Development Protein 1 (CFDP1) in Coronary Artery Disease (CAD). CFDP1 belongs to the evolutionary conserved Bucentaur (BCNT) family, and to date, its function and mechanism of action in Cardiovascular Development are still unclear. We utilized zebrafish to investigate the role of cfdp1 in the developing heart due to the high genomic homology, similarity in heart physiology, and ease of experimental manipulations. We showed that cfdp1 was expressed during development, and we tested two morpholinos and generated a cfdp1 mutant line. The cfdp1-/- embryos developed arrhythmic hearts and exhibited defective cardiac performance, which led to a lethal phenotype. Findings from both knockdown and knockout experiments showed that abrogation of cfdp1 leads to downregulation of Wnt signaling in embryonic hearts during valve development but without affecting Notch activation in this process. The cfdp1 zebrafish mutant line provides a valuable tool for unveiling the novel mechanism of regulating cardiac physiology and function. cfdp1 is essential for cardiac development, a previously unreported phenotype most likely due to early lethality in mice. The detected phenotype of bradycardia and arrhythmias is an observation with potential clinical relevance for humans carrying heterozygous CFDP1 mutations and their risk of developing CAD.

Role of short-wave-sensitive 1 (sws1) in cone development and first feeding in larval zebrafish.

Color vision is mediated by the expression of different major visual pigment proteins (opsins) on retinal photoreceptors. Vertebrates have four classes of cone opsins that are most sensitive to different wavelengths of light: short wavelength sensitive 1 (SWS1), short wavelength sensitive 2 (SWS2), medium wavelength sensitive (RH2), and long wavelength sensitive (LWS). UV wavelengths play important roles in foraging and communication. However, direct evidence provide links between sws1 and first feeding is lacking. Here, CRISPR/Cas9 technology was performed to generate mutant zebrafish lines with sws1 deletion. sws1 mutant zebrafish larvae exhibited decreased sws1, rh2-2, and lws1 expression, and increased rod gene (rho and gnat1) expression. Furthermore, the sws1-deficient larvae exhibited significantly reduced food intake, and the orexigenic genes npy and agrp signaling were upregulated at 6days postfertilization (dpf). The transcription expression of sws1 and rh2-3 genes decreased in sws1-/- adults compared to wild type. Surprisingly, the results of feeding at the adult stage were not the same with larvae. sws1 deficiency did not affect food intake and appetite gene expression at adult stages. These results reveal a role for sws1 in normal cone development and first feeding in larval zebrafish.

Precise mutagenesis in zebrafish using cytosine base editors.

Base editing is a powerful CRISPR-based technology for introducing precise substitutions into the genome. This technology greatly advances mutagenesis possibilities in vivo, particularly in zebrafish, for which the generation of precise point mutations is still challenging. Zebrafish have emerged as an important model for genetic studies and in vivo disease modeling. With the development of different base editor variants that recognize protospacer-adjacent motifs (PAMs) other than the classical 5'-NGG-3' PAM, it is now possible to design and test several guide RNAs to find the most efficient way to precisely introduce the desired substitution. Here, we describe the experimental design strategies and protocols for cytosine base editing in zebrafish, from guide RNA design and selection of base editor variants to generation of the zebrafish mutant line carrying the substitution of interest. By using co-selection by introducing a loss-of-function mutation in genes necessary for the formation of pigments, injected embryos with highly efficient base editing can be directly analyzed to determine the phenotypic impact of the targeted substitution. The generation of mutant embryos after base editor injections in zebrafish can be completed within 2 weeks.

COL11A2 as a candidate gene for vertebral malformations and congenital scoliosis.

Human vertebral malformations (VMs) have an estimated incidence of 1/2000 and are associated with significant health problems including congenital scoliosis (CS) and recurrent organ system malformation syndromes such as VACTERL (vertebral anomalies; anal abnormalities; cardiac abnormalities; tracheo-esophageal fistula; renal anomalies; limb anomalies). The genetic cause for the vast majority of VMs are unknown. In a CS/VM patient cohort, three COL11A2 variants (R130W, R1407L and R1413H) were identified in two patients with cervical VM. A third patient with a T9 hemivertebra and the R130W variant was identified from a separate study. These substitutions are predicted to be damaging to protein function, and R130 and R1407 residues are conserved in zebrafish Col11a2. To determine the role for COL11A2 in vertebral development, CRISPR/Cas9 was used to create a nonsense mutation (col11a2L642*) as well as a full gene locus deletion (col11a2del) in zebrafish. Both col11a2L642*/L642* and col11a2del/del mutant zebrafish exhibit vertebral fusions in the caudal spine, which form due to mineralization across intervertebral segments. To determine the functional consequence of VM-associated variants, we assayed their ability to suppress col11a2del VM phenotypes following transgenic expression within the developing spine. While wildtype col11a2 expression suppresses fusions in col11a2del/+ and col11a2del/del backgrounds, patient missense variant-bearing col11a2 failed to rescue the loss-of-function phenotype in these animals. These results highlight an essential role for COL11A2 in vertebral development and support a pathogenic role for two missense variants in CS.

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.

Stability and function of RCL1 are dependent on the interaction with BMS1.

During ribosome biogenesis, the small subunit (SSU) processome is responsible for 40S assembly. The BMS1/RCL1 complex is a core component of the SSU processome that plays an important role in 18S rRNA processing and maturation. Genetic studies using zebrafish mutants indicate that both Bms1-like (Bms1l) and Rcl1 are essential for digestive organ development. In spite of vital functions of this complex, the mutual dependence of these two nucleolar proteins for the stability and function remains elusive. In this study, we identified an RCL1-interacting domain in BMS1, which is conserved in zebrafish and humans. Moreover, both the protein stability and nucleolar entry of RCL1 depend on its interaction with BMS1, otherwise RCL1 degraded through the ubiquitination-proteasome pathway. Functional studies revealed that overexpression of RCL1 in BMS1-knockdown cells can partially rescue the defects in 18S rRNA processing and cell proliferation, and hepatocyte-specific overexpression of Rcl1 can resume zebrafish liver development in the bms1l substitution mutant bms1lsq163/sq163but not in the knockout mutant bms1lzju1/zju1, which is attributed to the nucleolar entry of Rcl1 in the former mutant. Our data demonstrate that BMS1 and RCL1 interaction is essential for not only pre-rRNA processing but also the communication between ribosome biogenesis and cell cycle regulation.

Dynamic regulation of inter-organelle communication by ubiquitylation controls skeletal muscle development and disease onset.

Ubiquitin-proteasome system (UPS) dysfunction is associated with the pathology of a wide range of human diseases, including myopathies and muscular atrophy. However, the mechanistic understanding of specific components of the regulation of protein turnover during development and disease progression in skeletal muscle is unclear. Mutations in KLHL40, an E3 ubiquitin ligase cullin3 (CUL3) substrate-specific adapter protein, result in severe congenital nemaline myopathy, but the events that initiate the pathology and the mechanism through which it becomes pervasive remain poorly understood. To characterize the KLHL40-regulated ubiquitin-modified proteome during skeletal muscle development and disease onset, we used global, quantitative mass spectrometry-based ubiquitylome and global proteome analyses of klhl40a mutant zebrafish during disease progression. Global proteomics during skeletal muscle development revealed extensive remodeling of functional modules linked with sarcomere formation, energy, biosynthetic metabolic processes, and vesicle trafficking. Combined analysis of klh40 mutant muscle proteome and ubiquitylome identified thin filament proteins, metabolic enzymes, and ER-Golgi vesicle trafficking pathway proteins regulated by ubiquitylation during muscle development. Our studies identified a role for KLHL40 as a regulator of ER-Golgi anterograde trafficking through ubiquitin-mediated protein degradation of secretion-associated Ras-related GTPase1a (Sar1a). In KLHL40-deficient muscle, defects in ER exit site vesicle formation and downstream transport of extracellular cargo proteins result in structural and functional abnormalities. Our work reveals that the muscle proteome is dynamically fine-tuned by ubiquitylation to regulate skeletal muscle development and uncovers new disease mechanisms for therapeutic development in patients.

CRISPR/Cas9-induced asap1a and asap1b co-knockout mutant zebrafish displayed abnormal embryonic development and impaired neutrophil migration

ASAP1 (Arf-GAP with SH3 domain, the ankyrin repeat and the PH domain) is the GTPase activating protein of the small G protein Arf. To understand more about the physiological functions of ASAP1 in vivo, we chose to use the zebrafish as an animal model, and analyzed the characterization of asap1 using loss-of-function studies. Here, two isoforms in zebrafish, asap1a and asap1b, were found to be homologous to human ASAP1, and the gene knockout zebrafish lines for asap1a and asap1b were established using the CRISPR/Cas9 technique with different insertions and deletions of bases. Zebrafish with asap1a and asap1b co-knockout showed a significant reduction in survival and hatching rates, as well as an increase in malformation rates during the early stages of development, while the asap1a or asap1b single knockout mutants did not affect the growth and development of individual zebrafish. Exploring the gene expression compensation between asap1a and asap1b using qRT-PCR, we found that asap1b had increased expression when asap1a was knocked out, showing a clear compensatory effect against asap1a knockout; In turn, asap1a did not have detectable compensating expression after asap1b knockout. Furthermore, the co-knockout homozygous mutants displayed impaired neutrophil migration to Mycobacterium marinum infection, and showed an increased bacterial load. Together, these are the first inherited asap1a and/or asap1b mutant zebrafish lines by the CRISPR/Cas9 gene editing approach, and by serving as useful models, they can significantly contribute to better annotation and follow-up physiological studies of human ASAP1.

The Tumor Suppressor Adenomatous Polyposis Coli (apc) Is Required for Neural Crest-Dependent Craniofacial Development in Zebrafish.

Neural crest (NC) is a unique vertebrate cell type arising from the border of the neural plate and epidermis that gives rise to diverse tissues along the entire body axis. Roberto Mayor and colleagues have made major contributions to our understanding of NC induction, delamination, and migration. We report that a truncating mutation of the classical tumor suppressor Adenomatous Polyposis Coli (apc) disrupts craniofacial development in zebrafish larvae, with a marked reduction in the cranial neural crest (CNC) cells that contribute to mandibular and hyoid pharyngeal arches. While the mechanism is not yet clear, the altered expression of signaling molecules that guide CNC migration could underlie this phenotype. For example, apcmcr/mcr larvae express substantially higher levels of complement c3, which Mayor and colleagues showed impairs CNC cell migration when overexpressed. However, we also observe reduction in stroma-derived factor 1 (sdf1/cxcl12), which is required for CNC migration into the head. Consistent with our previous work showing that APC directly enhances the activity of glycogen synthase kinase 3 (GSK-3) and, independently, that GSK-3 phosphorylates multiple core mRNA splicing factors, we identify 340 mRNA splicing variations in apc mutant zebrafish, including a splice variant that deletes a conserved domain in semaphorin 3f (sema3f), an axonal guidance molecule and a known regulator of CNC migration. Here, we discuss potential roles for apc in CNC development in the context of some of the seminal findings of Mayor and colleagues.

Gadusol is a maternally provided sunscreen that protects fish embryos from DNA damage

Exposure to ultraviolet radiation (UVR) is harmful to living cells, leading organisms to evolve protective mechanisms against UVR-induced cellular damage and stress.1,2 UVR, particularly UVB (280-320nm), can damageproteins and DNA, leading to errors during DNA repair and replication. Excessive UVR can induce cellulardeath. Aquatic organisms face risk of UV exposure as biologically harmful levels of UVB can penetrate >10m in clear water.3 While melanin is the only known sunscreen in vertebrates, it often emerges late in embryonic development, rendering embryos of many species vulnerable during the earlier stages. Algae and microbes produce a class of sunscreening compounds known as mycosporine-like amino acids (MAAs).4 Fish eggs contain a similar compound called gadusol, whose role as a sunscreen has yet to be tested despite its discovery over 40 years ago.5 The recent finding that many vertebrate genomescontain a biosynthetic pathway for gadusol suggests that many fish may produce and use this molecule as a sunscreen.6 We generated a gadusol-deficient mutant zebrafish to investigate the role of gadusol in protecting fish embryos and larvae from UVR. Our results demonstrate that maternally provided gadusol is the primary sunscreen in embryonic and larval development, while melanin provides modest secondary protection. The gadusol biosynthetic pathway is retained in the vast majority of teleost genomes but is repeatedly lost in species whose young are no longer exposed to UVR. Our data demonstrate that gadusol is a maternally provided sunscreen that is critical for early-life survival in the most species-rich branch of the vertebrate phylogeny.