Mutant Zebrafish Research Articles

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

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

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

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

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

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

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

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

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

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

Hypomyelinated vps16 Mutant Zebrafish Exhibit Systemic and Neurodevelopmental Pathologies.

Homotypic Fusion and Protein Sorting (HOPS) and Class C-core Vacuole/Endosome Tethering (CORVET) complexes regulate the correct fusion of endolysosomal bodies. Mutations in core proteins (VPS11, VPS16, VPS18, and VPS33) have been linked with multiple neurological disorders, including mucopolysaccharidosis (MPS), genetic leukoencephalopathy (gLE), and dystonia. Mutations in human Vacuolar Protein Sorting 16 (VPS16) have been associated with MPS and dystonia. In this study, we generated and characterized a zebrafish vps16(-/-) mutant line using immunohistochemical and behavioral approaches. The loss of Vps16 function caused multiple systemic defects, hypomyelination, and increased neuronal cell death. Behavioral analysis showed a progressive loss of visuomotor response and reduced motor response and habituation to acoustic/tap stimuli in mutants. Finally, using a novel multiple-round acoustic/tap stimuli test, mutants showed intermediate memory deficits. Together, these data demonstrate that zebrafish vps16(-/-) mutants show systemic defects, neurological and motor system pathologies, and cognitive impairment. This is the first study to report behavior abnormalities and memory deficiencies in a zebrafish vps16(-/-) mutant line. Finally, we conclude that the deficits observed in vps16(-/-) zebrafish mutants do not mimic pathologies associated with dystonia, but more align to abnormalities associated with MPS and gLE.

Characterisation of an Adult Zebrafish Model for SDHB-Associated Phaeochromocytomas and Paragangliomas.

Phaeochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumours arising from chromaffin cells. Pathogenic variants in the gene succinate dehydrogenase subunit B (SDHB) are associated with malignancy and poor prognosis. When metastases arise, limited treatment options are available. The pathomechanism of SDHB-associated PPGL remains largely unknown, and the lack of suitable models hinders therapy development. Germline heterozygous SDHB pathogenic variants predispose to developing PPGLs with a life-long penetrance of around 50%. To mimic the human disease phenotype, we characterised adult heterozygous sdhb mutant zebrafish as a potential model to study SDHB-related PPGLs. Adult sdhb mutant zebrafish did not develop an obvious tumour phenotype and were anatomically and histologically like their wild-type siblings. However, sdhb mutants showed significantly increased succinate levels, a major hallmark of SDHB-related PPGLs. While basal activity was increased during day periods in mutants, mitochondrial complex activity and catecholamine metabolite levels were not significantly different. In conclusion, we characterised an adult in vivo zebrafish model, genetically resembling human carriers. Adult heterozygous sdhb mutants mimicked their human counterparts, showing systemic elevation of succinate levels despite the absence of a tumour phenotype. This model forms a promising basis for developing a full tumour phenotype and gaining knowledge of the pathomechanism behind SDHB-related PPGLs.

Llgl1 mediates timely epicardial emergence and establishment of an apical laminin sheath around the trabeculating cardiac ventricle.

During heart development, the embryonic ventricle becomes enveloped by the epicardium, which adheres to the outer apical surface of the heart. This is concomitant with onset of ventricular trabeculation, where a subset of cardiomyocytes lose apicobasal polarity and delaminate basally from the ventricular wall. Llgl1 regulates the formation of apical cell junctions and apicobasal polarity, and we investigated its role in ventricular wall maturation. We found that llgl1 mutant zebrafish embryos exhibit aberrant apical extrusion of ventricular cardiomyocytes. While investigating apical cardiomyocyte extrusion, we identified a basal-to-apical shift in laminin deposition from the internal to the external ventricular wall. We find that epicardial cells express several laminin subunits as they adhere to the ventricle, and that the epicardium is required for laminin deposition on the ventricular surface. In llgl1 mutants, timely establishment of the epicardial layer is disrupted due to delayed emergence of epicardial cells, resulting in delayed apical deposition of laminin on the ventricular surface. Together, our analyses reveal an unexpected role for Llgl1 in correct timing of epicardial development, supporting integrity of the ventricular myocardial wall.

A novel homozygous variant of the PIGK gene caused by paternal disomy in a patient with neurodevelopmental disorder, cerebellar atrophy, and seizures.

Glycosylphosphatidylinositol (GPI)-anchored proteins are located at the cell surface by a covalent attachment between protein and GPI embedded in the plasma membrane. This attachment is catalyzed by GPI transamidase comprising five subunits (PIGK, PIGS, PIGT, PIGU, and GPAA1) in the endoplasmic reticulum. Loss of either subunit of GPI transamidase eliminates cell surface localization of GPI-anchored proteins. In humans, pathogenic variants in either subunit of GPI transamidase cause neurodevelopmental disorders. However, how the loss of GPI-anchored proteins triggers neurodevelopmental defects remains largely unclear. Here, we identified a novel homozygous variant of PIGK, NM_005482:c.481A > G,p. (Met161Val), in a Japanese female patient with neurodevelopmental delay, hypotonia, cerebellar atrophy, febrile seizures, hearing loss, growth impairment, dysmorphic facial features, and brachydactyly. The missense variant was found heterozygous in her father, but not in her mother. Zygosity analysis revealed that the homozygous PIGK variant in the patient was caused by paternal isodisomy. Rescue experiments using PIGK-deficient CHO cells revealed that the p.Met161Val variant of PIGK reduced GPI transamidase activity. Rescue experiments using pigk mutant zebrafish confirmed that the p.Met161Val variant compromised PIGK function in tactile-evoked motor response. We also demonstrated that axonal localization of voltage-gated sodium channels and concomitant generation of action potentials were impaired in pigk-deficient neurons in zebrafish, suggesting a link between GPI-anchored proteins and neuronal defects. Taken together, the missense p.Met161Val variant of PIGK is a novel pathogenic variant that causes the neurodevelopmental disorder.

Zebrafish Congenital Heart Disease Models: Opportunities and Challenges.

Congenital heart defects (CHDs) are common human birth defects. Genetic mutations potentially cause the exhibition of various pathological phenotypes associated with CHDs, occurring alone or as part of certain syndromes. Zebrafish, a model organism with a strong molecular conservation similar to humans, is commonly used in studies on cardiovascular diseases owing to its advantageous features, such as a similarity to human electrophysiology, transparent embryos and larvae for observation, and suitability for forward and reverse genetics technology, to create various economical and easily controlled zebrafish CHD models. In this review, we outline the pros and cons of zebrafish CHD models created by genetic mutations associated with single defects and syndromes and the underlying pathogenic mechanism of CHDs discovered in these models. The challenges of zebrafish CHD models generated through gene editing are also discussed, since the cardiac phenotypes resulting from a single-candidate pathological gene mutation in zebrafish might not mirror the corresponding human phenotypes. The comprehensive review of these zebrafish CHD models will facilitate the understanding of the pathogenic mechanisms of CHDs and offer new opportunities for their treatments and intervention strategies.

Arrhythmogenic cardiomyopathy related phenotypes are detected and pharmacologically modulated in Galectin-3 zebrafish models

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Italian Ministry of University and Research Introduction Arrhythmogenic Cardiomyopathy (AC) is a rare inherited cardiac disorder characterized by fibro-fatty replacement and progressive loss of the ventricular myocardium, causing life-threatening arrhythmias, syncope and sudden cardiac death in young and athletes. About 50% of AC cases carry one or more mutations in genes encoding for desmosomal proteins. However, mutations in non-desmosomal genes have been identified in 1-3% of AC patients, such as Galectin-3 (Gal-3). Purpose For a better understanding of Gal-3 role in AC pathogenesis, we have generated a stable Gal-3 knock-out (KO) zebrafish line, able to reproduce cardiac abnormalities and cell signaling dysregulation characteristic of AC, on which environmental factors and candidate drugs can be tested. Methods The zebrafish Gal-3 mutant line was generated by CRISPR/Cas9 strategy and then phenotypically characterized. Phenotyping included heart activity characterization, by pyHeart4Fish software, and gene expression analysis using Real Time PCR and signaling pathway reporters. Immunofluorescence analysis was performed using an antibody against L-plastin, a leukocyte marker for the detection of inflammatory cells, and Acridine Orange/Ethidium Bromide (AO/EB) staining to check for apoptotic and necrotic events. Transmission Electron Microscopy (TEM) analysis was carried out to detect possible ultrastructural alterations. A pathway-directed pharmacological treatment was applied to try to rescue pathological phenotypes. Results The Gal-3 KO zebrafish line, analysed at larval stage, presented structural abnormalities, pericardial effusion and/or hemopericardium associated with reduced contractility, bradycardia and presence of arrhythmia events. In addition to Wnt/β-catenin signaling dysregulation, as seen in other AC forms, we also observed cell death and inflammation. Specifically, the analysis of the L-plastin inflammatory marker showed remarkable infiltration of inflammatory cells in the cardiac region of Gal-3 mutants, whereas AO/EB staining demonstrated elevated number of apoptotic and necrotic cells. Moreover, TEM analysis revealed "pale", disorganized and delocalized desmosomes in mutant adult hearts. Of note, pharmacological treatment at larval stage, using a Wnt/β-catenin signaling agonist, partially rescued a set of AC cardiac phenotypes (contractility, bradycardia, arrhythmia). Conclusions Overall, the Gal-3 KO zebrafish line confirms in zebrafish the strict connection between Gal-3 mutation and AC-like cardiac alterations. Moreover, cell death and inflammation, known to be involved in the remodelling of the myocardium, were observed. The pharmacological rescue of the Wnt/β-catenin signaling partially recovered a set of cardiac phenotypes, confirming this pathway as a key modulator of AC progression. In conclusion, our results strongly support the usefulness of the Gal-3 zebrafish mutant to clarify Gal-3 involvement in AC, offering future perspectives for AC treatment.

Effect of nonsense-mediated mRNA decay factor SMG9 deficiency on premature aging in zebrafish

SMG9 is an essential component of the nonsense-mediated mRNA decay (NMD) machinery, a quality control mechanism that selectively degrades aberrant transcripts. Mutations in SMG9 are associated with heart and brain malformation syndrome (HBMS). However, the molecular mechanism underlying HBMS remains unclear. We generated smg9 mutant zebrafish (smg9oi7/oi7) that have a lifespan of approximately 6 months or longer, allowing for analysis of the in vivo function of Smg9 in adults in more detail. smg9oi7/oi7 zebrafish display congenital brain abnormalities and reduced cardiac contraction. Additionally, smg9oi7/oi7 zebrafish exhibit a premature aging phenotype. Analysis of NMD target mRNAs shows a trend toward increased mRNA levels in smg9oi7/oi7 zebrafish. Spermidine oxidase (Smox) is increased in smg9oi7/oi7 zebrafish, resulting in the accumulation of byproducts, reactive oxygen species, and acrolein. The accumulation of smox mRNA due to NMD dysregulation caused by Smg9 deficiency leads to increased oxidative stress, resulting in premature aging.

Ace Deficiency Induces Intestinal Inflammation in Zebrafish.

Inflammatory bowel disease (IBD) is a nonspecific chronic inflammatory disease resulting from an immune disorder in the intestine that is prone to relapse and incurable. The understanding of the pathogenesis of IBD remains unclear. In this study, we found that ace (angiotensin-converting enzyme), expressed abundantly in the intestine, plays an important role in IBD. The deletion of ace in zebrafish caused intestinal inflammation with increased expression of the inflammatory marker genes interleukin 1 beta (il1b), matrix metallopeptidase 9 (mmp9), myeloid-specific peroxidase (mpx), leukocyte cell-derived chemotaxin-2-like (lect2l), and chemokine (C-X-C motif) ligand 8b (cxcl8b). Moreover, the secretion of mucus in the ace-/- mutants was significantly higher than that in the wild-type zebrafish, validating the phenotype of intestinal inflammation. This was further confirmed by the IBD model constructed using dextran sodium sulfate (DSS), in which the mutant zebrafish had a higher susceptibility to enteritis. Our study reveals the role of ace in intestinal homeostasis, providing a new target for potential therapeutic interventions.

Macrophage transplantation rescues RNASET2-deficient leukodystrophy by replacing deficient microglia in a zebrafish model

RNASET2-deficient leukodystrophy is a rare infantile white matter disorder mimicking a viral infection and resulting in severe psychomotor impairments. Despite its severity, there is little understanding of cellular mechanisms of pathogenesis and no treatments. Recent research using the rnaset2 mutant zebrafish model has suggested that microglia may be the drivers of the neuropathology, due to their failure to digest apoptotic debris during neurodevelopment. Therefore, we developed a strategy for microglial replacement through transplantation of adult whole kidney marrow-derived macrophages into embryonic hosts. Using live imaging, we revealed that transplant-derived macrophages can engraft within host brains and express microglia-specific markers, suggesting the adoption of a microglial phenotype. Tissue-clearing strategies revealed the persistence of transplanted cells in host brains beyond embryonic stages. We demonstrated that transplanted cells clear apoptotic cells within the brain, as well as rescue overactivation of the antiviral response otherwise seen in mutant larvae. RNA sequencing at the point of peak transplant-derived cell engraftment confirms that transplantation can reduce the brain-wide immune response and particularly, the antiviral response, in rnaset2-deficient brains. Crucially, this reduction in neuroinflammation resulted in behavioral rescue-restoring rnaset2 mutant motor activity to wild-type (WT) levels in embryonic and juvenile stages. Together, these findings demonstrate the role of microglia as the cellular drivers of neuropathology in rnaset2 mutants and that macrophage transplantation is a viable strategy for microglial replacement in the zebrafish. Therefore, microglia-targeted interventions may have therapeutic benefits in RNASET2-deficient leukodystrophy.

A Simple Tube Escape Assay to Test Learning and Memory in Zebrafish with Minimized Habituation.

Various methods have been used in rodents to evaluate learning and memory. Although much less frequently used, the zebrafish emerges as an alternative model organism in this context. For example, it allows assessing potential behavioral deficits because of neurodevelopmental disorders or environmental neurotoxins. A variety of learning tasks have been employed in previous studies that required extensive habituation and training sessions. Here, we introduce a simpler and faster method to evaluate learning and memory of zebrafish with minimum habituation. A new apparatus, a transparent L-shaped tube, was developed in which we trained each zebrafish to swim through a long arm and measured the time to swim through this arm. We demonstrate that in this task, zebrafish could acquire both short-term (1 h) and long-term memory (4 days). We also studied learning and memory of a gene knockout (KO) zebrafish that showed social impairments related to autism. We found KO mutant zebrafish to show a quantitative impairment in habituation, learning, and memory performance compared with wild-type control fish. In conclusion, we established a novel learning apparatus and sensitive paradigm that allowed us to evaluate learning and memory of adult zebrafish that required only a brief habituation period and minimal training.

Somatic PDGFRB activating variants promote smooth muscle cell phenotype modulation in intracranial fusiform aneurysm

BackgroundThe fusiform aneurysm is a nonsaccular dilatation affecting the entire vessel wall over a short distance. Although PDGFRB somatic variants have been identified in fusiform intracranial aneurysms, the molecular and cellular mechanisms driving fusiform intracranial aneurysms due to PDGFRB somatic variants remain poorly understood.MethodsIn this study, single-cell sequencing and immunofluorescence were employed to investigate the phenotypic changes in smooth muscle cells within fusiform intracranial aneurysms. Whole-exome sequencing revealed the presence of PDGFRB gene mutations in fusiform intracranial aneurysms. Subsequent immunoprecipitation experiments further explored the functional alterations of these mutated PDGFRB proteins. For the common c.1684 mutation site of PDGFRβ, we established mutant smooth muscle cell lines and zebrafish models. These models allowed us to simulate the effects of PDGFRB mutations. We explored the major downstream cellular pathways affected by PDGFRBY562D mutations and evaluated the potential therapeutic effects of Ruxolitinib.ResultsSingle-cell sequencing of two fusiform intracranial aneurysms sample revealed downregulated smooth muscle cell markers and overexpression of inflammation-related markers in vascular smooth muscle cells, which was validated by immunofluorescence staining, indicating smooth muscle cell phenotype modulation is involved in fusiform aneurysm. Whole-exome sequencing was performed on seven intracranial aneurysms (six fusiform and one saccular) and PDGFRB somatic mutations were detected in four fusiform aneurysms. Laser microdissection and Sanger sequencing results indicated that the PDGFRB mutations were present in smooth muscle layer. For the c.1684 (chr5: 149505131) site mutation reported many times, further cell experiments showed that PDGFRBY562D mutations promoted inflammatory-related vascular smooth muscle cell phenotype and JAK-STAT pathway played a crucial role in the process. Notably, transfection of PDGFRBY562D in zebrafish embryos resulted in cerebral vascular anomalies. Ruxolitinib, the JAK inhibitor, could reversed the smooth muscle cells phenotype modulation in vitro and inhibit the vascular anomalies in zebrafish induced by PDGFRB mutation.ConclusionOur findings suggested that PDGFRB somatic variants played a role in regulating smooth muscle cells phenotype modulation in fusiform aneurysms and offered a potential therapeutic option for fusiform aneurysms.

Deciphering the function of the fifth class of Gα proteins: regulation of ionic homeostasis as unifying hypothesis

Trimeric G proteins transduce signals from a superfamily of receptors and each G protein controls a wide range of cellular and systemic functions. Their highly conserved alpha subunits fall in five classes, four of which have been well investigated (Gs, Gi, G12, Gq). In contrast, the function of the fifth class, Gv is completely unknown, despite its broad occurrence and evolutionary ancient origin (older than metazoans). Here we show a dynamic presence of Gv mRNA in several organs during early development of zebrafish, including the hatching gland, the pronephros and several cartilage anlagen, employing in situ hybridisation. Next, we generated a Gv frameshift mutation in zebrafish and observed distinct phenotypes such as reduced oviposition, premature hatching and craniofacial abnormalities in bone and cartilage of larval zebrafish. These phenotypes could suggest a disturbance in ionic homeostasis as a common denominator. Indeed, we find reduced levels of calcium, magnesium and potassium in the larvae and changes in expression levels of the sodium potassium pump atp1a1a.5 and the sodium/calcium exchanger ncx1b in larvae and in the adult kidney, a major osmoregulatory organ. Additionally, expression of sodium chloride cotransporter slc12a3 and the anion exchanger slc26a4 is altered in complementary ways in adult kidney. It appears that Gv may modulate ionic homeostasis in zebrafish during development and in adults. Our results constitute the first insight into the function of the fifth class of G alpha proteins.

Forward genetics combined with unsupervised classifications identified zebrafish mutants affecting biliary system formation

Impaired formation of the biliary network can lead to congenital cholestatic liver diseases; however, the genes responsible for proper biliary system formation and maintenance have not been fully identified. Combining computational network structure analysis algorithms with a zebrafish forward genetic screen, we identified 24 new zebrafish mutants that display impaired intrahepatic biliary network formation. Complementation tests suggested these 24 mutations affect 24 different genes. We applied unsupervised clustering algorithms to unbiasedly classify the recovered mutants into three classes. Further computational analysis revealed that each of the recovered mutations in these three classes has a unique phenotype on node-subtype composition and distribution within the intrahepatic biliary network. In addition, we found most of the recovered mutations are viable. In those mutant fish, which are already good animal models to study chronic cholestatic liver diseases, the biliary network phenotypes persist into adulthood. Altogether, this study provides unique genetic and computational toolsets that advance our understanding of the molecular pathways leading to biliary system malformation and cholestatic liver diseases.

Cachd1 interacts with Wnt receptors and regulates neuronal asymmetry in the zebrafish brain.

Neurons on the left and right sides of the nervous system often show asymmetric properties, but how such differences arise is poorly understood. Genetic screening in zebrafish revealed that loss of function of the transmembrane protein Cachd1 resulted in right-sided habenula neurons adopting left-sided identity. Cachd1 is expressed in neuronal progenitors, functions downstream of asymmetric environmental signals, and influences timing of the normally asymmetric patterns of neurogenesis. Biochemical and structural analyses demonstrated that Cachd1 can bind simultaneously to Lrp6 and Frizzled family Wnt co-receptors. Consistent with this, lrp6 mutant zebrafish lose asymmetry in the habenulae, and epistasis experiments support a role for Cachd1 in modulating Wnt pathway activity in the brain. These studies identify Cachd1 as a conserved Wnt receptor-interacting protein that regulates lateralized neuronal identity in the zebrafish brain.

Loss of αBa-crystallin, but not αA-crystallin, increases age-related cataract in the zebrafish lens

The vertebrate eye lens is an unusual organ in that most of its cells lack nuclei and the ability to replace aging protein. The small heat shock protein α-crystallins evolved to become key components of this lens, possibly because of their ability to prevent aggregation of aging protein that would otherwise lead to lens opacity. Most vertebrates express two α-crystallins, αA- and αB-crystallin, and mutations in each are linked to human cataract. In a mouse knockout model only the loss of αA-crystallin led to early-stage lens cataract. We have used the zebrafish as a model system to investigate the role of α-crystallins during lens development. Interestingly, while zebrafish express one lens-specific αA-crystallin gene (cryaa), they express two αB-crystallin genes, with one evolving lens specificity (cryaba) and the other retaining the broad expression of its mammalian ortholog (cryabb). In this study we used individual mutant zebrafish lines for all three α-crystallin genes to determine the impact of their loss on age-related cataract. Surprisingly, unlike mouse knockout models, we found that the loss of the αBa-crystallin gene cryaba led to an increase in lens opacity compared to cryaa null fish at 24 months of age. Loss of αA-crystallin did not increase the prevalence of cataract. We also used single cell RNA-Seq and RT-qPCR data to show a shift in the lens expression of zebrafish α-crystallins between 5 and 10 days post fertilization (dpf), with 5 and 6 dpf lenses expressing cryaa almost exclusively, and expression of cryaba and cryabb becoming more prominent after 10 dpf. These data show that cryaa is the primary α-crystallin during early lens development, while the protective role for cryaba becomes more important during lens aging. This study is the first to quantify cataract prevalence in wild-type aging zebrafish, showing that lens opacities develop in approximately 25% of fish by 18 months of age. None of the three α-crystallin mutants showed a compensatory increase in the expression of the remaining two crystallins, or in the abundant βB1-crystallin. Overall, these findings indicate an ontogenetic shift in the functional importance of individual α-crystallins during zebrafish lens development. Our finding that the lens-specific zebrafish αBa-crystallin plays the leading role in preventing age-related cataract adds a new twist to our understanding of vertebrate lens evolution.