Various states of cardiomyocyte (CM) polyploidy have been associated with cardiac injury responses, including regeneration and heart failure. However, our understanding of the comprehensive mechanisms governing CM ploidy and its relationship with heart physiology is limited. To address this issue and uncover genetic regulators, we surveyed CM ploidy across a new genetic resource known as the Hybrid Rat Diversity Panel (HRDP) and found significant variation in ploidy phenotypes across the panel. Using select rat strains with divergent displays of CM ploidy, we found that CM hyperpolyploidization (≥8 N) positively correlates with various physiological parameters, namely left ventricular dilation and reduced ejection fraction. Genome-wide association mapping identified several loci significantly associated with frequency of hyperpolyploid CMs. Investigation of genes harboring damaging protein coding variants within these loci identified enrichment of cytoarchitectural genes, of which the ACTIN-binding protein, Shroom3 , was found to be strongly and specifically expressed in CMs and harbors 7 damaging protein coding variants. CM-specific deletion of Shroom3 resulted in increased hyperpolyploidization and left ventricular dilation with reduced ejection fraction. Furthermore, functional characterization of single-nucleotide variants resulting in amino acid changes within SHROOM3 confirmed two protein coding variants that disrupted SHROOM3–ACTIN interaction and led to altered expression of genes involved in DNA replication. This study elucidates the genetic determinants of CM ploidy phenotypes and solidifies a correlative relationship between CM ploidy and left ventricular function. Importantly, CM intrinsic expression of at least one gene mapped in this study, Shroom3 , is confirmed to regulate CM hyperpolyploidization and cardiac function.

Background:Genome-wide studies in late-onset Alzheimer's disease (LOAD) have uncovered many risk loci, yet identifying the causal genes and clarifying how these genetic signals connect to molecular and cellular mechanisms relevant to AD pathogenesis in vivo remains challenging. Methods:Using Caenorhabditis elegans as a model to identify LOAD-associated genes that drive neurodegenerative processes, we focused on 14 understudied genes and their homologs: ABI3/abi-1, B4GALT3/bre-4, CCDC6/T09B9.4, CLPTM1 (two homologs C36B7.6 and R166.2), CNN2/cpn-2, DMWD/wdr-20, ECHDC3/ech-2, MADD/aex-3, NCK2/nck-1, RABEP1/rabn-5, RIN3/rin-1, SLC39A13/zipt-13, TRAM1/tram-1, and USP6NL/tbc-17. We knocked down these genes by RNAi and quantified lifespan, aging-associated degeneration of two neuron classes, PVD and PLM, and associative learning and short-term memory. Results:Lifespan was unaffected by most knockdowns, and only nck-1 and tbc-17 shortened lifespan. Across neuronal assays, multiple homologs modulated aging with clear neuron-class selectivity. Knockdown of aex-3, C36B7.6, cpn-2, ech-2, rabn-5, rin-1, T09B9.4, and zipt-13 attenuated late-life PVD degeneration, whereas R166.2 and tram-1 accelerated early PVD aging. Only two genes affected PLM aging: R166.2 knockdown exacerbated degeneration, while tbc-17 knockdown attenuated it despite its lifespan-shortening effect. In PLM neurons, tbc-17 knockdown, targeting a Rab GTPase-activating protein, also preserved mitochondrial architecture during early aging and shifted heat stress-induced mitochondrial remodeling toward a pattern consistent with improved quality control. In behavioral assays, ech-2 knockdown, targeting an enoyl-CoA-hydratase, enhanced short-term memory during early stages of aging. To further assess how LOAD-linked genes interact with Aβ-driven neurodegeneration, we developed a model that combines the PVD aging assay with a background expressing human Aβ1-42 pan-neuronally. In this model, Aβ expression accelerated age-dependent PVD degeneration, whereas ech-2 knockdown abolished this Aβ-induced effect. Conclusions:Our findings show that conserved homologs of several understudied LOAD risk genes causally modulate neuronal aging in vivo in a neuron-class-selective manner, often dissociable from organismal longevity. This C. elegans framework translates human genetic associations into quantitative, aging-linked neuronal phenotypes, and our results further emphasize early endosomal and lipid-related processes as key pathways that warrant functional testing in neuronal aging. This study also provides a tractable platform to prioritize targets for cross-species validation and to test synergy with established LOAD risk genes.

Mutations in CLRN1 cause Usher syndrome type IIIA (USH3A), an autosomal recessive disorder characterized by hearing and vision loss, and often accompanied by vestibular dysfunction. The identity of the cell types responsible for the pathology and mechanisms leading to vision loss in USH3A remains elusive. To address this, we employed CRISPR/Cas9 technology to delete a large region in the coding and untranslated (UTR) region of zebrafish clrn1. The retinas of clrn1 mutant larvae exhibited sensitivity to cell stress, along with age-dependent loss of function and degeneration in the photoreceptor layer. Investigation revealed disorganization in the outer retina in clrn1 mutants, including actin-based structures of the Müller glia and photoreceptor cells. To assess cell-specific contributions to USH3A pathology, we specifically re-expressed clrn1 in either Müller glia or photoreceptor cells. Müller glia re-expression of clrn1 prevented the elevated cell death observed in larval clrn1 mutant zebrafish exposed to high-intensity light. Notably, the degree of phenotypic rescue correlated with the level of Clrn1 re-expression. Surprisingly, high levels of Clrn1 expression enhanced cell death in both wild-type and clrn1 mutant animals. However, rod- or cone-specific Clrn1 re-expression did not reduce the extent of cell death. Taken together, our findings underscore three crucial insights. First, clrn1 mutant zebrafish exhibit key pathological features of USH3A; second, Clrn1 within Müller glia plays a pivotal role in photoreceptor maintenance, with its expression requiring controlled regulation; third, the reliance of photoreceptors on Müller glia suggests a structural support mechanism, possibly through direct interactions between Müller glia and photoreceptors mediated in part by Clrn1 protein.

Background: Cell polarity orchestrates spatial organization within tissues influencing cellular processes such as adhesion, proliferation, and differentiation. While the role of polarity proteins is well-established in epithelial systems, their function in mammalian cardiomyocytes remains poorly understood. Global deletion of the polarity protein, Llgl1, has been shown to impair cardiac trabeculation and disrupt cardiomyocyte junctions in zebrafish. Here, we investigate the role of Llgl1 in mammalian cardiomyocyte function. Methods: We developed a mouse model whereby Llgl1 is deleted from cardiomyocytes (Llgl1CKO) and assessed cardiomyocyte intercalated disc integrity, cell cycle activity, cardiac function, and electrophysiology. We subjected mice to myocardial infarction to test if permanent Llgl1 deletion, or inducible Llgl1 deletion in adult cardiomyocytes (Llgl1TamCKO), influences the cardiac injury response. We performed RNAsequencing and in vitro knockdown and viral overexpression experiments to delineate the mechanisms by which Llgl1 influences cardiomyocyte phenotypes. Results: Llgl1CKO mice display disrupted intercalated disc integrity indicated by reduced protein density at cardiomyocyte junctions, associated with electrical conduction defects that manifest as ventricular arrhythmia. Furthermore, Llgl1CKO mice display enhanced cardiomyocyte cell cycle activity extending into adulthood. Llgl1CKO mice and mice whereby Llgl1 is deleted by tamoxifen induction in adulthood both show improved cardiac function and reduced scar size following myocardial infarction. Mechanistically, Llgl1 alters mechanotransduction and cell cycle pathways in the heart, and regulates proper localization of N-cadherin to the cell membrane, a process that is optimized by phosphorylation of five serine residues at the C-terminus of Llgl1. Conclusions: We elucidate a key role for Llgl1 in establishing proper intercalated disc integrity in mammalian cardiomyocytes. Disruption of intercalated discs and mechanotransduction via Llgl1 knockout is associated with cardiac arrhythmia, elevated cell cycle activity and resistance to cardiac injury. These studies shed light on mechanisms underlying intercalated disc establishment and connect disrupted cardiomyocyte junctional integrity with enhanced cell cycle activity. ### Competing Interest Statement The authors have declared no competing interest.

Tunicamycins (TUN) are well-defined, Streptomyces-derived natural products that inhibit protein N-glycosylation in eukaryotes, and by a conserved mechanism also block bacterial cell wall biosynthesis. TUN inhibits the polyprenylphosphate-N-acetyl-hexosamine-1-phospho-transferases (PNPT), an essential family of enzymes found in both bacteria and eukaryotes. We have previously published the development of chemically modified TUN, called TunR1 and TunR2, that have considerably reduced activity on eukaryotes but that retain the potent antibacterial properties. A mechanism for this reduced toxicity has also been reported. TunR1 and TunR2 have been tested against mammalian cell lines in culture and against live insect cells but, until now, no in vivo evaluation has been undertaken for vertebrates. In the current work, TUN, TunR1, and TunR2 are investigated for their relative toxicity and antimycobacterial activity in zebrafish using a well-established Mycobacterium marinum (M. marinum) infection system, a model for studying human Mycobacterium tuberculosis infections. We also report the relative ability to activate the unfolded protein response (UPR), the known mechanism for the eukaryotic toxicity observed with TUN treatment. Importantly, TunR1 and TunR2 retained their antimicrobial properties, as evidenced by a reduction in M. marinum bacterial burden, compared to DMSO-treated zebrafish. In summary, findings from this study highlight the characteristics of recently developed TUN derivatives, mainly TunR2, and its potential for use as a novel anti-bacterial agent for veterinary and potential medical purposes.

Background:The goal of this study was to identify and characterize cell-cell interactions that facilitate endothelial tip cell fusion downstream of BMP (bone morphogenic protein)-mediated venous plexus formation. Methods:High resolution and time-lapse imaging of transgenic reporter lines and loss-of-function studies were carried out to study the involvement of mesenchymal stromal cells during venous angiogenesis. Results:BMP-responsive stromal cells facilitate timely and precise fusion of venous tip cells during developmental angiogenesis. Conclusions:Stromal cells are required for anastomosis of venous tip cells in the embryonic caudal hematopoietic tissue.

IntroductionWhile Yap and Wwtr1 regulate resident cardiac fibroblast to myofibroblast differentiation following cardiac injury, their role specifically in activated myofibroblasts remains unexplored. MethodsWe assessed the pathophysiological and cellular consequence of genetic depletion of Yap alone (Yap(fl/fl);Postn(MCM)) or Yap and Wwtr1 (Yap(fl/fl);Wwtr1(fl/+);Postn(MCM)) in adult mouse myofibroblasts following myocardial infarction and identify and validate novel downstream factors specifically in cardiac myofibroblasts that mediate pathological remodeling. ResultsFollowing myocardial infarction, depletion of Yap in myofibroblasts had minimal effect on heart function while depletion of Yap/Wwtr1 resulted in smaller scars, reduced interstitial fibrosis, and improved ejection fraction and fractional shortening. Single cell RNA sequencing of interstitial cardiac cells 7 days post infarction showed suppression of pro-fibrotic genes in fibroblasts derived from Yap(fl/fl),Wwtr1(fl/+);Postn(MCM) hearts. In vivo myofibroblast depletion of Yap/Wwtr1 as well in vitro knockdown of Yap/Wwtr1 dramatically decreased RNA and protein expression of the matricellular factor Ccn3. Administration of recombinant CCN3 to adult mice following myocardial infarction remarkably aggravated cardiac function and scarring. CCN3 administration drove myocardial gene expression of pro-fibrotic genes in infarcted left ventricles implicating CCN3 as a novel driver of cardiac fibrotic processes following myocardial infarction. DiscussionYap/Wwtr1 depletion in myofibroblasts attenuates fibrosis and significantly improves cardiac outcomes after myocardial infarction and we identify Ccn3 as a factor downstream of Yap/Wwtr1 that contributes to adverse cardiac remodeling post MI. Myofibroblast expression of Yap, Wwtr1, and Ccn3 could be further explored as potential therapeutic targets for modulating adverse cardiac remodeling post injury.

Hereditary hemorrhagic telangiectasia (HHT) is associated with defective capillary network, leading to dilated superficial vessels and arteriovenous malformations (AVMs) in which arteries connect directly to the veins. Loss or haploinsufficiency of components of TGF-β signaling, ALK1, ENG, SMAD4, and BMP9, have been implicated in the pathogenesis AVMs. Emerging evidence suggests that the inability of endothelial cells to detect, transduce and respond to blood flow, during early development, is an underpinning of AVM pathogenesis. Therefore, components of endothelial flow detection may be instrumental in potentiating TGF-β signaling in perfused blood vessels. Here, we argue that endothelial cilium, a microtubule-based and flow-sensitive organelle, serves as a signaling hub by coupling early flow detection with potentiation of the canonical TGF-β signaling in nascent endothelial cells. Emerging evidence from animal models suggest a role for primary cilia in mediating vascular development. We reason, on recent observations, that endothelial cilia are crucial for vascular development and that embryonic loss of endothelial cilia will curtail TGF-β signaling, leading to associated defects in arteriovenous development and impaired vascular stability. Loss or dysfunction of endothelial primary cilia may be implicated in the genesis of AVMs due, in part, to inhibition of ALK1/SMAD4 signaling. We speculate that AVMs constitute part of the increasing spectrum of ciliopathy-associated vascular defects.

Coat protein complex II (COPII) factors mediate cargo export from the endoplasmic reticulum (ER), but bulky collagens and lipoproteins are too large for traditional COPII vesicles. Mammalian CTAGE5 and TANGO1 have been well characterized individually as specialized cargo receptors at the ER that function with COPII coats to facilitate trafficking of bulky cargoes. Here, we present a genetic interaction study in zebrafish of deletions in ctage5, tango1, or both to investigate their distinct and complementary potential functions. We found that Ctage5 and Tango1 have different roles related to organogenesis, collagen versus lipoprotein trafficking, stress-pathway activation, and survival. While disruption of both ctage5 and tango1 compounded phenotype severity, mutation of either factor alone revealed novel tissue-specific defects in the building of heart, muscle, lens, and intestine, in addition to previously described roles in the development of neural and cartilage tissues. Together, our results demonstrate that Ctage5 and Tango1 have overlapping functions, but also suggest divergent roles in tissue development and homeostasis.

Introduction Usher syndrome (USH) represents a group of genetically heterogenous autosomal recessive disorders, characterized by combined vision and sensorineural hearing loss, and in some cases vestibular dysfunction.1–3 It primarily affects the light-sensitive photoreceptor cells in the retina and the auditory hair cells in the cochlea. Vision loss in all cases is progressive, and manifests as retinitis pigmentosa (RP), characterized by the gradual degeneration of the photoreceptor cells.4 Peripheral rod function is lost first, leading to night blindness and constricted visual fields, followed by the death of cones and severe visual impairment. There are currently no drugs or biological therapies proven to be effective in treating USH syndrome. Cochlear implantation, which bypasses the damaged hair cells and stimulates the primary auditory neurons directly, is an effective approach to alleviate the hearing impairment in patients. However, there is no remedy for the progressive loss of the photoreceptor neurons in the retina, and thus a critical unmet need exists to develop therapeutic strategies to prevent blindness in USH syndrome. The generation of animal models that faithfully mimic the human USH disorder, the timing of gene therapy interventions, the identification of correct target cells and patient selection are key factors in successfully reaching this goal. USH syndrome was named after Dr Charles Howard Usher, a Scottish ophthalmologist who described 69 patients with the disease in 1914.5 As is commonly the case in medicine, the disease was first described much earlier (1858) in brothers suffering from blindness and deafness by von Gräfe6 and was further characterized by his student, Liebreich,7 3 years later in a larger population of patients. From its discovery until recently, USH syndrome and its subgroups were delineated by clinical characteristics.8 Now the disease is recognized by symptomology and classified by genotyping. To date, there are at least 10 causative genes that are associated with USH syndrome, summarized in previous reviews.1,2,9 In general, based on its diverse clinical symptoms, particularly the onset and severity of the sensorineural hearing impairment and the presence of vestibular dysfunction, USH syndrome is grouped into 3 clinical subtypes. Type I disease is associated with severe-to-profound prelingual hearing loss, vestibular abnormalities and prepubescent onset of RP symptoms.10,11 USH1 results from mutations in MYO7A (USH1B), HARMONIN (USH1C), CDH23 (USH1D), PCDH15 (USH1F), SANS (USH1G), and CIB2 (USH1J). Patients with type II disease display moderate-to-severe congenital hearing loss without vestibular dysfunction, and account for >50% of all USH cases. Their RP symptoms generally begin in the second decade.12 USH2 is caused by mutations in USH2A (USH2A), ADGRV1 (USH2C), and WHRN (USH2D). Patients with type III disease caused by mutations in CLRN1 (USH3A) gene have postlingual progressive hearing loss, variable vestibular dysfunction, and variable onset of RP that rapidly progresses to legal blindness by the fourth decade of life.13–17 Mutations in the HARS gene (Histidyl-tRNA synthetase) are associated with the ultra-rare form USH3B.18,19 Other genes have been described in cases of atypical USH, including: CEP250 and CEP78, encoding members of the CEP family of centrosome-associated proteins; ESPN, encoding the F-actin cross-linker espin; ARSG, encoding the arylsulfatase G enzyme.20 Gene Therapy for USH Syndrome: Current Challenges The success of gene therapy for Leber Congenital Amaurosis (LCA2) due to defects in the RPE65 gene and subsequent FDA and EMA approval of Luxturna (voretigene neparvovec) have sparked hope that many other autosomal recessive retinal (and neurological) diseases may also be treated using a vector-based gene delivery approach.21,22 USH syndrome was no exception and several laboratories embarked on USH gene transfer studies in animal models. Conventional recombinant adeno-associated viral vectors (AAVs) contain a single-stranded DNA expression cassette, which includes a cell-specific or ubiquitous promoter, the transgene and polyadenylation signal, flanked by palindromic inverted terminal repeats at both ends. In contrast to other gene therapy systems, AAV vectors have low immunogenicity (especially when given subretinally), display long-lasting expression in postmitotic cells due to their ability to primarily persist as episomes, and efficiently transduce both photoreceptors and the retinal pigmented epithelium (RPE), where most RP-associated genes are expressed.23 Overall, AAV vectors hold tremendous potential to prevent retinal degeneration in USH syndrome. However, there are numerous challenges in the field, which become apparent by acknowledging the critical disease-specific parameters that account for the success of AAV-based gene augmentation therapy in RPE65 LCA. These include the availability of small and large animal models of RPE65-deficiency displaying a robust phenotype, a slow degenerative component of the disease accompanying a marked retinal dysfunction, a large window for treatment, and the lack of detrimental effects following long-term AAV-mediated transgene overexpression in RPE.24 In contrast to ocular diseases in USH syndrome, the molecular basis of retinal dysfunction in LCA2 is well understood. Moreover, the lack of RPE65 causes a substantial loss of visual function in murine and canine animal models, as demonstrated by the significantly reduced or nonrecordable electroretinograms.25–27 The marked loss of vision in animal models, which contrasts sharply with the remarkable preservation of retinal architecture, has allowed testing of long-term gene therapy effects.28–30 The RPE65 protein is a 65 kDa retinoid isomerohydrolase, which plays an essential role in the retinoid visual cycle by converting all-trans-retinyl ester to 11-cis-retinol in RPE cells.31–33 The 11-cis retinal chromophore deficiency in LCA2 photoreceptor outer segments can be corrected by restoring RPE65 enzyme to RPE, either by using a cell-specific human RPE65 promoter or a modified version of the ubiquitous chicken-beta actin promoter. Furthermore, one RPE cells interdigitates with 20 to 40 photoreceptor cells at the apical interface, amplifying the effects of gene therapy while minimizing the potential downside of nonuniform AAV-mediated transgene expression in neighboring RPE cells. Subretinal injection of an AAV2 vector delivering the canine wild-type RPE65 cDNA in dog models has produced long-term beneficial effects on visual function and protection from degeneration especially when treatment was initiated early.29,30,34,35 Ultimately, the successful restoration of vision in a large animal model paved the way towards an AAV-based gene therapy for LCA2 patients.36–40 Unlike the case of RPE65-associated disease, the majority of the genetically engineered mouse models of USH syndrome do not exhibit a visual phenotype, although they all display hearing loss.41,42 Some of these models were reported to display only a progressive retinal dysfunction with aging.42 It has been suggested that the lack of a retinal phenotype in mouse models could result from a distinct species-specific cell pattern of gene expression or to morphologic differences between human and mouse photoreceptors, for example, mice lack the well-developed, actin-filled calyceal processes, the subcellular structures extending from the inner segments in frogs, pigs, zebrafish, and primates.43,44 The absence of a robust ocular phenotype in animal models has severely limited the ability to perform therapeutic, preclinical proof-of-concept testing for vision loss in USH syndrome. However, gene replacement, gene editing, and small molecule therapy have been used to preserve hearing in many USH animal models with positive results.9,45–53 There are several recent, in-depth reviews of these therapies.9,54,55 These data, combined with the progressive nature of the vision loss due to USH syndrome, have motivated a few companies to empirically study treatments on human subjects. One example is the lentiviral-mediated MYO7A gene delivery in USH1B.56,57 This approach was shown to correct the melanosome mislocalization and opsin accumulation at the photoreceptor connecting cilium in the Myo7a-deficient shaker1 mouse model of USH1B.57 Conventional AAV has a small packaging capacity compared with lentiviral vector (4.7 vs. 8.5 kb). MYO7A cDNA, which is expressed in photoreceptors and the RPE cells, is too large (~6.7 kb) to be packaged in AAV vectors.58 Therefore, an equine infectious anemia virus-based lentiviral vector (equine infectious anemia virus-CMV-MYO7A, UshStat) was subretinally delivered to USH1B patients with the goal to prevent or slow down the RP progression (ClinicalTrials.gov identifier: NCT01505062). Long-term safety evaluation of this treatment in patients who received UshStat is currently being performed in a clinical trial (NCT02065011, results pending). Another clinical trial (UshTher) is currently underway, which uses a dual AAV vector approach to express MYO7A in the retina of patients following subretinal delivery (https://cordis.europa.eu/project/id/754848/it). Dual AAV vectors take advantage of the ability of the 2 distinct AAV genomes containing the 5′ and 3′ half of MYO7A transgene cDNA to undergo intermolecular concatamerization, followed by homologous recombination to reconstitute the whole large gene expression cassette.59,60 To date, there has been no clinically positive data reported from either one of these trials. The disease associated with much larger genes, for example, USH2A (cDNA~15.6 kb), may benefit from genome editing approaches, such as CRISPR-Cas9-sgRNAs, and those that promote exon skipping to restore functional protein restoration.61–66 A splicing-modulating antisense oligonucleotide approach designed to cause in-frame skipping of the mutated exon 13 of the USH2A disease-associated transcripts results in a shortened but functional usherin protein, which localizes correctly in photoreceptors and hair cells.66–68 Interestingly, recent studies have shown that zebrafish models of USH2A disease display early-onset retinal dysfunction, indicating they may represent useful tools to investigate the pathophysiology underlying USH syndrome and test therapeutic strategies.69,70 Morpholino antisense oligonucleotides were shown to mediate ush2a exon 13 skipping and rescue the visual dysfunction in a zebrafish USH2A model.66 This mutation-specific therapeutic strategy is being evaluated in a clinical trial using an antisense oligonucleotide (QR-421a) administered through intravitreal injections, with early evidence of efficacy reported at the 3-month interim analysis (phase 1/2 Stellar trial of QR-421a, ProQR Therapeutics, Clinicaltrials.gov ID: NCT03780257). Critical for the success of future therapeutic approaches is the generation and characterization of novel animal models that mimic the human ocular USH disease. These models will not only serve as preclinical platforms to test potential therapies, including viral-based gene delivery strategies, but can also be used to gain insight into the molecular mechanisms underlying retinal dysfunction and degeneration in USH syndrome. Interestingly, a pig model of USH1C lacking harmonin, USH1C (R31X), was recently reported to mimic the human disease, displaying combined deafness, changes in photoreceptor architecture, and significantly reduced visual function.71 USH syndrome has an estimated prevalence ranging from 4 to 17 cases per 100,000 individuals worldwide.72,73 Approximately 98% of USH syndrome patients belong to either type 1 or type 2 subgroups of disease. In contrast, USH3 is relatively rare, accounting for ~2% of all cases.74 However, it is the most prevalent form of USH disease in the Finnish population, where it represents an estimated 40% of all USH cases, and it also segregates to a high degree in patients of Ashkenazi-Jewish descent.16,17,74 Through funding from the Usher III initiative, a team of researchers was brought together in 2007 to uncover the roles of CLRN1 in sensory organs and the pathology that ensues when the gene is defective. Several therapeutic approaches were developed as a result of this sustained effort, with the ultimate goal of treating USH3A patients. An offshoot of this effort also resulted in several NIH-funded grants specifically directed towards understanding the complex pathophysiology of USH3 in the retina and cochlea. The rest of this review will highlight important findings of this research, and discuss strategies for developing a potentially effective gene therapy approach for USH syndrome. The USH Protein Network The lack of therapeutic approaches to prevent vision loss in USH patients has motivated a considerable amount of research on the pathophysiology of disease. Fluorescence colocalization studies on tissue sections from different species and reciprocal pull-down assays suggest that all proteins encoded by USH genes interact with each other and organize into large complexes within specific subcellular regions in photoreceptors and hair cells.75 They form highly dynamic networks, which consist of proteins from different classes with critical roles in transport, adhesion, and signaling processes in both sensory cell types. USH proteins include the actin-based motor protein myosin VIIa (MYO7A, USH1B); the cell-adhesion transmembrane proteins cadherin 23 (CDH23, USH1D), protocadherin 15 (PCDH15, USH1F), and usherin (USH2A); the very-large G protein-coupled receptor-1 VLGR1 (ADGRV1, GPR98, USH2C); the calcium and integrin binding protein 2 (CIB2, USH1J); the PDZ-domain scaffolding proteins harmonin (USH1C) and whirlin (WHRN, USH2D); the ankyrin and SAM-domain containing scaffolding protein SANS (USH1G); the tetraspanin-like protein clarin-1 (CLRN1, USH3A). In the retina, USH proteins were found to concentrate in specialized regions of photoreceptor cells, the connecting cilium, calyceal processes, and periciliary membrane.2 Some of the USH proteins were also detected at ribbon synapses in both photoreceptors and hair cells, which enable a rapid and sustained release of neurotransmitters necessary for the high frequency transmission of signals in sensory cells.76 The auditory system has often served as a mirror to gain insight into potential functions of USH proteins in the retina. In the cochlea, USH proteins were detected in the hair cell bundle, a mechanosensory antenna containing numerous F-actin rich stereocilia projecting from the apical surface of these auditory cells. As an elegant example, PCDH15 and CDH23 form both transient and permanent extracellular filamentous links between neighboring stereocilia, while harmonin, Sans, and myosin VIIa anchor these links to the intracellular actin core.77–80 In adult stages, PCDH15 and CDH23 form tip links, suggested to act similar to gating springs and open the sensory mechanoelectrical transduction ion channels in response to mechanical stimuli provided by sound waves.81,82 In the retina, USH1 proteins were detected at the fragile actin-rich calyceal processes in macaque, zebrafish, and humans, and may play a role in their development and maintenance.43,83 Similar to the interstereociliary links found in the hair bundle, USH1 proteins may form and stabilize membrane-membrane connection sites linking the calyceal processes to photoreceptor outer segments, allowing them to withstand daily mechanical stress.43 Consistent with this possibility, USH proteins organize and stabilize interactions between microvilli of absorptive intestinal cells.84,85 In contrast to USH1, the USH2 proteins usherin, VLGR1, and whirlin are mainly located in the periciliary ridge membrane complex region surrounding the photoreceptor connecting cilium, where they provide structural support and may also participate in vesicle docking with the plasma membrane during protein translocation to the outer segment.63,86 Pathogenic mutations leading to dysfunction or absence of a particular USH protein were found to alter the localization of other members in the network, a property which has been exploited to evaluate the efficacy of gene therapy experiments in animal models.87 Considering their subcellular localization to specific regions critical for transport and structural maintenance, careful AAV vector dosing and choice of regulatable promoters in gene therapy studies will be needed to ensure that long-term vector-mediated USH proteins expression mimics endogenous levels and does not lead to detrimental effects on photoreceptors structure and function.88 The use of novel penetrating AAV capsids with higher transduction efficiency may enable photoreceptor targeting following a surgically safer intravitreal approach, reducing the concentration of vector reaching the outer retina, and avoiding potential overexpression effects.89,90 CLRN1: an “Invisible” Tetraspanin Protein of Unknown Biological Function A major challenge in the USH field lies in the difficulty of reliably detecting the USH proteins in the retina, due to limited epitope availability, a consequence of USH protein complexes and their low levels of expression. This aspect, combined with the lack of thoroughly validated antibody reagents and proper negative controls in certain cases, has generated conflicting information regarding the cellular expression pattern of some of the USH proteins in the retina. This is especially true for CLRN1, a tetraspanin-like glycoprotein whose biological function is currently unknown.91 The main CLRN1 isoform encodes a 232 amino acid protein that contains a single glycosylation site at asparagine 48 (N48) in the first extracellular loop and a potential PDZ binding motif within its C-terminal end.91,92 In vitro experiments using a hemagglutinin (HA)-epitope tagged CLRN1 have shown that following its overexpression in HEK cells, the protein concentrates at specific regions within plasma membrane and forms CLRN1-enriched microdomains, recruiting diverse proteins involved in cell-adhesion, focal adhesion, and tight junctions, with an essential role in organizing the actin cytoskeleton.93 In the cochlea, CLRN1 has been suggested to interact with USH1 proteins harmonin and PCDH15.47,94Clrn1 knockout and the N48K knock-in mouse models of USH3 do not develop a retinal degeneration phenotype, but display an early-onset hearing loss and disorganized F-actin-rich stereocilia, indicating that one function of CLRN1 in the cochlea is to maintain the structural integrity of the hair bundle.95–97 A zebrafish model further established that CLRN1 is as an essential hair bundle protein.98 Other studies reported that CLRN1 absence also causes synaptic defects in cochlea hair cells.47,94 Three recent studies have shown that gene therapy can successfully prevent hearing loss in different mouse models of USH3 following early postnatal AAV-mediated Clrn1 cochlear delivery, when the sensory hair cell structure is preserved.47–49 These proof of principle studies raise hope that AAV-mediated CLRN1 expression to the correct retinal target cells will also be successful, providing that therapy is initiated before significant structural damage to photoreceptor cells occurs. Studies aimed to define the cellular expression of endogenous CLRN1 in the eye detected it either in the inner or outer retinal cells, or both, depending on the method used (in situ hybridization for mRNA or immunostaining for protein).97,99–101 Localization was further confounded by differences in RNA hybridization probes or antibodies used, variability in sample processing conditions, as well as the particular species analyzed. In one study, Clrn1 transcripts were transiently detected during postnatal development in the inner retina by in situ hybridization (ISH), in a pattern consistent with progenitor cells and Müller glia expression.97 By using rodless aged rd1 mice and laser capture microdissection, that study further showed that in adult mouse retinas Clrn1 mRNA was confined to the inner nuclear layer, detectable only by RT-PCR. In the same year, another group reported that CLRN1 protein was expressed in photoreceptor synapses and connecting cilium regions following immunostaining of the mouse retina with a custom made anti-CLRN1 antibody.101 In zebrafish, CLRN1 was detected in both the inner and outer retina.99 However, several studies concluded that endogenous CLRN1 protein remains below the levels of detection of immunostaining.47,49,97,100 Recently, we have generated and characterized a novel N-terminus epitope-tagged CLRN1 knock-in mouse to facilitate the detection of endogenous CLRN1 protein expression in retinal sections with a high affinity anti-HA antibody.100 We noted a similar background labeling pattern in both HA-tagged CLRN1 knock-in and the C57BL/6J controls. Importantly, the HA-tagged CLRN1 in this mouse was only detectable by western blot analysis of retinal homogenates, and was expressed continuously during postnatal development and adulthood.100 The HA-tagged CLRN1 protein in retinal sections could only be successfully detected following its viral-mediated overexpression using a subretinal or intravitreal delivery approach.100,102 The availability of single-cell RNA sequencing (scRNAseq) technology has provided an unprecedented opportunity to firmly map the localization of CLRN1 and other USH gene transcripts in the retina in multiple species, providing a complementary tool to immunodetection.103,104 In our recently published study, by using scRNAseq analysis of large data sets, we bypassed the challenges of immunostaining experiments, and convincingly demonstrated that CLRN1 transcripts in adult retina are present in Müller glia in human, mouse and non-human primates.100 Müller glia enrichment of CLRN1 transcripts in the retina has been corroborated in human samples by other scRNAseq studies.105–108 Interestingly, scRNAseq data sets have provided evidence that HARMONIN (USH1C), CDH23 (USH1D), and SANS (USH1G) transcripts are also present Müller glia, supporting the need to consider the involvement of multiple cell types in USH disease.108 Furthermore, by using the highly sensitive RNAscope ISH assay we detected CLRN1 transcripts in the inner retina and not photoreceptors, both in the adult mouse and human tissue, a result consistent with the scRNAseq data.100 No signal was detected when the ISH Clrn1 target probes were used on Clrn1 knockout mouse sections, confirming the high specificity of the assay.100 We have recently examined CLRN1 mRNA expression in the pig retina using a similar RNAscope ISH chromogenic assay. We determined that CLRN1 transcripts are abundantly expressed early in postnatal development, before the photoreceptor outer segments are fully developed, concentrating within the middle part of the inner nuclear layer of piglet retinas (Fig. 1). Taken together, these experiments provide solid evidence that CLRN1 transcripts are expressed in the inner retina in multiple species, and highlight the complexities inherent in understanding the molecular pathophysiology of vision loss in USH syndrome and potentially, a novel biology associated with retinal degeneration caused by Müller glia dysfunction. However, these studies do not completely rule out the possibility that CLRN1 might also be expressed in photoreceptor cells at very low levels, undetectable with our current technologies.Figure 1: Detection of CLRN1 transcripts in piglet retina at postnatal day P2 using the RNAscope ISH assay (red punctate staining). GCL indicates ganglion cell layer; INL, inner nuclear layer; ISH, in situ hybridization; ONL, outer nuclear layer; RPE, retinal pigment epithelium. The paraffin-embedded retina tissue sections were counterstained with Gill hematoxylin to visualize nuclei (blue). Scale bar: 20 µm.The potential roles of CLRN1 in protein scaffolding and signaling within the retina, as well as its specific molecular impact on Müller glia and photoreceptor neurons are currently unknown. Müller glia are involved in many different aspects of retinal biology, including neuronal development, modulation of neuronal synaptic activity and structural support of photoreceptor cells, and there are species-specific differences between the density and architecture of these cells in the retina.109,110 Previous studies have also suggested a possible function for Müller glia as radial tension sensors, showing that these extraordinary cells are sensitive to mechanically enforced changes in retinal structure.111,112 They have been compared with taut springs, providing mechanical resilience to the retina, and protecting the retina from ripping apart.111 Under normal physiological conditions, the retina is subjected to daily mechanical stress, including during its postnatal expansion. As a tetraspanin, CLRN1 may be involved in maintaining adhesion links between Müller glia and retinal neurons, protecting photoreceptors from mechanical stress, thus enabling their proper function throughout development and with aging. USH3 patients often display a severe impairment in retinal function at very early stages of the disease, with electroretinogram amplitudes being notably decreased in children as young as 3 to 5 years of age.16 Novel animal models of USH3 that mimic human retinal disease could be used to answer several critical questions with regard to gene therapy approaches. First, is presymptomatic transgene expression of CLRN1 required within photoreceptors, Müller glia, or both cell types to prevent blindness. Second, does ectopic CLRN1 expression in either photoreceptors or Müller glia actually promote degeneration. This could happen if exogenous CLRN1 protein prevents normal USH complexes formation in those cells. Finally, does CLRN1 delivery in adult stages, following symptomatic onset, restore vision loss and slow down the disease progression in USH3 patients. A variety of AAV-based gene therapy vector capsids and promoters targeting either photoreceptors or Müller glia are already available, and more are currently being generated and continuously optimized.23,89,113–116 The development and characterization of large animal models of USH syndrome is urgently needed in the field, as it will contribute greatly to the advancement of our understanding of the pathophysiology of this disease, and critically, provide an opportunity to study the cellular autonomy of retinal degeneration in USH patients. Conclusions On the surface, gene replacement appears to be a logical path to treat autosomal recessive retinal pathologies. This has led to the many laboratories and companies moving directly to gene therapeutic strategies to treat certain USH subtypes. The success of this path is yet to be determined. This review has highlighted several hurdles that should be overcome to optimize development of gene therapy towards specific inherited diseases. These fall into 2 general areas, (1) understanding the molecular pathology of the specific disease, and (2) developing gene therapy vectors that safely reach the target cells and express in a manner that effectively corrects biochemical defects without causing collateral damage. In LCA2, the detailed understanding of RPE65 role in vision, the simplicity of the gene structure and availability of both engineered and naturally occurring small (mouse) and large (dog) animal models enabled many researchers to successfully treat these animals, leading to the regulatory approvals of Luxturna. This review and others painfully demonstrate the complexities inherent in understanding the molecular pathophysiology of vision loss secondary to USH disease. Both the lack of solid understanding of the normal function of the associated proteins, and how the omission of these proteins specifically impacts the retina, limit the ability to develop USH therapies. This is true for all forms of USH, but perhaps is most evident for USH3, caused by mutations in an “invisible” CLRN1 protein. A key tool necessary to reveal this information is the generation of suitable animal models displaying a robust visual dysfunction and retinal degeneration phenotype. These models will allow basic scientists to better understand the molecular basis of disease and translational laboratories to develop effective therapies.

PURPOSE. Proper refractive development of the eye, termed emmetropization, is critical for focused vision and is impacted by both genetic determinants and several visual environment factors. Improper emmetropization caused by genetic variants can lead to congenital hyperopia, which is characterized by small eyes and relatively short ocular axial length. To date, variants in only four genes have been firmly associated with human hyperopia, one of which is MFRP. Zebrafish mfrp mutants also have hyperopia and, similar to reports in mice, exhibit increased macrophage recruitment to the retina. The goal of this research was to examine the effects of macrophage ablation on emmetropization and mfrp-related hyperopia. METHODS. We utilized a chemically inducible, cell-specific ablation system to deplete macrophages in both wild-type and mfrp mutant zebrafish. Spectral-domain optical coherence tomography was then used to measure components of the eye and determine relative refractive state. Histology, immunohistochemistry, and transmission electron microscopy were used to further study the eyes. RESULTS. Although macrophage ablation does not cause significant changes to the relative refractive state of wild-type zebrafish, macrophage ablation in mfrp mutants significantly exacerbates their hyperopic phenotype, resulting in a relative refractive error 1.3 times higher than that of non-ablated mfrp siblings. CONCLUSIONS. Genetic inactivation of mfrp leads to hyperopia, as well as abnormal accumulation of macrophages in the retina. Ablation of the mpeg1-positive macrophage population exacerbates the hyperopia, suggesting that macrophages may be recruited in an effort help preserve emmetropization and ameliorate hyperopia.

Interkinetic nuclear migration (IKNM) is the process in which pseudostratified epithelial nuclei oscillate from the apical to basal surface and in phase with the mitotic cycle. In the zebrafish retina, neuroepithelial retinal progenitor cells (RPCs) increase Notch activity with apical movement of the nuclei, and the depth of nuclear migration correlates with the probability that the next cell division will be neurogenic. This study focuses on the mechanisms underlying the relationships between IKNM, cell signaling, and neurogenesis. In particular, we have explored the role IKNM has on endosome biology within RPCs. Through genetic manipulation and live imaging in zebrafish, we find that early (Rab5-positive) and recycling (Rab11a-positive) endosomes polarize in a dynamic fashion within RPCs and with reference to nuclear position. Functional analyses suggest that dynamic polarization of recycling endosomes and their activity within the neuroepithelia modulates the subcellular localization of Crb2a, consequently affecting multiple signaling pathways that impact neurogenesis including Notch, Hippo, and Wnt activities. As nuclear migration is heterogenous and asynchronous among RPCs, Rab11a-affected signaling within the neuroepithelia is modulated in a differential manner, providing mechanistic insight to the correlation of IKNM and selection of RPCs to undergo neurogenesis.

Capillary malformation-arteriovenous malformation (CM-AVM) syndrome is a class of capillary anomalies that are associated with arteriovenous malformations and arteriovenous fistulas, which carry a risk of hemorrhages. There are no broadly effective pharmacological therapies currently available. Most CM-AVMs are associated with a loss of RASA1, resulting in constitutive activation of RAS signaling. However, protein interaction analysis revealed that RASA1 forms a complex with Rho GTPase-activating protein (RhoGAP), a negative regulator of RhoA signaling. Herein, we propose that loss of RASA1 function results in constitutive activation of RhoA signaling in endothelial cells, resulting in enhanced vascular permeability. Therefore, strategies aimed at curtailing RhoA activity should be tested as an adjunctive therapeutic approach in cell culture studies and animal models of RASA1 deficiency.

The development of the biliary system is a complex yet poorly understood process, with relevance to multiple diseases including biliary atresia, choledochal cysts, and gallbladder agenesis. We present here a crucial role for Hippo-Yap/Taz signaling in this context. Analysis of sav1 mutant zebrafish revealed dysplastic morphology and expansion of both intrahepatic and extrahepatic biliary cells, and ultimately larval lethality. Biliary dysgenesis, but not larval lethality, is driven primarily by Yap signaling. Re-expression of Sav1 protein in sav1-/- hepatocytes is able to overcome these initial deficits and allow sav1-/- fish to survive, suggesting cell non-autonomous signaling from hepatocytes. Examination of sav1-/- rescued adults reveals loss of gallbladder and formation of dysplastic cell masses expressing biliary markers suggesting roles for Hippo signaling in extrahepatic biliary carcinomas. Deletion of stk3 revealed the phenotypes observed in sav1 mutant fish function primarily through canonical Hippo signaling and supports a role for phosphatase PP2A, but also suggests Sav1 has functions in addition to facilitating Stk3 activity. Overall, this study defines a role for Hippo-Yap signaling in the maintenance of both intra- and extrahepatic biliary ducts.

Adherens junctions remodeling regulated by apical polarity proteins constitutes a major driving force for tissue morphogenesis, though the precise mechanism remains inconclusive. Here we reported that Crumbs complex component MPP5a interacts with small GTPase Rab11 in Golgi to synergistically transport cadherin and Crumbs components to the apical domain, thus establishing the apical epithelial polarity and adherens junctions. In contrast, Par complex recruited by MPP5a is incapable to interact with Rab11 but may assemble cytoskeleton to facilitate the cadherin exocytosis. In accordance, dysfunction of MPP5a induced an invasive migration of epithelial cells. This adherens junctions remodeling pattern is frequently observed in zebrafish lens epithelial cells and neuroepithelial cells. The data identified an unrecognized MPP5a/Rab11 complex and described its essential role in guiding the apical polarization and zonula adherens formation in epithelial cells.

Induction of endoplasmic reticulum (ER) stress is associated with diverse developmental and degenerative diseases. Modified ER homeostasis causes activation of conserved stress pathways at the ER called the unfolded protein response (UPR). ATF6 is a transcription factor activated during ER stress as part of a coordinated UPR. ATF6 resides at the ER and, upon activation, is transported to the Golgi apparatus, where it is cleaved by proteases to create an amino-terminal cytoplasmic fragment (ATF6f). ATF6f translocates to the nucleus to activate transcriptional targets. Here, we describe the establishment and validation of zebrafish reporter lines for ATF6 activity. These transgenic lines are based on a defined and multimerized ATF6 consensus site, which drives either eGFP or destabilized eGFP, enabling dynamic study of ATF6 activity during development and disease. The results show that the reporter is specific for the ATF6 pathway, active during development and induced in disease models known to engage UPR. Specifically, during development, ATF6 activity is highest in the lens, skeletal muscle, fins and gills. The reporter is also activated by common chemical inducers of ER stress, including tunicamycin, thapsigargin and brefeldin A, as well as by heat shock. In models for amyotrophic lateral sclerosis and cone dystrophy, ATF6 reporter expression is induced in spinal cord interneurons or photoreceptors, respectively, suggesting a role for ATF6 response in multiple neurodegenerative diseases. Collectively our results show that these ATF6 reporters can be used to monitor ATF6 activity changes throughout development and in zebrafish models of disease.This article has an associated First Person interview with the first author of the paper.

The Hippo-Yap pathway regulates multiple cellular processes in response to mechanical and other stimuli. In Drosophila, the polarity protein Lethal (2) giant larvae [L(2)gl], negatively regulates Hippomediated transcriptional output. However, in vertebrates, little is known about its homolog Llgl1. Here, we define a novel role for vertebrate Llgl1 in regulating Yap stability in cardiomyocytes, which impacts heart development. In contrast to the role of Drosophila L(2)gl, Llgl1 depletion in cultured rat cardiomyocytes decreased Yap protein levels and blunted target gene transcription without affecting Yap transcript abundance. Llgl1 depletion in zebrafish resulted in larger and dysmorphic cardiomyocytes, pericardial effusion, impaired blood flow and aberrant valvulogenesis. Cardiomyocyte Yap protein levels were decreased in llgl1 morphants, whereas Notch, which is regulated by hemodynamic forces and participates in valvulogenesis, was more broadly activated. Consistent with the role of Llgl1 in regulating Yap stability, cardiomyocytespecific overexpression of Yap in Llgl1-depleted embryos ameliorated pericardial effusion and restored blood flow velocity. Altogether, our data reveal that vertebrate Llgl1 is crucial for Yap stability in cardiomyocytes and its absence impairs cardiac development.

The cellular signaling pathways underlying peripheral nerve sheath tumor (PNST) formation are poorly understood. Hippo signaling has been recently implicated in the biology of various cancers, and is thought to function downstream of mutations in the known PNST driver, NF2. Utilizing CRISPR-Cas9 gene editing, we targeted the canonical Hippo signaling kinase Lats2. We show that, while germline deletion leads to early lethality, targeted somatic mutations of zebrafish lats2 leads to peripheral nerve sheath tumor formation. These peripheral nerve sheath tumors exhibit high levels of Hippo effectors Yap and Taz, suggesting that dysregulation of these transcriptional co-factors drives PNST formation in this model. These data indicate that somatic lats2 deletion in zebrafish can serve as a powerful experimental platform to probe the mechanisms of PNST formation and progression.