The USH3A causative gene clarin1 functions in Müller glia to maintain retinal photoreceptors.

Mutations in CLRN1 cause Usher syndrome type IIIA (USH3A), an autosomal recessive disorder characterized by hearing and vision loss, and often accompanied by vestibular balance issues. 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. Retina 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 rescue 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.

The tunicamycin derivative TunR2 exhibits potent antibiotic properties with low toxicity in an in vivo Mycobacterium marinum-zebrafish TB infection model.

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.

Mesenchymal Stromal Cells Facilitate Tip Cell Fusion Downstream of BMP-Mediated Venous Angiogenesis-Brief Report.

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.

Myofibroblast Ccn3 is regulated by Yap and Wwtr1 and contributes to adverse cardiac outcomes.

Introduction: While Yap and Wwtr1 regulate resident cardiac fibroblast to myofibroblast differentiation following cardiac injury, their role specifically in activated myofibroblasts remains unexplored. Methods: We 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. Results: Following 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. Discussion: Yap/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.

Endothelial cilia dysfunction in pathogenesis of hereditary hemorrhagic telangiectasia.

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-ß 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-ß 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-ß 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-ß 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.

Ablation of mpeg+ Macrophages Exacerbates mfrp-Related Hyperopia.

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.

Complementary and divergent functions of zebrafish Tango1 and Ctage5 in tissue development and homeostasis.

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.

RhoA activation-mediated vascular permeability in capillary malformation-arteriovenous malformation syndrome: a hypothesis.

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.

Interplay of MPP5a with Rab11 synergistically builds epithelial apical polarity and zonula adherens.

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

Llgl1 regulates zebrafish cardiac development by mediating Yap stability in cardiomyocytes.

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 Hippo-mediated 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, cardiomyocyte-specific 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.

Core Hippo pathway components act as a brake on Yap and Taz in the development and maintenance of the biliary network.

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 allows 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 that 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.

Dynamic Polarization of Rab11a Modulates Crb2a Localization and Impacts Signaling to Regulate Retinal Neurogenesis.

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.

Establishment and validation of an endoplasmic reticulum stress reporter to monitor zebrafish ATF6 activity in development and disease.

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.

Upstream regulation of the Hippo-Yap pathway in cardiomyocyte regeneration.

The response of the adult mammalian heart to injury such as myocardial infarction has long been described as primarily fibrotic scarring and adverse remodeling with little to no regeneration of cardiomyocytes. Emerging studies have challenged this paradigm by demonstrating that, indeed, adult mammalian cardiomyocytes are capable of completing cytokinesis albeit at levels vastly insufficient to compensate for the loss of functional cardiomyocytes following ischemic injury. Thus, there is great interest in identifying mechanisms to guide adult cardiomyocyte cell cycle re-entry and facilitate endogenous heart regeneration. The Hippo signaling pathway is a core kinase cascade that functions to suppress the transcriptional co-activators Yap and Taz by phosphorylation and therefore cytoplasmic retention or phospho-degradation. This pathway has recently sparked interest in the field of cardiac regeneration as inhibition of Hippo kinase signaling or overdriving the transcriptional co-activator, Yap, significantly promotes proliferation of terminally differentiated adult mammalian cardiomyocytes and can restore function in failing mouse hearts. Thus, the Hippo pathway is an attractive therapeutic target for promoting cardiomyocyte renewal and cardiac regeneration. Although the core kinases and transcriptional activators of the Hippo pathway have been studied extensively over the last twenty years, the regulatory inputs of this pathway, particularly in vertebrates, are poorly understood. Recent studies have elucidated several upstream regulatory inputs to the Hippo pathway in adult mammalian cardiomyocytes that influence cell proliferation and heart regeneration. Considering upstream inputs to the Hippo pathway are thought to be context and cell type specific, targeting these various components could serve as a therapeutic approach for refining Hippo-Yap signaling in the heart. Here, we provide an overview of the emerging regulatory inputs to the Hippo pathway as they relate to mammalian cardiomyocytes and heart regeneration.

Precise Short Sequence Insertion in Zebrafish Using a CRISPR/Cas9 Approach to Generate a Constitutively Soluble Lrp2 Protein.

LRP2 is a large transmembrane receptor expressed on absorptive epithelia where it binds many extracellular ligands to control several signaling pathways. Mutations in LRP2 are associated with buphthalmic eye enlargement, myopia and other non-ocular symptoms. Though studies have clearly shown that absence of LRP2 causes these phenotypes, and that overexpression of individual LRP2 domains can exacerbate eye enlargement caused by the absence of Lrp2, the relationship between soluble LRP2 fragments and full-length membrane-bound LRP2 is not completely understood. Here we use a CRISPR/Cas9 approach to insert a stop codon cassette into zebrafish lrp2 to prematurely truncate the protein before its transmembrane domain while leaving the entire extracellular domain intact. The resulting mutant line will be a useful tool for examining Lrp2 function in the eye, and testing hypotheses regarding its extracellular processing.

Somatic Mutations of lats2 Cause Peripheral Nerve Sheath Tumors in Zebrafish.

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.

The Roles of Hippo Signaling Transducers Yap and Taz in Chromatin Remodeling.

Hippo signaling controls cellular processes that ultimately impact organogenesis and homeostasis. Consequently, disease states including cancer can emerge when signaling is deregulated. The major pathway transducers Yap and Taz require cofactors to impart transcriptional control over target genes. Research into Yap/Taz-mediated epigenetic modifications has revealed their association with chromatin-remodeling complex proteins as a means of altering chromatin structure, therefore affecting accessibility and activity of target genes. Specifically, Yap/Taz have been found to associate with factors of the GAGA, Ncoa6, Mediator, Switch/sucrose nonfermentable (SWI/SNF), and Nucleosome Remodeling and Deacetylase (NuRD) chromatin-remodeling complexes to alter the accessibility of target genes. This review highlights the different mechanisms by which Yap/Taz collaborate with other factors to modify DNA packing at specific loci to either activate or repress target gene transcription.

Regeneration of the zebrafish retinal pigment epithelium after widespread genetic ablation.

The retinal pigment epithelium (RPE) is a specialized monolayer of pigmented cells within the eye that is critical for maintaining visual system function. Diseases affecting the RPE have dire consequences for vision, and the most prevalent of these is atrophic (dry) age-related macular degeneration (AMD), which is thought to result from RPE dysfunction and degeneration. An intriguing possibility for treating RPE degenerative diseases like atrophic AMD is the stimulation of endogenous RPE regeneration; however, very little is known about the mechanisms driving successful RPE regeneration in vivo. Here, we developed a zebrafish transgenic model (rpe65a:nfsB-eGFP) that enabled ablation of large swathes of mature RPE. RPE ablation resulted in rapid RPE degeneration, as well as degeneration of Bruch's membrane and underlying photoreceptors. Using this model, we demonstrate for the first time that zebrafish are capable of regenerating a functional RPE monolayer after RPE ablation. Regenerated RPE cells first appear at the periphery of the RPE, and regeneration proceeds in a peripheral-to-central fashion. RPE ablation elicits a robust proliferative response in the remaining RPE. Subsequently, proliferative cells move into the injury site and differentiate into RPE. BrdU incorporation assays demonstrate that the regenerated RPE is likely derived from remaining peripheral RPE cells. Pharmacological disruption using IWR-1, a Wnt signaling antagonist, significantly reduces cell proliferation in the RPE and impairs overall RPE recovery. These data demonstrate that the zebrafish RPE possesses a robust capacity for regeneration and highlight a potential mechanism through which endogenous RPE regenerate in vivo.

The Hippo Pathway Regulates Caveolae Expression and Mediates Flow Response via Caveolae.

The Hippo pathway plays major roles in development, regeneration, and cancer. Its activity is tightly regulated by both diffusible chemical ligands and mechanical stimuli. The pathway consists of a series of kinases that can control the sub-cellular localization and stability of YAP or TAZ, homologous transcriptional co-factors. Caveolae, small (60-100 nm) bulb-like invaginations of the plasma membrane, are comprised predominantly of caveolin and cavin proteins and can respond to mechanical stimuli. Here, we show that YAP/TAZ, the major transcriptional mediators of the Hippo pathway, are critical for expression of caveolae components and therefore caveolae formation in both mammalian cells and zebrafish. In essence, without YAP/TAZ, the cell loses an entire organelle. CAVEOLIN1 and CAVIN1, the two essential caveolar genes, are direct target genes of YAP/TAZ, regulated via TEA domain (TEAD) transcription factors. Notably, YAP/TAZ become nuclear enriched and facilitate target gene transcription in cells with diminished levels of caveolae. Furthermore, caveolar-mediated shear stress response activates YAP/TAZ. These data link caveolae to Hippo signaling in the context of cellular responses to mechanical stimuli and suggest activity-based feedback regulation between components of caveolae and the outputs of the Hippo pathway.