Type I IFN in Glomerular Disease: Scarring beyond the STING.

The field of nephrology has recently directed a considerable amount of attention towards the stimulator of interferon genes (STING) molecule since it appears to be a potent driver of chronic kidney disease (CKD). STING and its activator, the cyclic GMP-AMP synthase (cGAS), along with intracellular RIG-like receptors (RLRs) and toll-like receptors (TLRs), are potent inducers of type I interferon (IFN-I) expression. These cytokines have been long recognized as part of the mechanism used by the innate immune system to battle viral infections; however, their involvement in sterile inflammation remains unclear. Mounting evidence pointing to the involvement of the IFN-I pathway in sterile kidney inflammation provides potential insights into the complex interplay between the innate immune system and damage to the most sensitive segment of the nephron, the glomerulus. The STING pathway is often cited as one cause of renal disease not attributed to viral infections. Instead, this pathway can recognize and signal in response to host-derived nucleic acids, which are also recognized by RLRs and TLRs. It is still unclear, however, whether the development of renal diseases depends on subsequent IFN-I induction or other processes involved. This review aims to explore the main endogenous inducers of IFN-I in glomerular cells, to discuss what effects autocrine and paracrine signaling have on IFN-I induction, and to identify the pathways that are implicated in the development of glomerular damage.

The small GTPase regulatory protein Rac1 drives podocyte injury independent of cationic channel protein TRPC5.

Transient receptor potential canonical channels (TRPCs) are non-selective cationic channels that play a role in signal transduction, especially in G -protein-mediated signaling cascades. TRPC5 is expressed predominantly in the brain but also in the kidney. However, its role in kidney physiology and pathophysiology is controversial. Some studies have suggested that TRPC5 drives podocyte injury and proteinuria, particularly after small GTPase Rac1 activation to induce the trafficking of TRPC5 to the plasma membrane. Other studies using TRPC5 gain-of-function transgenic mice have questioned the pathogenic role of TRPC5 in podocytes. Here, we show that TRPC5 over-expression or inhibition does not ameliorate proteinuria induced by the expression of constitutively active Rac1 in podocytes. Additionally, single-cell patch-clamp studies did not detect functional TRPC5 channels in primary cultures of podocytes. Thus, we conclude that TRPC5 plays a role redundant to that of TRPC6 in podocytes and is unlikely to be a useful therapeutic target for podocytopathies.

Deiodinase-3 is a thyrostat to regulate podocyte homeostasis.

BACKGROUND: Nephrotic syndrome (NS) is associated with kidney podocyte injury and may occur as part of thyroid autoimmunity such as Graves' disease. Therefore, the present study was designed to ascertain if and how podocytes respond to and regulate the input of biologically active thyroid hormone (TH), 3,5,3'-triiodothyronine (T3); and also to decipher the pathophysiological role of type 3 deiodinase (D3), a membrane-bound selenoenzyme that inactivates TH, in kidney disease. METHODS: To study D3 function in healthy and injured (PAN, puromycin aminonucleoside and LPS, Lipopolysaccharide-mediated) podocytes, immunofluorescence, qPCR and podocyte-specific D3 knockout mouse were used. Surface plasmon resonance (SPR), co-immunoprecipitation and Proximity Ligation Assay (PLA) were used for the interaction studies. FINDINGS: Healthy podocytes expressed D3 as the predominant deiodinase isoform. Upon podocyte injury, levels of Dio3 transcript and D3 protein were dramatically reduced both in vitro and in the LPS mouse model of podocyte damage. D3 was no longer directed to the cell membrane, it accumulated in the Golgi and nucleus instead. Further, depleting D3 from the mouse podocytes resulted in foot process effacement and proteinuria. Treatment of mouse podocytes with T3 phenocopied the absence of D3 and elicited activation of αvß3 integrin signaling, which led to podocyte injury. We also confirmed presence of an active thyroid stimulating hormone receptor (TSH-R) on mouse podocytes, engagement and activation of which resulted in podocyte injury. INTERPRETATION: The study provided a mechanistic insight into how D3-αvß3 integrin interaction can minimize T3-dependent integrin activation, illustrating how D3 could act as a renoprotective thyrostat in podocytes. Further, injury caused by binding of TSH-R with TSH-R antibody, as found in patients with Graves' disease, explained a plausible link between thyroid disorder and NS. FUNDING: This work was supported by American Thyroid Association (ATA-2018-050.R1).

The Effect of Fluence on Macrophage Kinetics, Oxidative Stress, and Wound Closure Using Real-Time In Vivo Imaging.

Objective: The aim of our study was to quantify the effect of doses delivered by a He:Ne laser on individual macrophage kinetics, tissue oxidative stress, and wound closure using real-time in vivo imaging. Background: Photobiomodulation has been reported to reduce tissue inflammation and accelerate wound closure; however, precise parameters of laser settings to optimize macrophage behavior have not been established. We hypothesized that quantitative and real-time in vivo imaging could identify optimal fluence for macrophage migration, reduction of reactive oxygen species, and acceleration of wound closure. Methods: Larval zebrafish Tg(mpeg-dendra2) were loaded with dihydroethidium for oxidative stress detection. Fish were caudal fin injured, treated with 635 nm continuous 5 mW He:Ne laser irradiation at 3, 9, or 18 J/cm2 and time-lapsed imaged within the first 120 min postinjury. Images taken 1 and 24-h postinjury were compared for percentage wound closure. Results: A fluence of 3 J/cm2 demonstrated significant increases in macrophage migration speed, fewer stops along the way, and greatest directed migration toward the wound. These findings were associated with a significant reduction in wound content reactive oxygen species when compared with control wounded fins. Both 3 and 9 J/cm2 significantly accelerated wound closure when compared with nonirradiated control fish. Conclusions: Wound macrophage activity could be manipulated by applied fluence, leading to reduced levels of wound reactive oxygen species and accelerated wound closure. The zebrafish model provides a means to quantitatively compare wound macrophage behavior in response to a variety of laser treatment parameters in real time.

Nonimmune cell-derived ICOS ligand functions as a renoprotective αvß3 integrin-selective antagonist.

Soluble urokinase receptor (suPAR) is a circulatory molecule that activates αvß3 integrin on podocytes, causes foot process effacement, and contributes to proteinuric kidney disease. While active integrin can be targeted by antibodies and small molecules, endogenous inhibitors haven't been discovered yet. Here we report what we believe is a novel renoprotective role for the inducible costimulator ligand (ICOSL) in early kidney disease through its selective binding to podocyte αvß3 integrin. Contrary to ICOSL's immune-regulatory role, ICOSL in nonhematopoietic cells limited the activation of αvß3 integrin. Specifically, ICOSL contains the arginine-glycine-aspartate (RGD) motif, which allowed for a high-affinity and selective binding to αvß3 and modulation of podocyte adhesion. This binding was largely inhibited either by a synthetic RGD peptide or by a disrupted RGD sequence in ICOSL. ICOSL binding favored the active αvß3 rather than the inactive form and showed little affinity for other integrins. Consistent with the rapid induction of podocyte ICOSL by inflammatory stimuli, glomerular ICOSL expression was increased in biopsies of early-stage human proteinuric kidney diseases. Icosl deficiency in mice resulted in an increased susceptibility to proteinuria that was rescued by recombinant ICOSL. Our work identified a potentially novel role for ICOSL, which serves as an endogenous αvß3-selective antagonist to maintain glomerular filtration.

An automated quantitative image analysis pipeline of in vivo oxidative stress and macrophage kinetics.

Macrophage behavior is of great interest in response to tissue injury and promotion of regeneration. With increasing numbers of zebrafish reporter-based assays, new capabilities now exist to characterize macrophage migration, and their responses to biochemical cues, such as reactive oxygen species. Real time detection of macrophage behavior in response to oxidative stress using quantitative measures is currently beyond the scope of commercially available software solutions, presenting a gap in understanding macrophage behavior. To address this gap, we developed an image analysis pipeline solution to provide real time quantitative measures of cellular kinetics and reactive oxygen species content in vivo after tissue injury. This approach, termed Zirmi, differs from current software solutions that may only provide qualitative, single image analysis, or cell tracking solutions. Zirmi is equipped with user-defined algorithm parameters to customize quantitative data measures with visualization checks for an analysis pipeline of time-based changes. Moreover, this pipeline leverages open-source PhagoSight, as an automated keyhole cell tracking solution, to avoid parallel developments and build upon readily available tools. This approach demonstrated standardized space- and time-based quantitative measures of (1) fluorescent probe based oxidative stress and (2) macrophage recruitment kinetic based changes after tissue injury. Zirmi image analysis pipeline performed at execution speeds up to 10-times faster than manual image-based approaches. Automated segmentation methods were comparable to manual methods with a DICE Similarity coefficient > 0.70. Zirmi provides an open-source, quantitative, and non-generic image analysis pipeline. This strategy complements current wide-spread zebrafish strategies, for automated standardizations of analysis and data measures.

A Podocyte-Based Automated Screening Assay Identifies Protective Small Molecules.

Podocyte injury and loss mark an early step in the pathogenesis of various glomerular diseases, making these cells excellent targets for therapeutics. However, cell-based high-throughput screening assays for the rational development of podocyte-directed therapeutics are currently lacking. Here, we describe a novel high-content screening-based phenotypic assay that analyzes thousands of podocytes per assay condition in 96-well plates to quantitatively measure dose-dependent changes in multiple cellular features. Our assay consistently produced a Z' value >0.44, making it suitable for compound screening. On screening with >2100 pharmacologically active agents, we identified 24 small molecules that protected podocytes against injury in vitro (1% hit rate). Among the identified hits, we confirmed an ß1-integrin agonist, pyrintegrin, as a podocyte-protective agent. Treatment with pyrintegrin prevented damage-induced decreases in F-actin stress fibers, focal adhesions, and active ß1-integrin levels in cultured cells. In vivo, administration of pyrintegrin protected mice from LPS-induced podocyte foot process effacement and proteinuria. Analysis of the murine glomeruli showed that LPS administration reduced the levels of active ß1 integrin in the podocytes, which was prevented by cotreatment with pyrintegrin. In rats, pyrintegrin reduced peak proteinuria caused by puromycin aminonucleoside-induced nephropathy. Our findings identify pyrintegrin as a potential therapeutic candidate and show the use of podocyte-based screening assays for identifying novel therapeutics for proteinuric kidney diseases.

In vivo imaging and characterization of actin microridges.

Actin microridges form labyrinth like patterns on superficial epithelial cells across animal species. This highly organized assembly has been implicated in mucus retention and in the mechanical structure of mucosal surfaces, however the mechanisms that regulate actin microridges remain largely unknown. Here we characterize the composition and dynamics of actin microridges on the surface of zebrafish larvae using live imaging. Microridges contain phospho-tyrosine, cortactin and VASP, but not focal adhesion kinase. Time-lapse imaging reveals dynamic changes in the length and branching of microridges in intact animals. Transient perturbation of the microridge pattern occurs before cell division with rapid re-assembly during and after cytokinesis. Microridge assembly is maintained with constitutive activation of Rho or inhibition of myosin II activity. However, expression of dominant negative RhoA or Rac alters microridge organization, with an increase in distance between microridges. Latrunculin A treatment and photoconversion experiments suggest that the F-actin filaments are actively treadmilling in microridges. Accordingly, inhibition of Arp2/3 or PI3K signaling impairs microridge structure and length. Taken together, actin microridges in zebrafish represent a tractable in vivo model to probe pattern formation and dissect Arp2/3-mediated actin dynamics in vivo.

odd-skipped related 2 is required for fin chondrogenesis in zebrafish.

BACKGROUND: odd-skipped related 2 (osr2) encodes a vertebrate ortholog of the Drosophila odd-skipped zinc-finger transcription factor. Osr2 in mouse is required for proper palate, eyelid, and bone development. Zebrafish knock-down experiments have also suggested a role for osr2, along with its paralog osr1, in early pectoral fin specification and pronephric development. RESULTS: We show here that osr2 has a specific function later in development, independent of osr1, in the regulation of sox9a expression and promoting fin chondrogenesis. mRNA in situ hybridization demonstrated osr2 expression in the developing floorplate and later during organogenesis in the pronephros and gut epithelium. In the pectoral fin buds, osr2 was specifically expressed in fin mesenchyme. osr2 knock down in zebrafish embryos disrupted both three and five zinc finger alternatively spliced osr2 isoforms and eliminated wild-type osr2 mRNA. osr2 morphants exhibited normal pectoral fin bud specification but exhibited defective fin chondrogenesis, with loss of differentiated chondrocytes. Defects in chondrogenesis were paralleled by loss of sox9a as well as subsequent col2a1 expression, linking osr2 function to essential regulators of chondrogenesis. CONCLUSIONS: The zebrafish odd-skipped related 2 gene regulates sox9a and col2a1 expression in chondrocyte development and is specifically required for zebrafish fin morphogenesis.

Expression of fgf23 and αklotho in developing embryonic tissues and adult kidney of the zebrafish, Danio rerio.

Fibroblast growth factor 23 (FGF23) is an endocrine hormone that is secreted by bone and acts on the kidney and parathyroid glands to regulate phosphate homeostasis. The effects of FGF23 on phosphate homeostasis are mediated by binding to FGF receptors and their coreceptor, αklotho, which are abundantly expressed in the kidney and parathyroid glands. However, the mechanisms of how FGF23 regulates phosphate handling in the proximal tubule are unclear because αklotho is primarily expressed in the distal nephron in humans and rodents. The purpose of this study was to gain additional insight into the FGF23-αklotho system by investigating the spatial and temporal aspects of the expression of fgf23 and αklotho in the zebrafish, Danio rerio. Here, we report that zebrafish fgf23 begins to be expressed after organogenesis and is continually expressed into adulthood in the corpuscles of Stannius, which are endocrine glands that lie in close proximity to the nephron and are thought to contribute to calcium and phosphate homeostasis in fish. Zebrafish αklotho expression can be detected by 24-h postfertilization in the brain, pancreas and the distal pronephros, and by 56-h postfertilization in liver. Expression in the distal pronephros persists throughout development, and by Day 5, there is also strong expression in the proximal pronephros. αklotho continues to be expressed in the tubules of the metanephros of the adult kidney. These data indicate conservation of the FGF23-αklotho system across species and suggest a likely role for αklotho in the proximal and distal tubules.

Inhibitory effects of Robo2 on nephrin: a crosstalk between positive and negative signals regulating podocyte structure.

Robo2 is the cell surface receptor for the repulsive guidance cue Slit and is involved in axon guidance and neuronal migration. Nephrin is a podocyte slit-diaphragm protein that functions in the kidney glomerular filtration barrier. Here, we report that Robo2 is expressed at the basal surface of mouse podocytes and colocalizes with nephrin. Biochemical studies indicate that Robo2 forms a complex with nephrin in the kidney through adaptor protein Nck. In contrast to the role of nephrin that promotes actin polymerization, Slit2-Robo2 signaling inhibits nephrin-induced actin polymerization. In addition, the amount of F-actin associated with nephrin is increased in Robo2 knockout mice that develop an altered podocyte foot process structure. Genetic interaction study further reveals that loss of Robo2 alleviates the abnormal podocyte structural phenotype in nephrin null mice. These results suggest that Robo2 signaling acts as a negative regulator on nephrin to influence podocyte foot process architecture.

Genome-wide studies of copy number variation and exome sequencing identify rare variants in BAG3 as a cause of dilated cardiomyopathy.

Dilated cardiomyopathy commonly causes heart failure and is the most frequent precipitating cause of heart transplantation. Familial dilated cardiomyopathy has been shown to be caused by rare variant mutations in more than 30 genes but only ~35% of its genetic cause has been identified, principally by using linkage-based or candidate gene discovery approaches. In a multigenerational family with autosomal dominant transmission, we employed whole-exome sequencing in a proband and three of his affected family members, and genome-wide copy number variation in the proband and his affected father and unaffected mother. Exome sequencing identified 428 single point variants resulting in missense, nonsense, or splice site changes. Genome-wide copy number analysis identified 51 insertion deletions and 440 copy number variants > 1 kb. Of these, a 8733 bp deletion, encompassing exon 4 of the heat shock protein cochaperone BCL2-associated athanogene 3 (BAG3), was found in seven affected family members and was absent in 355 controls. To establish the relevance of variants in this protein class in genetic DCM, we sequenced the coding exons in BAG3 in 311 other unrelated DCM probands and identified one frameshift, two nonsense, and four missense rare variants absent in 355 control DNAs, four of which were familial and segregated with disease. Knockdown of bag3 in a zebrafish model recapitulated DCM and heart failure. We conclude that new comprehensive genomic approaches have identified rare variants in BAG3 as causative of DCM.

The ADPKD genes pkd1a/b and pkd2 regulate extracellular matrix formation.

Mutations in polycystin1 (PKD1) account for the majority of autosomal dominant polycystic kidney disease (ADPKD). PKD1 mutations are also associated with vascular aneurysm and abdominal wall hernia, suggesting a role for polycystin1 in extracellular matrix (ECM) integrity. In zebrafish, combined knockdown of the PKD1 paralogs pkd1a and pkd1b resulted in dorsal axis curvature, hydrocephalus, cartilage and craniofacial defects, and pronephric cyst formation at low frequency (10-15%). Dorsal axis curvature was identical to the axis defects observed in pkd2 knockdown embryos. Combined pkd1a/b, pkd2 knockdown demonstrated that these genes interact in axial morphogenesis. Dorsal axis curvature was linked to notochord collagen overexpression and could be reversed by knockdown of col2a1 mRNA or chemical inhibition of collagen crosslinking. pkd1a/b- and pkd2-deficient embryos exhibited ectopic, persistent expression of multiple collagen mRNAs, suggesting a loss of negative feedback signaling that normally limits collagen gene expression. Knockdown of pkd1a/b also dramatically sensitized embryos to low doses of collagen-crosslinking inhibitors, implicating polycystins directly in the modulation of collagen expression or assembly. Embryos treated with wortmannin or LY-29400 also exhibited dysregulation of col2a1 expression, implicating phosphoinositide 3-kinase (PI3K) in the negative feedback signaling pathway controlling matrix gene expression. Our results suggest that pkd1a/b and pkd2 interact to regulate ECM secretion or assembly, and that altered matrix integrity may be a primary defect underlying ADPKD tissue pathologies.

Collective cell migration drives morphogenesis of the kidney nephron.

Tissue organization in epithelial organs is achieved during development by the combined processes of cell differentiation and morphogenetic cell movements. In the kidney, the nephron is the functional organ unit. Each nephron is an epithelial tubule that is subdivided into discrete segments with specific transport functions. Little is known about how nephron segments are defined or how segments acquire their distinctive morphology and cell shape. Using live, in vivo cell imaging of the forming zebrafish pronephric nephron, we found that the migration of fully differentiated epithelial cells accounts for both the final position of nephron segment boundaries and the characteristic convolution of the proximal tubule. Pronephric cells maintain adherens junctions and polarized apical brush border membranes while they migrate collectively. Individual tubule cells exhibit basal membrane protrusions in the direction of movement and appear to establish transient, phosphorylated Focal Adhesion Kinase-positive adhesions to the basement membrane. Cell migration continued in the presence of camptothecin, indicating that cell division does not drive migration. Lengthening of the nephron was, however, accompanied by an increase in tubule cell number, specifically in the most distal, ret1-positive nephron segment. The initiation of cell migration coincided with the onset of fluid flow in the pronephros. Complete blockade of pronephric fluid flow prevented cell migration and proximal nephron convolution. Selective blockade of proximal, filtration-driven fluid flow shifted the position of tubule convolution distally and revealed a role for cilia-driven fluid flow in persistent migration of distal nephron cells. We conclude that nephron morphogenesis is driven by fluid flow-dependent, collective epithelial cell migration within the confines of the tubule basement membrane. Our results establish intimate links between nephron function, fluid flow, and morphogenesis.

Mouse G-protein gamma3 expression in the developing CNS and neural crest cell derivatives.

Heterotrimeric G-protein signaling, involving alpha, beta and gamma subunits, plays a number of roles in differentiation and development. Individual gamma subunits interact with a beta subunit and as a heterodimer, is responsible for modulating many G protein-mediated cellular responses. The 12 gamma subunits in mammals have highly variable distribution and expression patterns in adult tissues. gamma3 is abundantly and widely expressed in the brain and when its expression is knocked-out, the mice show increased susceptibility to seizures, reduced body weights and decreased adiposity compared to the wild-type littermates (Schwindinger et al., 2004). Recent evidence has shown the Gng3 gene being strongly induced in activated CD4+ T-cells (Dubeykovskiy et al., 2006) and its involvement in the developing mammalian enteric nervous system (Heanue and Pachnis, 2006). Given this diversity in expression and interest in finding models of human disease, and to extend our previous investigation with zebrafish gamma3 (Kelly et al., 2001), we undertook an analysis to report the temporal and spatial expression patterns of gamma3 mRNA during mouse embryogenesis. Analysis reveals that gamma3 transcripts were first expressed in mid-late embryonic stages. Specifically, signals were predominant in the CNS and in neural crest cell derivatives including but not limited to the trigeminal and dorsal root (spinal) ganglia, and in cells of the adrenal medulla. These data indicate that G protein coupled signaling involving gamma3 participates in a number of physiological roles, not only in the CNS, but also in numerous cells derived from the neural crest.

Expression of trpC1 and trpC6 orthologs in zebrafish.

Transient receptor potential (TRP) genes encode subunits that form cation-selective ion channels in a variety of organisms and cell types. TRP channels serve diverse functions ranging from thermal, tactile, taste, and osmolar sensing to fluid flow sensing. TRPC1 and TRPC6 belong to the TRPC subfamily, members of which are thought to contribute to several cellular events such as regulated migration of neuronal dendrites, contractile responses of smooth muscle cells and maintenance of the structural integrity of kidney podocytes. Pathogenic roles have been suggested for TRPC1 in asthma and chronic obstructive pulmonary disease, and TRPC6 dysfunction was recently linked to proteinuric kidney disease. To explore the potential roles for TRPC channels in zebrafish organ function, we cloned zebrafish trpC1 and trpC6 cDNAs, and investigated their expression during zebrafish development. We detected trpC1 expression in the head, in cells surrounding the outflow tract of the heart, and in the ganglion cells as well as the inner nuclear layer of the eye. trpC6 expression was detected in the head, pectoral fins, aortic endothelial cells, and gastrointestinal smooth muscle cells. Our results point to roles of TRPC channels in several tissues during zebrafish development, and suggest that the zebrafish may be a suitable model system to study the pathophysiology of TRPC1 and TRPC6 in specific cell types.

The zebrafish fleer gene encodes an essential regulator of cilia tubulin polyglutamylation.

Cilia and basal bodies are essential organelles for a broad spectrum of functions, including the development of left-right asymmetry, kidney function, cerebrospinal fluid transport, generation of photoreceptor outer segments, and hedgehog signaling. Zebrafish fleer (flr) mutants exhibit kidney cysts, randomized left-right asymmetry, hydrocephalus, and rod outer segment defects, suggesting a pleiotropic defect in ciliogenesis. Positional cloning flr identified a tetratricopeptide repeat protein homologous to the Caenorhabditis elegans protein DYF1 that was highly expressed in ciliated cells. flr pronephric cilia were shortened and showed a reduced beat amplitude, and olfactory cilia were absent in mutants. flr cilia exhibited ultrastructural defects in microtubule B-tubules, similar to axonemes that lack tubulin posttranslational modifications (polyglutamylation or polyglycylation). flr cilia showed a dramatic reduction in cilia polyglutamylated tubulin, indicating that flr encodes a novel modulator of tubulin polyglutamylation. We also found that the C. elegans flr homologue, dyf-1, is also required for tubulin polyglutamylation in sensory neuron cilia. Knockdown of zebrafish Ttll6, a tubulin polyglutamylase, specifically eliminated tubulin polyglutamylation and cilia formation in olfactory placodes, similar to flr mutants. These results are the first in vivo evidence that tubulin polyglutamylation is required for vertebrate cilia motility and structure, and, when compromised, results in failed ciliogenesis.

Dynamic expression of the osmosensory channel trpv4 in multiple developing organs in zebrafish.

Transient receptor potential channels function in a wide spectrum of tissues and transduce sensory stimuli. The vanilloid (capsaicin) channel TRPV4 is sensitive to osmotic changes and plays a central role in osmoregulatory responses in a variety of organisms. We cloned a zebrafish trpv4 cDNA and assayed its expression during embryogenesis. trpv4 is expressed as maternal mRNA in 4-cell embryos and later zygotic expression is first observed in the forming notochord at the one somite stage. Notochord expression persists to 24 hpf when broad expression in the brain is observed. At 32 hpf trpv4 expression is observed in the endocardium, restricted primarily to the ventricular endothelium. Low level expression of trpv4 is also seen from 32-48 hpf in the pronephric kidney with strongest expression in the most distal nephron segment and in the cloaca. Expression is also observed in lateral line organs starting at 32 hpf, primarily in the hair cells. At 72 hpf, expression of trpv4 in heart, kidney, brain, and lateral line organs persists while expression in the notochord is down-regulated.