Steve Mangos

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.

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.

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.

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.

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.

Fishing for New Glomerular Disease-Related Genes

Identification of new disease-related genes contributing to various forms of glomerular pathobiology is a critical step leading to the development of novel treatments and therapies for kidney-related disorders. Significant progress toward this end has been realized by contributions from the Matrix

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.

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. Developmental Dynamics 242:1284–1292, 2013.