Exocrine pancreas proteases regulate ß-cell proliferation in zebrafish ciliopathy models and in murine systems.

Pancreatic ß-cells are a critical cell type in the pathology of diabetes. Models of genetic syndromes featuring diabetes can provide novel mechanistic insights into regulation of ß-cells in the context of disease. We previously examined ß-cell mass in models of two ciliopathies, Alström Syndrome (AS) and Bardet-Biedl Syndrome (BBS), which are similar in the presence of metabolic phenotypes, including obesity, but exhibit strikingly different rates of diabetes. Zebrafish models of these disorders show deficient ß-cells with diabetes in AS models and an increased ß-cells absent diabetes in BBS models, indicating ß-cell generation or maintenance that correlates with disease prevalence. Using transcriptome analyses, differential expression of several exocrine pancreas proteases with directionality that was consistent with ß-cell numbers were identified. Based on these lines of evidence, we hypothesized that pancreatic proteases directly impact ß-cells. In the present study, we examined this possibility and found that pancreatic protease genes contribute to proper maintenance of normal ß-cell numbers, proliferation in larval zebrafish, and regulation of AS and BBS ß-cell phenotypes. Our data suggest that these proteins can be taken up directly by cultured ß-cells and ex vivo murine islets, inducing proliferation in both. Endogenous uptake of pancreatic proteases by ß-cells was confirmed in vivo using transgenic zebrafish and in intact murine pancreata. Taken together, these findings support a novel proliferative signaling role for exocrine pancreas proteases through interaction with endocrine ß-cells.

ZRANB3 is an African-specific type 2 diabetes locus associated with beta-cell mass and insulin response.

Genome analysis of diverse human populations has contributed to the identification of novel genomic loci for diseases of major clinical and public health impact. Here, we report a genome-wide analysis of type 2 diabetes (T2D) in sub-Saharan Africans, an understudied ancestral group. We analyze ~18 million autosomal SNPs in 5,231 individuals from Nigeria, Ghana and Kenya. We identify a previously-unreported genome-wide significant locus: ZRANB3 (Zinc Finger RANBP2-Type Containing 3, lead SNP p = 2.831 × 10-9). Knockdown or genomic knockout of the zebrafish ortholog results in reduction in pancreatic ß-cell number which we demonstrate to be due to increased apoptosis in islets. siRNA transfection of murine Zranb3 in MIN6 ß-cells results in impaired insulin secretion in response to high glucose, implicating Zranb3 in ß-cell functional response to high glucose conditions. We also show transferability in our study of 32 established T2D loci. Our findings advance understanding of the genetics of T2D in non-European ancestry populations.

Genomic knockout of alms1 in zebrafish recapitulates Alström syndrome and provides insight into metabolic phenotypes.

Alström syndrome (OMIM #203800) is an autosomal recessive obesity ciliopathy caused by loss-of-function mutations in the ALMS1 gene. In addition to multi-organ dysfunction, such as cardiomyopathy, retinal degeneration and renal dysfunction, the disorder is characterized by high rates of obesity, insulin resistance and early-onset type 2 diabetes mellitus (T2DM). To investigate the underlying mechanisms of T2DM phenotypes, we generated a loss-of-function deletion of alms1 in the zebrafish. We demonstrate conservation of hallmark clinical characteristics alongside metabolic syndrome phenotypes, including a propensity for obesity and fatty livers, hyperinsulinemia and glucose response defects. Gene expression changes in ß-cells isolated from alms1-/- mutants revealed changes consistent with insulin hypersecretion and glucose sensing failure, which were corroborated in cultured murine ß-cells lacking Alms1. We also found evidence of defects in peripheral glucose uptake and concomitant hyperinsulinemia in the alms1-/- animals. We propose a model in which hyperinsulinemia is the primary and causative defect underlying generation of T2DM associated with alms1 deficiency. These observations support the alms1 loss-of-function zebrafish mutant as a monogenic model for mechanistic interrogation of T2DM phenotypes.

Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish.

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an excellent model for rapid assessment of whole transcriptome from whole animal or individual cell populations due to the ease of isolation of RNA from large numbers of animals. Here a protocol for global gene expression analysis in zebrafish embryos using RNA sequencing (RNASeq) is presented. We describe preparation of RNA from whole embryos or from cell populations obtained using cell sorting in transgenic animals. We also describe an approach for analysis of RNASeq data to identify enriched pathways and Gene Ontology (GO) terms in global gene expression data sets. Finally, we provide a protocol for validation of gene expression changes using quantitative reverse transcriptase PCR (qRT-PCR). These protocols can be used for comparative analysis of control and experimental sets of zebrafish to identify novel gene expression changes, and provide molecular insight into phenotypes of interest.

New var reconstruction algorithm exposes high var sequence diversity in a single geographic location in Mali.

BACKGROUND: Encoded by the var gene family, highly variable Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) proteins mediate tissue-specific cytoadherence of infected erythrocytes, resulting in immune evasion and severe malaria disease. Sequencing and assembling the 40-60 var gene complement for individual infections has been notoriously difficult, impeding molecular epidemiological studies and the assessment of particular var elements as subunit vaccine candidates. METHODS: We developed and validated a novel algorithm, Exon-Targeted Hybrid Assembly (ETHA), to perform targeted assembly of var gene sequences, based on a combination of Pacific Biosciences and Illumina data. RESULTS: Using ETHA, we characterized the repertoire of var genes in 12 samples from uncomplicated malaria infections in children from a single Malian village and showed them to be as genetically diverse as vars from isolates from around the globe. The gene var2csa, a member of the var family associated with placental malaria pathogenesis, was present in each genome, as were vars previously associated with severe malaria. CONCLUSION: ETHA, a tool to discover novel var sequences from clinical samples, will aid the understanding of malaria pathogenesis and inform the design of malaria vaccines based on PfEMP1. ETHA is available at: https://sourceforge.net/projects/etha/ .

Whole organism transcriptome analysis of zebrafish models of Bardet-Biedl Syndrome and Alström Syndrome provides mechanistic insight into shared and divergent phenotypes.

BACKGROUND: Bardet-Biedl Syndrome (BBS) and Alström Syndrome are two pleiotropic ciliopathies with significant phenotypic overlap between them across many tissues. Although BBS and Alström genes are necessary for the proper function of primary cilia, their role in defects across multiple organ systems is unclear. METHODS: To provide insight into the pathways underlying BBS and Alström phenotypes, we carried out whole organism transcriptome analysis by RNA sequencing in established zebrafish models of the syndromes. RESULTS: We analyzed all genes that were significantly differentially expressed and found enrichment of phenotypically significant pathways in both models. These included multiple pathways shared between the two disease models as well as those unique to each model. Notably, we identified significant downregulation of genes in pathways relevant to visual system deficits and obesity in both disorders, consistent with those shared phenotypes. In contrast, neuronal pathways were significantly downregulated only in the BBS model but not in the Alström model. Our observations also suggested an important role for G-protein couple receptor and calcium signaling defects in both models. DISCUSSION: Pathway network analyses of both models indicate that visual system defects may be driven by genetic mechanisms independent of other phenotypes whereas the majority of other phenotypes are a result of genetic players that contribute to multiple pathways simultaneously. Additionally, examination of genes differentially expressed in opposing directions between the two models suggest a deficit in pancreatic function in the Alström model, that is not present in the BBS model. CONCLUSIONS: These findings provide important novel insight into shared and divergent phenotypes between two similar but distinct genetic syndromes.

Differential effects on ß-cell mass by disruption of Bardet-Biedl syndrome or Alstrom syndrome genes.

Rare genetic syndromes characterized by early-onset type 2 diabetes have revealed the importance of pancreatic ß-cells in genetic susceptibility to diabetes. However, the role of genetic regulation of ß-cells in disorders that are also characterized by highly penetrant obesity, a major additional risk factor, is unclear. In this study, we investigated the contribution of genes associated with two obesity ciliopathies, Bardet-Biedl Syndrome and Alstrom Syndrome, to the production and maintenance of pancreatic ß-cells. Using zebrafish models of these syndromes, we identified opposing effects on production of ß-cells. Loss of the Alstrom gene, alms1, resulted in a significant decrease in ß-cell production whereas loss of BBS genes, bbs1 or bbs4, resulted in a significant increase. Examination of the regulatory program underlying ß-cell production suggested that these effects were specific to ß-cells. In addition to the initial production of ß-cells, we observed significant differences in their continued maintenance. Under prolonged exposure to high glucose conditions, alms1-deficient ß-cells were unable to continually expand as a result of decreased proliferation and increased cell death. Although bbs1-deficient ß-cells were similarly susceptible to apoptosis, the overall maintenance of ß-cell number in those animals was sustained likely due to increased proliferation. Taken together, these findings implicate discrepant production and maintenance of ß-cells in the differential susceptibility to diabetes found between these two genetic syndromes.