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.

Improving Biomedical Engineering Undergraduate Learning Through Use of Online Graduate Engineering Courses During the COVID-19 Pandemic.

In order to provide undergraduate students with a full, rich online learning experience we adapted pre-existing online content including graduate courses from Johns Hopkins University Engineering for Professionals (JHU EP) program. These online courses were designed using published methodologies and held to a high level of rigor of a Masters-level curriculum. Adapting pre-existing online course material enabled us to more rapidly adapt to the COVID-19 shutdown of in-person education. We adapted content to meet the majority of lab-based learning objectives rather than generating self-recorded lecture material and allowing us to focus faculty time on addressing student needs. Here we discuss benefits, challenges, and methods for replicating these courses, and lessons to be applied in future offerings from this experience.

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.

Blood vessel anastomosis is spatially regulated by Flt1 during angiogenesis.

Blood vessel formation is essential for vertebrate development and is primarily achieved by angiogenesis - endothelial cell sprouting from pre-existing vessels. Vessel networks expand when sprouts form new connections, a process whose regulation is poorly understood. Here, we show that vessel anastomosis is spatially regulated by Flt1 (VEGFR1), a VEGFA receptor that acts as a decoy receptor. In vivo, expanding vessel networks favor interactions with Flt1 mutant mouse endothelial cells. Live imaging in human endothelial cells in vitro revealed that stable connections are preceded by transient contacts from extending sprouts, suggesting sampling of potential target sites, and lowered Flt1 levels reduced transient contacts and increased VEGFA signaling. Endothelial cells at target sites with reduced Flt1 and/or elevated protrusive activity were more likely to form stable connections with incoming sprouts. Target cells with reduced membrane-localized Flt1 (mFlt1), but not soluble Flt1, recapitulated the bias towards stable connections, suggesting that relative mFlt1 expression spatially influences the selection of stable connections. Thus, sprout anastomosis parameters are regulated by VEGFA signaling, and stable connections are spatially regulated by endothelial cell-intrinsic modulation of mFlt1, suggesting new ways to manipulate vessel network formation.

Flt-1 (VEGFR-1) coordinates discrete stages of blood vessel formation.

AIMS: In developing blood vessel networks, the overall level of vessel branching often correlates with angiogenic sprout initiations, but in some pathological situations, increased sprout initiations paradoxically lead to reduced vessel branching and impaired vascular function. We examine the hypothesis that defects in the discrete stages of angiogenesis can uniquely contribute to vessel branching outcomes. METHODS AND RESULTS: Time-lapse movies of mammalian blood vessel development were used to define and quantify the dynamics of angiogenic sprouting. We characterized the formation of new functional conduits by classifying discrete sequential stages-sprout initiation, extension, connection, and stability-that are differentially affected by manipulation of vascular endothelial growth factor-A (VEGF-A) signalling via genetic loss of the receptor flt-1 (vegfr1). In mouse embryonic stem cell-derived vessels genetically lacking flt-1, overall branching is significantly decreased while sprout initiations are significantly increased. Flt-1(-/-) mutant sprouts are less likely to retract, and they form increased numbers of connections with other vessels. However, loss of flt-1 also leads to vessel collapse, which reduces the number of new stable conduits. Computational simulations predict that loss of flt-1 results in ectopic Flk-1 signalling in connecting sprouts post-fusion, causing protrusion of cell processes into avascular gaps and collapse of branches. Thus, defects in stabilization of new vessel connections offset increased sprout initiations and connectivity in flt-1(-/-) vascular networks, with an overall outcome of reduced numbers of new conduits. CONCLUSIONS: These results show that VEGF-A signalling has stage-specific effects on vascular morphogenesis, and that understanding these effects on dynamic stages of angiogenesis and how they integrate to expand a vessel network may suggest new therapeutic strategies.

An RNAi-based suppressor screen identifies interactors of the Myt1 ortholog of Caenorhabditis elegans.

Oocyte maturation in all species is controlled by a protein complex termed the maturation promoting factor (MPF). MPF comprises a cyclin-dependent kinase (CDK) and its partner cyclin, and it is regulated by dueling regulatory phosphorylation events on the CDK. In Caenorhabditis elegans, the Wee1/Myt1 ortholog WEE-1.3 provides the inhibitory phosphorylations on CDK-1 that keep MPF inactive and halt meiosis. Prior work has shown that depletion of WEE-1.3 in C. elegans results in precocious oocyte maturation in vivo and a highly penetrant infertility phenotype. This study sought to further define the precocious maturation phenotype and to identify novel interactors with WEE-1.3. We found that WEE-1.3 is expressed throughout the germline and in developing embryos in a perinuclear pattern, and demonstrated that oocytes in WEE-1.3-depleted germlines have begun to transcribe embryonic genes and exhibit inappropriate expression of proteins normally restricted to fertilized eggs. In addition, we performed an RNAi suppressor screen of the infertile phenotype to identify novel factors that, when co-depleted with WEE-1.3, restore fertility to these animals. We screened ∼1900 essential genes by RNAi feeding and identified 44 (∼2% of the tested genes) that are suppressors of the WEE-1.3 depletion phenotype. The suppressors include many previously unidentified players in the meiotic cell cycle and represent a pool of potential WEE-1.3 interacting proteins that function during C. elegans oocyte maturation and zygotic development.

The tailless ortholog nhr-67 functions in the development of the C. elegans ventral uterus.

The development of the C. elegans uterus provides a model for understanding the regulatory pathways that control organogenesis. In C. elegans, the ventral uterus develops through coordinated signaling between the uterine anchor cell (AC) and a ventral uterine (VU) cell. The nhr-67 gene encodes the nematode ortholog of the tailless nuclear receptor gene. Fly and vertebrate tailless genes function in neuronal and ectodermal developmental pathways. We show that nhr-67 functions in multiple steps in the development of the C. elegans uterus. First, it functions in the differentiation of the AC. Second, it functions in reciprocal signaling between the AC and an equipotent VU cell. Third, it is required for a later signaling event between the AC and VU descendants. nhr-67 is required for the expression of both the lag-2/Delta signal in the AC and the lin-12/Notch receptor in all three VU cells and their descendants, suggesting that nhr-67 may be a key regulator of Notch-signaling components. We discuss the implications of these findings for proposed developmental regulatory pathways that include the helix-loop-helix regulator hlh-2/daughterless and transcription factor egl-43/Evi1 in the differentiation of ventral uterine cell types.

Functional redundancy of paralogs of an anaphase promoting complex/cyclosome subunit in Caenorhabditis elegans meiosis.

The anaphase promoting complex/cyclosome (APC/C) mediates the metaphase-to-anaphase transition by instructing the ubiquitination and turnover of key proteins at this stage of the cell cycle. We have recovered a gain-of-function allele in an APC5 subunit of the anaphase promoting complex/cyclosome. This finding led us to investigate further the role of APC5 in Caenorhabditis elegans, which contains two APC5 paralogs. We have shown that these two paralogs, such-1 and gfi-3, are coexpressed in the germline but have nonoverlapping expression patterns in other tissues. Depletion of such-1 or gfi-3 alone does not have a notable effect on the meiotic divisions; however, codepletion of these two factors results in meiotic arrest. In sum, the two C. elegans APC5 paralogs have a redundant function during the meiotic divisions.