• Corrigendum: Transcriptomic profiling of zebrafish hair cells using RiboTag [Front. Cell Dev. Biol., 6, 47, (2018)] doi: 10.3389/fcell.2018.00047

      Matern, M.S.; Beirl, A.; Ogawa, Y. (Frontiers Media S.A., 2018)
      Although the reported fold change values and statistics were accurate, there was a mistake in the CPM values shown in Supplementary Dataset 1 in the original article. This mistake has been corrected and it has resulted in a slight change in the number of transcripts meeting the CPM cutoff for expression (n = 17,164), enrichment (n = 2,379), and depletion (n = 2,258). These corrected numbers of expressed, enriched and depleted transcripts are now reflected in the text, as well as in Figure 3a. The gene ontology and ZEOGS analyses have been redone and changes have been made to Tables 1-3, and Supplementary Table 2. The genes chosen for validation now fall within the top 100 enriched transcripts rather than the top 50; therefore, Supplementary Table 3 has been changed to show the top 100 enriched transcripts. The scientific conclusions made from these analyses have not changed in any way. Copyright 2018 Matern, Beirl, Ogawa, Song, Paladugu, Kindt and Hertzano.
    • Exocrine pancreas proteases regulate β-cell proliferation in zebrafish ciliopathy models and in murine systems

      Hostelley, Timothy L; Nesmith, Jessica E; Larkin, Emily; Jones, Amanda; Boyes, Daniel; Leitch, Carmen C; Fontaine, Magali; Zaghloul, Norann A (The Company of Biologists Ltd., 2021-06-14)
      Pancreatic β-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 β-cells in the context of disease. We previously examined β-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 β-cells with diabetes in AS models and an increased β-cells absent diabetes in BBS models, indicating β-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 β-cell numbers were identified. Based on these lines of evidence, we hypothesized that pancreatic proteases directly impact β-cells. In the present study, we examined this possibility and found that pancreatic protease genes contribute to proper maintenance of normal β-cell numbers, proliferation in larval zebrafish, and regulation of AS and BBS β-cell phenotypes. Our data suggest that these proteins can be taken up directly by cultured β-cells and ex vivo murine islets, inducing proliferation in both. Endogenous uptake of pancreatic proteases by β-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 β-cells.
    • Survey of human chromosome 21 gene expression effects on early development in Danio rerio

      Edie, S.; Zaghloul, N.A.; Leitch, Carmen C. (Genetics Society of America, 2018)
      Trisomy for human chromosome 21 (Hsa21) results in Down syndrome (DS), one of the most genetically complex conditions compatible with human survival. Assessment of the physiological consequences of dosage-driven overexpression of individual Hsa21 genes during early embryogenesis and the resulting contributions to DS pathology in mammals are not tractable in a systematic way. A recent study looked at loss-of-function of a subset of Caenorhabditis elegans orthologs of Hsa21 genes and identified ten candidates with behavioral phenotypes, but the equivalent over-expression experiment has not been done. We turned to zebrafish as a developmental model and, using a number of surrogate phenotypes, we screened Hsa21 genes for effects on early embyrogenesis. We prepared a library of 164 cDNAs of conserved protein coding genes, injected mRNA into early embryos and evaluated up to 5 days post-fertilization (dpf). Twenty-four genes produced a gross morphological phenotype, 11 of which could be reproduced reliably. Seven of these gave a phenotype consistent with down regulation of the sonic hedgehog (Shh) pathway; two showed defects indicative of defective neural crest migration; one resulted consistently in pericardial edema; and one was embryonic lethal. Combinatorial injections of multiple Hsa21 genes revealed both additive and compensatory effects, supporting the notion that complex genetic relationships underlie end phenotypes of trisomy that produce DS. Together, our data suggest that this system is useful in the genetic dissection of dosage-sensitive gene effects on early development and can inform the contribution of both individual loci and their combinatorial effects to phenotypes relevant to the etiopathology of DS. Copyright 2018 Edie et al.
    • Systematic identification and characterization of cardiac long intergenic noncoding RNAs in zebrafish

      Wang, L.; Ma, X.; Xu, X. (Nature Publishing Group, 2017)
      Long intergenic noncoding RNAs (lincRNAs) are increasingly recognized as potential key regulators of heart development and related diseases, but their identities and functions remain elusive. In this study, we sought to identify and characterize the cardiac lincRNA transcriptome in the experimentally accessible zebrafish model by integrating bioinformatics analysis with experimental validation. By conducting genome-wide RNA sequencing profiling of zebrafish embryonic hearts, adult hearts, and adult muscle, we generated a high-confidence set of 813 cardiac lincRNA transcripts, 423 of which are novel. Among these lincRNAs, 564 are expressed in the embryonic heart, and 730 are expressed in the adult heart, including 2 novel lincRNAs, TCONS-00000891 and TCONS-00028686, which exhibit cardiac-enriched expression patterns in adult heart. Using a method similar to a fetal gene program, we identified 51 lincRNAs with differential expression patterns between embryonic and adult hearts, among which TCONS-00009015 responded to doxorubicin-induced cardiac stress. In summary, our genome-wide systematic identification and characterization of cardiac lincRNAs lays the foundation for future studies in this vertebrate model to elucidate crucial roles for cardiac lincRNAs during heart development and cardiac diseases. Copyright 2017 The Author(s).
    • Transcriptomic profiling of zebrafish hair cells using RiboTag

      Matern, M.S.; Beirl, A.; Ogawa, Y. (Frontiers Media S.A., 2018)
      The zebrafish inner ear organs and lateral line neuromasts are comprised of a variety of cell types, including mechanosensitive hair cells. Zebrafish hair cells are evolutionarily homologous to mammalian hair cells, and have been particularly useful for studying normal hair cell development and function. However, the relative scarcity of hair cells within these complex organs, as well as the difficulty of fine dissection at early developmental time points, makes hair cell-specific gene expression profiling technically challenging. Cell sorting methods, as well as single-cell RNA-Seq, have proved to be very informative in studying hair cell-specific gene expression. However, these methods require that tissues are dissociated, the processing for which can lead to changes in gene expression prior to RNA extraction. To bypass this problem, we have developed a transgenic zebrafish model to evaluate the translatome of the inner ear and lateral line hair cells in their native tissue environment; the Tg(myo6b:RiboTag) zebrafish. This model expresses both GFP and a hemagglutinin (HA) tagged rpl10a gene under control of the myo6b promoter (myo6b:GFP-2A-rpl10a-3xHA), resulting in HA-tagged ribosomes expressed specifically in hair cells. Consequently, intact zebrafish larvae can be used to enrich for actively translated hair cell mRNA via an immunoprecipitation protocol using an antibody for the HA-tag (similar to the RiboTag mice). We demonstrate that this model can be used to reliably enrich for actively translated zebrafish hair cell mRNA. Additionally, we perform a global hair cell translatome analysis using RNA-Seq and show enrichment of known hair cell expressed transcripts and depletion of non-hair cell expressed transcripts in the immunoprecipitated material compared with mRNA extracted from whole fish (input). Our results show that our model can identify novel hair cell expressed genes in intact zebrafish, without inducing changes to gene expression that result from tissue dissociation and delays during cell sorting. Overall, we believe that this model will be highly useful for studying changes in zebrafish hair cell-specific gene expression in response to developmental progression, mutations, as well as hair cell damage by noise or ototoxic drug exposure. Copyright 2018 Matern, Beirl, Ogawa, Song, Paladugu, Kindt and Hertzano.