Down-regulation of inhibitor of apoptosis levels provides competence for steroid-triggered cell death

A pulse of the steroid hormone ecdysone triggers the destruction of larval salivary glands during Drosophila metamorphosis through a transcriptional cascade that converges on reaper (rpr) and head involution defective (hid) induction, resulting in caspase activation and cell death. We identify the CREB binding protein (CBP) transcriptional cofactor as essential for salivary gland cell death. We show that CBP acts 1 d before the onset of metamorphosis in apparent response to a mid-third instar ecdysone pulse, when CBP is necessary and sufficient for down-regulation of the Drosophila inhibitor of apoptosis 1 (DIAP1). It is only after DIAP1 levels are reduced that salivary glands become competent to die through rpr/hid-mediated cell death. Before this time, high levels of DIAP1 block salivary gland cell death, even in the presence of ectopic rpr expression. This study shows that naturally occurring changes in inhibitor of apoptosis levels can be critical for regulating cell death during development. It also provides a molecular mechanism for the acquisition of competence in steroid signaling pathways

Leucine-Rich Repeat Containing Protein LRRC8A Is Essential for Swelling-Activated Cl\u3csup\u3e−\u3c/sup\u3e Currents and Embryonic Development in Zebrafish

Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society. A volume-regulated anion channel (VRAC) has been electrophysiologically characterized in innumerable mammalian cell types. VRAC is activated by cell swelling and mediates the volume regulatory efflux of Cl− and small organic solutes from cells. Two groups recently identified the mammalian leucine-rich repeat containing protein LRRC8A as an essential VRAC component. LRRC8A must be coexpressed with at least one of the other four members of this gene family, LRRC8B-E, to reconstitute VRAC activity in LRRC8−/− cells. LRRC8 genes likely arose with the origin of chordates. We identified LRRC8A and LRRC8C-E orthologs in the zebrafish genome and demonstrate that zebrafish embryo cells and differentiated adult cell types express a swelling-activated Cl− current indistinguishable from mammalian VRAC currents. Embryo cell VRAC currents are virtually eliminated by morpholino knockdown of the zebrafish LRRC8A ortholog lrrc8aa. VRAC activity is fully reconstituted in LRRC8−/− human cells by coexpression of zebrafish lrrc8aa and human LRRC8C cDNAs. lrrc8aa expression varies during zebrafish embryogenesis and lrrc8aa knockdown causes pericardial edema and defects in trunk elongation and somatogenesis. Our studies provide confirmation of the importance of LRRC8A in VRAC activity and establish the zebrafish as a model system for characterizing the molecular regulation and physiological roles of VRAC and LRRC8 proteins

Emerging Roles for Immune Cells and MicroRNAs in Modulating the Response to Cardiac Injury

Stimulating cardiomyocyte regeneration after an acute injury remains the central goal in cardiovascular regenerative biology. While adult mammals respond to cardiac damage with deposition of rigid scar tissue, adult zebrafish and salamander unleash a regenerative program that culminates in new cardiomyocyte formation, resolution of scar tissue, and recovery of heart function. Recent studies have shown that immune cells are key to regulating pro-inflammatory and pro-regenerative signals that shift the injury microenvironment toward regeneration. Defining the genetic regulators that control the dynamic interplay between immune cells and injured cardiac tissue is crucial to decoding the endogenous mechanism of heart regeneration. In this review, we discuss our current understanding of the extent that macrophage and regulatory T cells influence cardiomyocyte proliferation and how microRNAs (miRNAs) regulate their activity in the injured heart

A Conserved MicroRNA Regulatory Circuit Is Differentially Controlled during Limb/Appendage Regeneration.

Although regenerative capacity is evident throughout the animal kingdom, it is not equally distributed throughout evolution. For instance, complex limb/appendage regeneration is muted in mammals but enhanced in amphibians and teleosts. The defining characteristic of limb/appendage regenerative systems is the formation of a dedifferentiated tissue, termed blastema, which serves as the progenitor reservoir for regenerating tissues. In order to identify a genetic signature that accompanies blastema formation, we employ next-generation sequencing to identify shared, differentially regulated mRNAs and noncoding RNAs in three different, highly regenerative animal systems: zebrafish caudal fins, bichir pectoral fins and axolotl forelimbs.These studies identified a core group of 5 microRNAs (miRNAs) that were commonly upregulated and 5 miRNAs that were commonly downregulated, as well as 4 novel tRNAs fragments with sequences conserved with humans. To understand the potential function of these miRNAs, we built a network of 1,550 commonly differentially expressed mRNAs that had functional relationships to 11 orthologous blastema-associated genes. As miR-21 was the most highly upregulated and most highly expressed miRNA in all three models, we validated the expression of known target genes, including the tumor suppressor, pdcd4, and TGFβ receptor subunit, tgfbr2 and novel putative target genes such as the anti-apoptotic factor, bcl2l13, Choline kinase alpha, chka and the regulator of G-protein signaling, rgs5.Our extensive analysis of RNA-seq transcriptome profiling studies in three regenerative animal models, that diverged in evolution ~420 million years ago, reveals a common miRNA-regulated genetic network of blastema genes. These comparative studies extend our current understanding of limb/appendage regeneration by identifying previously unassociated blastema genes and the extensive regulation by miRNAs, which could serve as a foundation for future functional studies to examine the process of natural cellular reprogramming in an injury context

Eleven blastema-associated genes significantly differentially expressed during regenerating zebrafish caudal fin, bichir pectoral fins and axolotl forelimbs.

<p>Eleven blastema-associated genes significantly differentially expressed during regenerating zebrafish caudal fin, bichir pectoral fins and axolotl forelimbs.</p

Expression of miRNAs following blastema formation in regenerating bichir pectoral fins and axolotl forelimbs with log₂(fold change) greater than +0.58 or less than -0.58 sorted by descending fold change.

<p>The first set of miRNAs were those that were commonly up- or down-regulated in bichir and axolotl. The second and third sets of miRNAs were those that were up- or down-regulated in bichir or axolotl, but not commonly up- or down-regulated.</p

Expression of miRNAs during blastema formation in zebrafish caudal fin, bichir pectoral fins and axolotl forelimbs.

<p>Expression of miRNAs during blastema formation in zebrafish caudal fin, bichir pectoral fins and axolotl forelimbs.</p

Vertebrate models used to study limb/appendage regeneration.

<p><b>(a)</b> Phylogenetic relationship among three vertebrate taxa studied to determine conserved gene expression patterns in regenerating limb/appendages following blastema formation. These animal systems last shared a common ancestor ~420 million years ago. <b>(b)</b> Blastema tissues as shown by hematoxylin and eosin staining on paraffin tissue sections of regenerating zebrafish caudal fin, bichir pectoral fins and axolotl forelimbs. (* = blastema; dpa = days post-amputation).</p

miRNA expression profiling by small RNA sequencing.

<p><b>(a)</b> Overview of shared and unique miRNAs detected in regenerating limb/appendages following blastema formation in zebrafish caudal fin, bichir pectoral fins and axolotl forelimbs. <b>(b)</b> Gene expression patterns of differentially expressed zebrafish miRNAs shown as a heat map of log₂-transformed read counts per million, in triplicate. <b>(c)</b> Relationship among fold-change and average level of expression for each miRNA in each taxa with miR-21 and miR-133. (dpa = days post-amputation).</p