miR-1/206 downregulates splicing factor Srsf9 to promote C2C12 differentiation

Background Myogenesis is driven by specific changes in the transcriptome that occur during the different stages of muscle differentiation. In addition to controlled transcriptional transitions, several other post-transcriptional mechanisms direct muscle differentiation. Both alternative splicing and miRNA activity regulate gene expression and production of specialized protein isoforms. Importantly, disruption of either process often results in severe phenotypes as reported for several muscle diseases. Thus, broadening our understanding of the post-transcriptional pathways that operate in muscles will lay the foundation for future therapeutic interventions. Methods We employed bioinformatics analysis in concert with the well-established C2C12 cell system for predicting and validating novel miR-1 and miR-206 targets engaged in muscle differentiation. We used reporter gene assays to test direct miRNA targeting and studied C2C12 cells stably expressing one of the cDNA candidates fused to a heterologous, miRNA-resistant 3&prime; UTR. We monitored effects on differentiation by measuring fusion index, myotube area, and myogenic gene expression during time course differentiation experiments. Results Gene ontology analysis revealed a strongly enriched set of putative miR-1 and miR-206 targets associated with RNA metabolism. Notably, the expression levels of several candidates decreased during C2C12 differentiation. We discovered that the splicing factor Srsf9 is a direct target of both miRNAs during myogenesis. Persistent Srsf9 expression during differentiation impaired myotube formation and blunted induction of the early pro-differentiation factor myogenin as well as the late differentiation marker sarcomeric myosin, Myh8. Conclusions Our data uncover novel miR-1 and miR-206 cellular targets and establish a functional link between the splicing factor Srsf9 and myoblast differentiation. The finding that miRNA-mediated clearance of Srsf9 is a key myogenic event illustrates the coordinated and sophisticated interplay between the diverse components of the gene regulatory network. </div

Sexually dimorphic cardiac adaptation is mediated by Cre expression, independent of estrogen-receptor-α expression

The mammalian heart is a remarkably adaptable organ. In particular, the contractile cells of the heart, the cardiac myocytes can respond to dramatic changes in metabolic and functional demand. Both clinical data and murine genetic studies suggest fundamental differences in male and female cardiac biology, including at the cellular level of the myocyte. In this thesis, I address the clinical question of why cardiovascular disease differs in males and females at the cardiac myocyte level. Specifically, I elucidate the importance and mechanism of estrogen signaling in male and female cardiac myocytes. Upon identifying Estrogen Receptor-α (ERα) as the predominant estrogen receptor in cardiac myocytes, I generated a cardiac myocyte-specific ERα knockout mouse using an established cardiac myocyte-specific Cre recombinase driver. I characterized the molecular and functional consequences of deleting ERα in cardiac myocytes and in doing so uncovered a novel, cardiotoxic effect of Cre recombinase expression in cardiac myocytes. I therefore performed a molecular, functional, and bioinformatic analysis of the most commonly used cardiac myocyte-specific Cre expression model. In parallel with my in vivo experiments, I assessed subcellular localization, signaling activation, and transcriptional competency of full-length ERα and two alternate spliceforms in isolated cardiac myocytes to address the cellular mechanism of ERα activity in cardiac myocytes. Altogether, this thesis sheds light on the sexually dimorphic nature of cardiac myocyte biology and the influence of such on overall cardiovascular function

Ubiquitous transgene expression and Cre-based recombination driven by the ubiquitin promoter in zebrafish

Molecular genetics approaches in zebrafish research are hampered by the lack of a ubiquitous transgene driver element that is active at all developmental stages. Here, we report the isolation and characterization of the zebrafish ubiquitin (ubi) promoter, which drives constitutive transgene expression during all developmental stages and analyzed adult organs. Notably, ubi expresses in all blood cell lineages, and we demonstrate the application of ubi-driven fluorophore transgenics in hematopoietic transplantation experiments to assess true multilineage potential of engrafted cells. We further generated transgenic zebrafish that express ubiquitous 4-hydroxytamoxifen-controlled Cre recombinase activity from a $ubi:cre^{ERt2}$ transgene, as well as ubi:loxP-EGFP-loxP-mCherry (ubi:Switch) transgenics and show their use as a constitutive fluorescent lineage tracing reagent. The ubi promoter and the transgenic lines presented here thus provide a broad resource and important advancement for transgenic applications in zebrafish.Stem Cell and Regenerative Biolog

The Effect of a Depth Gradient on the Mating Behavior, Oviposition Site Preference, and Embryo Production in the Zebrafish, Danio rerio

Captive zebrafish (Danio rerio) exhibit a limited repertoire of mating behaviors, likely due to the somewhat unnatural environment of aquaria. Observations in their natural habitat led us to believe that a depth gradient within the mating setup would positively affect fish mating. By tilting the tank to produce a depth gradient, we observed novel behaviors along with a preference for oviposition in the shallow area. Although we did not see an increase in the likelihood of a pair of fish to mate, we did see an increase in the embryo output in both adults and juveniles. In the adults, tilting led to a significant increase in embryo production (436 ± 35 tilted vs. 362 ± 34 untilted; p < 0.05). A similar effect was seen in juvenile fish as they progressed through sexual maturity. These results suggest that tilting of mating cages in the laboratory setting will lead to demonstrable improvements in embryo production for zebrafish researchers, and highlights the possibility of other manipulations to increase fecundity

Epoxyeicosatrienoic Acids Enhance Embryonic Haematopoiesis and Adult Marrow Engraftment

Haematopoietic stem and progenitor cell (HSPC) transplant is a widely used treatment for life-threatening conditions including leukemia; however, the molecular mechanisms regulating HSPC engraftment of the recipient niche remain incompletely understood. Here, we developed a competitive HSPC transplant method in adult zebrafish, using in vivo imaging as a non-invasive readout. We used this system to conduct a chemical screen and identified epoxyeicosatrienoic acids (EET) as a family of lipids1,2 that enhance HSPC engraftment. EETs’ pro-haematopoietic effects were conserved in the developing zebrafish embryo, where 11,12-EET promoted HSPC specification by activating a unique AP-1/runx1 transcription program autonomous to the haemogenic endothelium. This effect required the activation of the PI3K pathway, specifically PI3Kγ. In adult HSPCs, 11,12-EET induced transcriptional programs, including AP-1 activation, which modulate multiple cellular processes, such as migration, to promote engraftment. Finally, we demonstrated that the EET effects on enhancing HSPC homing and engraftment are conserved in mammals. Our study established a novel method to explore the molecular mechanisms of HSPC engraftment, and discovered a previously unrecognized, evolutionarily conserved pathway regulating multiple haematopoietic generation and regeneration processes. EETs may have clinical application in marrow or cord blood transplantation