UCSF

The earliest transcriptional events during vertebrate heart development are thought to occur during gastrulation. Mesp1, a bHLH transcription factor that binds DNA to regulate gene expression, has been described as an early regulator of mesoderm development. However, the regulation and contribution to heart development by Mesp proteins in vivo remains poorly understood. In this study, we use zebrafish as a model organism to investigate the role of Mespaa, a Mesp1 homolog. We identified regions in the mespaa promoter that specifically drive reporter gene expression in the heart indicating Mespaa's contribution to cardiac cell fate.

To investigate the role of Mespaa, we knocked down mespaa transcripts using morpholinos at the 1-cell stage and found partial to complete reversal of heart laterality. Additionally, overexpression of mespaa mRNA at the 1-cell stage resulted in zebrafish embryos with cardia bifida and tail blisters. To identify downstream target genes of Mespaa, we performed RNA-seq analysis in embryos knocked down for mespaa transcripts during gastrulation and identified the miR-430 locus as a potential target. Interestingly, knockdown or overexpression of miR-430 resulted in similar phenotypes compared to mespaa overexpression or knockdown, respectively. Here we investigate the role of Mespaa and miR-430 in cardiac cell migration to the midline and cardiac L/R asymmetry. We hypothesize that Mespaa regulates miR-430 for proper heart tube formation and laterality. We also investigate downstream signaling pathways involved in cardiac cell migration to the midline (S1P signaling), and in cardiac L/R asymmetry (Nodal signaling). Here, we found a decrease in Nodal antagonist, lefty2, expression in mespaa-MO-injected embryos or miR-430a overexpressing embryos, compared to controls. Additionally, when Mespaa is overexpressed, regulation of Nodal signaling via lefty2 3'UTR increases compared to controls. Together, this suggests that regulation of Nodal signaling occurs through Mespaa and miR-430 for proper L/R heart asymmetry.

Finally, we also found an increase in S1P signaling through s1pr2 levels in miR-430 knocked down embryos or when targeting the 3'UTR miR-430 seed sequence using CRISPR-Cas9. Together this suggests that miR-430 is contributing to heart tube formation through regulation of S1P signaling through S1pr2 which is essential for cardiac migration to the midline.