Abstract
MicroRNAs have been explored in different organisms and are involved as molecular switches modulating cellular specification and differentiation during the embryonic development, including the cardiovascular system. In this study, we analyze the expression profiles of different microRNAs during early cardiac development. By using whole mount in situ hybridization in developing chick embryos, with microRNA-specific LNA probes, we carried out a detailed study of miR-23b, miR-130a, miR-106a, and miR-100 expression during early stages of embryogenesis (HH3 to HH17). We also correlated those findings with putative microRNA target genes by means of mirWalk and TargetScan analyses. Our results demonstrate a dynamic expression pattern in cardiac precursor cells from the primitive streak to the cardiac looping stages for miR-23b, miR-130a, and miR-106a. Additionally, miR-100 is later detectable during cardiac looping stages (HH15-17). Interestingly, the sinus venosus/inflow tract was shown to be the most representative cardiac area for the convergent expression of the four microRNAs. Through in silico analysis we revealed that distinct Hox family members are predicted to be targeted by the above microRNAs. We also identified expression of several Hox genes in the sinus venosus at stages HH11 and HH15. In addition, by means of gain-of-function experiments both in cardiomyoblasts and sinus venosus explants, we demonstrated the modulation of the different Hox clusters, Hoxa, Hoxb, Hoxc, and Hoxd genes, by these microRNAs. Furthermore, we correlated the negative modulation of several Hox genes, such as Hoxa3, Hoxa4, Hoxa5, Hoxc6, or Hoxd4. Finally, we demonstrated through a dual luciferase assay that Hoxa1 is targeted by miR-130a and Hoxa4 is targeted by both miR-23b and miR-106a, supporting a possible role of these microRNAs in Hox gene modulation during differentiation and compartmentalization of the posterior structures of the developing venous pole of the heart.
Highlights
During chick gastrulation the primitive streak precardiac cells invaginate and migrate anterolaterally to form the precardiac mesoderm, between the ectoderm and the inductive adjacent endoderm (Garcia-Martinez and Schoenwolf, 1993; Schultheiss et al, 1995; Garcia-Martinez et al, 1997; Redkar et al, 2001; Lopez-Sanchez et al, 2001, 2009, 2018), at both sides of the embryo, determining the first heart field (FHF; Harvey, 2002)
We have identified that several Hox genes targeted by these microRNAs are expressed in the HH11 and HH15 sinus venosus
During early cardiac looping (HH11), miR-23b is expressed in both inflow and outflow tracts, subsequently being more evident on the dorsal surface of the cardiac sinus venosus and the dorsal mesocardium (HH14)
Summary
During chick gastrulation (stage HH3; Hamburger and Hamilton, 1951, 1992) the primitive streak precardiac cells invaginate and migrate anterolaterally to form the precardiac mesoderm, between the ectoderm and the inductive adjacent endoderm (Garcia-Martinez and Schoenwolf, 1993; Schultheiss et al, 1995; Garcia-Martinez et al, 1997; Redkar et al, 2001; Lopez-Sanchez et al, 2001, 2009, 2018), at both sides of the embryo (stages HH5-7), determining the first heart field (FHF; Harvey, 2002). A complex network of signaling pathways and transcriptional factors is required to regulate early cardiac development In this sense, the FGF, TGF, and Wnt pathways, among others, have been widely involved in early cardiogenesis. RA signaling is required for SHF differentiation, modulating the expression of specific marker genes, including
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