Fig. 6

gata6 positively regulates bmp4 and shha expression, and negatively regulates epithelial and smooth muscle proliferation. (A–F): Expression of bmp4, shha and ihha is decreased in the gut of gata6 morphants at 96 hpf by in situ hybridization (A–F) and qPCR of embryonic trunk (G) as compared to wild type (WT). A2–F2 are longitudinal sections of stained embryos in the midgut region. (H–I) Transverse sections of the gut show strong EDU staining in both epithelial and smooth muscle layers in WT embryos (H) and gata6 morphants (I) at 96 hpf. (J) Quantification of EdU positive epithelial and smooth muscle cells shows significantly increased proliferative cells in both layers of the gata6 morphant gut. (* indicates p<0.05; ** indicates p<0.01). (K) Model for miR-145 regulation of visceral paracrine pathways. The visceral smooth muscle cells (SMCs) are shown in light blue and the gut epithelium in dark green. In wild type embryos, Hedgehog family members are expressed in the epithelium and responsible for the induction of the bmp4 in the adjacent smooth muscle layers. MiR-145 normally inhibits expression of gata6, maintaining low bmp4 expression levels in smooth muscle cells. When miR-145 is downregulated, gata6 expression increases, promotes bmp4 expression, and via paracrine secretion, activates higher expression of both shh and ihh in the epithelial layer in a positive feedback loop, leading to decrease proliferation of both layers. The magnification for A–F is shown in F, for A2–F2 is shown in F2 and for H, I is shown in I.