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Fig. 4. Lhx9 regulates integrin α4-paxillin signaling in PE clusters. (A-D) Whole embryo in situ hybridization for itga4, stages 36 to 46. Lateral views of anterior
portion (left panels) with magnified image of cardiac region (right panels). Red arrowheads indicate PE cluster; asterisks indicates heart. (E,F) In situ
hybridization for itga4 in control (E) and Lhx9-depleted embryos (F) at stage 41. Representative cardiac region shown, anterior to right, dorsal to top; red
arrowhead marks PE cluster. (O) Quantification of observed itga4 expression (reduced expression denoting ≤50% stain intensity compared with controls)
and clustering defects shown in E,F (see Fig. 2M for phenotype assessment), embryos taken from six independent experiments, P≤0.0001 by Chi-square test.
(G-J′) Transverse cardiac region agarose sections from control (G-H′) and Lhx9-depleted (I-J′) stage 41 embryos post-in situ hybridization for tcf21 (G,I), nuclei
expression with DAPI (H,J, blue) and Itgβ1 immunohistochemistry (H′,J′, magenta). White outlines (H,H′,J,J′) indicate PE cluster as depicted by tcf21 and Itgβ1.
(K-N′) Transverse cardiac region agarose sections from control (K-L′) and Lhx9-depleted (M-N′) stage 45 Xla.Tg(Cardiac-actin:GFP)Mohun embryos depicting
representative immunohistochemical analysis for DAPI (blue), GFP (green) to label cardiomyocytes, Itgβ1 (magenta) to label epicardial cells and phosphorylated
Y118-paxillin (red). Magnified images (L,L′,N,N′) from white boxes in K,M. White arrowheads indicate PE cluster. (P) Pixel intensity (integrated density) levels for
phosphorylated Y118-paxillin from five control and 10 Lhx9-depleted embryos; P=0.0134 by two-tailed Student’s t-test. v, ventricle. |
