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Fig. 2. Lhx9 is required for proper epicardial layer and PE cluster formation. Epicardial layer formation (as shown with tbx18) analysis in control (A,C) and
Lhx9-depleted (B,D) embryos at stage 45. (A,B) Images show lateral view of cardiac region, dorsal to the top and anterior to right. (C,D) Transverse gelatin
sections through representative embryo cardiac regions showing tbx18 expression, dorsal to the top. PE cell clustering (red arrowheads) and epicardial cell layer
on the ventricular surface is shown. (E) Epicardial layer formation defects were quantified as abnormal when≤50% of the ventricle is covered, as depicted in
schematic. (F) Quantification of observed epicardial layer formation defects, as represented in A-D, from three independent experiments, P≤0.0001 by two-tailed
Fisher’s exact test. Proepicardial clustering morphology was analyzed using in situ hybridization for tbx18 in control (G,I,K) and Lhx9-depleted (H,J,L) embryos at
stage 38 (G,H), stage 41 (I,J) and stage 42 (K,L). Ventral view showing cardiac region, anterior to the top. Tbx18 expression is detected as a cluster of cells in
control embryos on the right of the embryo near the atrioventricular sulcus (I,K, red arrowheads), whereas in Lhx9-depleted embryos, the cluster is either not
detected and tbx18 expression remains throughout the septum transversum region or is mis-positioned to the caudal side of the heart (abnormal clustering) (J,L,
green arrowheads). (M) Quantification of PE clustering defects is depicted in the schematic as being abnormal by bilateral tbx18 expression retention on septum
transversum and/or cluster mis-positioning of ≥45° caudal to the AVS compared with controls. (N) Quantification of observed clustering phenotype at stage 41
represented in C and D. Data taken from seven independent experiments, P≤0.0001 by two-tailed Fisher’s exact test. (O-T) In situ hybridization analysis for tcf21
(O,P,U) and wt1 (Q-T) proepicardial expression at stage 41. Images depict lateral view of the cardiac region, dorsal to the top and anterior to the right (M-P) or
ventral view, dorsal to top (Q,R). Red arrowheads indicate clustered PE cells. (U) Quantification of observed phenotypes as depicted in M, data taken from six
independent experiments, P≤0.0001 by two-tailed Fisher’s exact test. avs, atrioventricular sulcus; pe/peo, proepicardial organ; st, septum transversum; v,
ventricle. |
