Figure 8. Effect of Bmpr1a knock-in into Acvr1 locus (Bmpr1a-KI) on gene expression and OFT morphology.
A, qRT-PCR on E9 RV+OFT: controls (no Cre, no Bmpr1a-KI), blue columns; Acvr1-cKO with Bmpr1a-KI mutants (ie Acvr1F, Bmpr1a-KI, Mef2c[AHF]-Cre+), purple columns; and B, Acvr1-cKO (red columns) vs Acvr1-cKO with Bmpr1a-KI (purple columns) showing that when Acvr1 was replaced by expression from one allele of Bmpr1a cDNA in the Acvr1 locus average Tbx1 and Wnt2 expression became normal, and Tgfb2 expression was over rather than under expressed (arrows) but expression of other genes shown remained only partially normalized (Smad6) or abnormal in a similar way to Acvr1 cKO. Each bar average of 3, +/− SEM, * p≤0.05.
E12 4-chamber views: Gross morphology of Control, mild and severe Acvr1-cKO and Bmpr1a-cKO without (C, Ja, Jb, Q) and with Bmpr1a-KI (D, Ka, Kb, R) dissected to reveal trunks where possible. Presence of Bmpr1a-KI in Acvr1-cKO improved morphology of RV, OFT in some (Ka vs Ja) but not all (Kb vs Jb) embryos, enlarging RV size and improving OFT orientation (R vs Q, green area) in Bmpr1a-cKO. Blood in arterial trunks shows septation in controls and mild Acvr1-cKO (Ka).
E12 sections, transverse to OFT, H&E: Control (I-U), Acvr1-cKO mild (Ja-Pa) and severe (Jb-Pb) phenotype and Bmpr1a-cKO (Q-W), all withBmpr1a-KI. OFT, from arterial trunk level (top row, sections) to proximal OFT (bottom row). In presence of Bmpr1a-KI, all cKO showed some improved wall thickness and cushion organization; but uneven wall thickness and distal epicardium (ep) was still seen where distal septation failed. In mild Acvr1-cKO with Bmpr1a-KI, trunk septation and mid-OFT cushion formation including condensed areas (blue *, arrows), myocardial growth into cushion (red arrowhead) more similar to control, but proximal cushion morphology (black*) and continuity with AV cushion (open arrow, Pa) absent. In the severe mutant with Bmpr1-KI, trunk septation failed (Lb), uneven trunk wall (Lb, short arrow), RVO lumen displaced (open arrowhead), distal (Mb) and proximal cushions abnormal (Pb), mid- to proximal OFT anterior wall too thick (red lines), some wall very thin (arrowheads Mb, Nb) but mid-OFT orientation relatively normal. In Bmpr1-cko with Bmpr1a-KI, trunk septation only partial, uneven trunk wall (K, short arrows), separate RVO lumen lost (open arrow) but mid- and proximal orientation, condensation (blue arrows), intercalated leaflet (green*) and proximal cushion continuity with AV cushion (W, black arrow), wall thickness except proximally (red lines) similar to control. Compare with Acvr1- and Bmpr1a-cKO E12 morphology in Figures 1–2 and Online Figures 2–4. Spaces between cushions and walls are artifacts.
Ao, ‘aortic’ lumen; AV, atrioventricular cushion; ep, epicardium; my, myocardium; P, ‘pulmonary’ lumen. Scale bar, 200µm.