Abstract Body: Loss of the transcription factor MEF2C leads to segment-specific heart tube defects, despite uniform expression in the developing heart. By embryonic day (E) 9 in Mef2c-null embryos, the prospective right ventricle is absent and the remaining single ventricle is severely hypoplastic, but by contrast, the inflow tract exhibits an expanded mis-patterned morphology. To explain these distinct defects in different segments of the heart tube, we hypothesized that MEF2C controls unique gene regulatory programs in each segment. To address this, we performed a time course of single-nucleus RNA- and ATAC-sequencing from E7.5 to E9 in wild-type and Mef2c-null embryos (n = 2 embryos per timepoint, per genotype; 12 embryos total). We computationally identified segment-specific, MEF2C-dependent enhancers, and validated that seven of eleven candidates demonstrated enhancer activity in the embryonic zebrafish heart (Figure 1; n >= 50 embryos per candidate). Using inferred gene regulatory networks, we uncovered a novel genetic interaction between Mef2c and the nuclear hormone receptor gene Nr2f2. We found that Nr2f2 exhibits increased and aberrant regulatory activity in the transcriptional networks of Mef2c-/- embryos. Specifically, we observed nearly twice as many regulatory interactions between Nr2f2 and putative target genes in the Mef2c-null networks relative to wild-type (Figure 2).

In validating this genetic interaction between Mef2c and Nr2f2, we discovered that compound Mef2c-/-; Nr2f2+/- mutants exhibited rescued expression of genes encoding contractile proteins at E8.5 (e.g. Ttn and Myl2; n = 3-6 embryos per genotype) and improved heart tube morphology by E9.5 (n = 5-7 embryos per genotype), compared to Mef2c-/- embryos (Figure 3). This raises the intriguing possibility that the contractile forces generated by the initiation of beating in the linear heart tube (~E8) are critical for subsequent development and looping of the heart tube. My future work is aimed at uncovering the mechanisms by which beating forces may regulate the progression of cardiomyocyte differentiation and heart tube morphogenesis.

In conclusion, we have shown that MEF2C controls distinct transcriptional networks in the developing heart tube, in part by employing segment-specific enhancers, and have identified a previously unknown genetic interaction between Mef2c and Nr2f2 that underlies normal heart tube development.