Deletion of a Hand1 lncRNA-Containing Septum Transversum Enhancer Alters lncRNA Expression but Is Not Required for Hand1 Expression

We have previously identified a Hand1 transcriptional enhancer that drives expression within the septum transversum, the origin of the cells that contribute to the epicardium. This enhancer directly overlaps a common exon of a predicted family of long non-coding RNAs (lncRNA) that are specific to mice. To interrogate the necessity of this Hand1 enhancer, as well as the importance of these novel lncRNAs, we deleted the enhancer sequences, including the common exon shared by these lncRNAs, using genome editing. Resultant homozygous Hand1 enhancer mutants (Hand1(Δ)(ST/ΔST)) present with no observable phenotype. Assessment of lncRNA expression reveals that Hand1(Δ)(ST/ΔST) mutants effectively eliminate detectable lncRNA expression. Expression analysis within Hand1(Δ)(ST/ΔST) mutant hearts indicates higher levels of Hand1 than in controls. The generation of Hand1 compound heterozygous mutants with the Hand1(LacZ) null allele (Hand1(Δ)(ST/LacZ)) also did not reveal any observable phenotypes. Together these data indicate that deletion of this Hand1 enhancer and by consequence a family of murine-specific lncRNAs does not impact embryonic development in observable ways.