The long noncoding RNAHand2asorchestrates heart development through regulation of precise expression ofHAND2

Rigorous exploration and dissection of potential actions and effects of long noncoding RNA (lncRNA) in animals remain challenging. Here using multiple knockout mouse models and single- cell RNA sequencing, we demonstrate that the divergent lncRNA Hand2as has a key, complex modulatory effect on the expression of its neighboring gene HAND2 and subsequently on heart development and function, largely independent of Hand2as transcription and transcripts. Full-length deletion of Hand2as in mouse causes moderate yet prevalent upregulation of HAND2 in hundreds of cardiac cells, resulting in profound biological consequences, including dysregulated cardiac gene programs, congenital heart defects and perinatal lethality. We propose a cis -functional role for the Hand2as locus in dampening HAND2 expression to restrain cardiomyocyte proliferation, thereby orchestrating a balanced development of cardiac cell lineages. This study highlights the need for complementary genetic and single-cell approaches to delineate the function and primary molecular effects of an lncRNA in animals. Impact statement The long noncoding RNA Hand2as critically controls the precise expression of its neighboring gene HAND2 , thereby balancing cardiac lineages and expression programs that are essential for heart development and function.