Expression level of Hand2 affects specification of enteric neurons and gastrointestinal function in mice

Background & Aims: Hand2 is a basic helix-loop-helix transcription factor required for terminal differentiation of enteric neurons. We studied Hand2 haploinsufficient mice, to determine whether reduced expression of Hand2 allows sufficient enteric neurogenesis for survival, but not for development of a normal enteric nervous system (ENS). Methods: Enteric transcripts that encode Hand2 and the neuron-specific embryonic lethal abnormal vision proteins HuB, HuC, and HuD were quantified. Immunocytochemistry was used to identify and quantify neurons. Apoptosis was analyzed with the terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate nick-end labeling procedure. Intracellular microelectrodes were used to record inhibitory junction potentials. Gastrointestinal transit and colonic motility were measured in vivo. Results: Levels of of enteric Hand2 transcripts were associated with genotypes of mice, in the following order: Hand2^+/+ > Hand2^LoxP/+ > Hand2^+/- > Hand2^LoxP/-. Parallel reductions were found in expression of HuD and in regional and phenotypic manners. Numbers of neurons, numbers of neuronal nitric oxide synthase⁺ and calretinin⁺, but not substance P⁺ or vasoactive intestinal peptide⁺ neurons, decreased. No effects were observed in stomach or cecum. Apoptosis was not detected, consistent with the concept that Hand2 inhibits neuronal differentiation, rather than regulates survival. The amplitude of inhibitory junction potentials in colonic circular muscle was similar in Hand2 wild-type and haploinsufficient mice, although in haploinsufficient mice, the purinergic component was reduced and a nitrergic component appeared. The abnormal ENS of haploinsufficient mice slowed gastrointestinal motility but protected mice against colitis. Conclusions: Reduced expression of factors required for development of the ENS can cause defects in the ENS that are subtle enough to escape detection yet cause significant abnormalities in bowel function.