Expression Level of Hand2 Affects GI Function in Mice
Expression Level of Hand2 Affects GI 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 transferase–mediated 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 > Hand2 > Hand2 . 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.
Hand1 and Hand2, which encode basic helix-loop-helix transcription factors, are expressed in developing gut. Enteric expression of Hand2 in mice is developmentally regulated and restricted to crest-derived cells, while that of Hand1 occurs in muscle and interstitial cells of Cajal. Although deletion of Hand2 does not interfere with the colonization of the bowel by crest-derived cells, these cells are unable to form neurons in vitro. Transfection of enteric crest-derived cells (ENCDC) with small interfering RNA to silence Hand2 in vitro also prevents neuronal differentiation. The Wnt1-Cre−mediated conditional inactivation of Hand2 in migrating crest-derived cells interferes with the terminal differentiation of HuC/D-expressing enteric neurons; nevertheless, enteric crest-derived precursors express early pan-neuronal markers, such as β3-tubulin. It has been suggested that Hand2 expression is required for terminal differentiation of enteric neurons, albeit not for ENCDC to colonize the bowel, commit to a neuronal lineage, or form glia.
Exon 1 of Hand2 was flanked with loxP sites to generate conditional knockout mice. Although Hand2 mice appear normal, a cleft palate develops in Hand2 fetuses, suggesting that flanking intron 1 of the Hand2 gene with loxP sites generates a hypomorphic allele that impairs development, at least of the palate. Similarly, in vitro experiments with small interfering RNA have suggested that there is a minimum essential threshold of Hand2 expression that must be exceeded for terminal differentiation of enteric neurons.
We now show that enteric neurogenesis is a quantitative function of Hand2 expression, which is reduced in the bowel of Hand2 and further reduced in Hand2 mice. Parallel reductions occur in the enteric expression of the neuron-specific gene, HuD, and in the numbers of enteric neurons, especially those containing neuronal nitric oxide synthase (nNOS) and calretinin. The gene-dosage of Hand2 appears to be critical for the acquisition of a normal complement of enteric neurons. These molecular defects have functional consequences. Gastrointestinal motility is reduced in Hand2 haploinsufficient mice, which are also, paradoxically, resistant to intestinal inflammation.
Abstract and Introduction
Abstract
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 transferase–mediated 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 > Hand2 > Hand2 . 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.
Introduction
Hand1 and Hand2, which encode basic helix-loop-helix transcription factors, are expressed in developing gut. Enteric expression of Hand2 in mice is developmentally regulated and restricted to crest-derived cells, while that of Hand1 occurs in muscle and interstitial cells of Cajal. Although deletion of Hand2 does not interfere with the colonization of the bowel by crest-derived cells, these cells are unable to form neurons in vitro. Transfection of enteric crest-derived cells (ENCDC) with small interfering RNA to silence Hand2 in vitro also prevents neuronal differentiation. The Wnt1-Cre−mediated conditional inactivation of Hand2 in migrating crest-derived cells interferes with the terminal differentiation of HuC/D-expressing enteric neurons; nevertheless, enteric crest-derived precursors express early pan-neuronal markers, such as β3-tubulin. It has been suggested that Hand2 expression is required for terminal differentiation of enteric neurons, albeit not for ENCDC to colonize the bowel, commit to a neuronal lineage, or form glia.
Exon 1 of Hand2 was flanked with loxP sites to generate conditional knockout mice. Although Hand2 mice appear normal, a cleft palate develops in Hand2 fetuses, suggesting that flanking intron 1 of the Hand2 gene with loxP sites generates a hypomorphic allele that impairs development, at least of the palate. Similarly, in vitro experiments with small interfering RNA have suggested that there is a minimum essential threshold of Hand2 expression that must be exceeded for terminal differentiation of enteric neurons.
We now show that enteric neurogenesis is a quantitative function of Hand2 expression, which is reduced in the bowel of Hand2 and further reduced in Hand2 mice. Parallel reductions occur in the enteric expression of the neuron-specific gene, HuD, and in the numbers of enteric neurons, especially those containing neuronal nitric oxide synthase (nNOS) and calretinin. The gene-dosage of Hand2 appears to be critical for the acquisition of a normal complement of enteric neurons. These molecular defects have functional consequences. Gastrointestinal motility is reduced in Hand2 haploinsufficient mice, which are also, paradoxically, resistant to intestinal inflammation.
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