Expression of HAND Gene Products May Be Sufficient for the Differentiation of Avian Neural Crest-Derived Cells into Catecholaminergic Neurons in Culture

Abstract

Members of the basic helix-loop-helix family of DNA binding proteins have important roles in the development of subpopulations of neural crest-derived neurons. We have cloned the chicken homologues of dHAND (HAND2) and eHAND (HAND1), basic helix-loop-helix DNA binding proteins whose neuronal expression is restricted to sympathetic and enteric neural crest-derived ganglia. Transcripts encoding dHAND and eHAND are expressed in sympathetic ganglia beginning at Hamburger and Hamilton stage 17–18. Antisense blockade of transcripts encoding HAND genes in neural crest-derived cells in vitro results in a significant reduction in neurogenesis. Differentiation of catecholaminergic neurons is also reduced by 52% if the expression of transcripts encoding dHAND and eHAND is reduced using antisense oligonucleotide blockade. The effect on neurogenesis and phenotypic expression of neural crest-derived neurons is specific; blockade of HAND gene expression has no apparent influence on the differentiation in vitro of neural tube-derived neurons. Use of a replication-competent avian retrovirus to constitutively express HAND genes in neural crest-derived cells in vitro, under nonpermissive growth conditions in medium supplemented with 2% chick embryo extract (CEE), induced precocious catecholaminergic differentiation. Constitutive expression of HAND gene products resulted in a significant increase in catecholaminergic differentiation of cells grown in medium supplemented with 10% CEE, a permissive growth condition for catecholaminergic development. These results suggest that the expression by neural crest cells of dHAND and eHAND may be both sufficient and necessary for catecholaminergic phenotypic expression.