Cloning and characterization of Xenopus Id4 reveals differing roles for Id genes.

We have identified Xenopus Id4, a member of the Id (inhibitor of differentiation/DNA binding) class of helix-loop-helix proteins. Id factors dimerize with general bHLH factors, preventing their interaction with tissue-specific bHLH factors, to inhibit premature differentiation. The presence of several Id proteins could reflect simple redundancy in function, or more interestingly, might suggest different activities for these proteins. During embryonic development, Xenopus Id4 is expressed in a number of neural tissues, including Rohon-Beard neurons, olfactory placode, eye primordia, and the trigeminal ganglia. It is also expressed in other organs, such as the pronephros and liver primordium. As embryogenesis progresses, it is expressed in the migrating melanocytes and lateral line structures. We compare the expression of Id4 mRNA with that of Id2 and Id3 and find that the Id genes are expressed in complementary patterns during neurogenesis, myogenesis, kidney development, in the tailbud, and in the migrating neural crest. To examine the regulation of Id gene expression during Xenopus neural development, we show that expression of Id3 and Id4 can be induced by overexpression of BMP4 in the whole embryo and in ectodermal explants. Expression of Id2, Id3, and Id4 in these explants is unaffected by the expression of FGF-8 or a dominant-negative Ras (N17ras), suggesting that Id genes are not regulated by the FGF signaling pathway in naive ectoderm. We also show that Notch signaling can activate Id2 and Id3 expression in the whole embryo. In contrast, Id4 expression in the Rohon-Beard cells is inhibited by activated Notch and increased by a dominant-negative Delta. This may reflect an increase in Rohon-Beard cells in response to inhibition of Notch signaling rather than transcriptional regulation of Id4. Finally, to compare the activities of Id2, Id3, and Id4, we use animal cap explants and in vivo overexpression to show that Id proteins can differentially inhibit the activities of neurogenin and neuroD, both neurogenic bHLH molecules and MyoD, a myogenic bHLH protein. Id4 is able to inhibit the activity all these bHLH molecules, Id2 inhibits MyoD and neuroD, while Id3 blocks only neuroD activity in our assays.