Misexpression of basic helix-loop-helix genes in the murine cerebral cortex affects cell fate choices and neuronal survival

To investigate the role(s) of basic helix-loop-helix genes (bHLH) genes in the developing murine cerebral cortex, Mash1, Math2, Math3, Neurogenin1 (Ngn1), Ngn2, NeuroD, NeuroD2 and Id1 were transduced in vivo into the embryonic and postnatal cerebral cortex using retrovirus vectors. The morphology and location of infected cells were analyzed at postnatal stages. The data indicate that a subset of bHLH genes are capable of regulating the choice of neuronal versus glial fate and that, when misexpressed, they can be deleterious to the survival of differentiating neurons, but not glia.     

A network of yeast basic helix-loop-helix interactions

The Ino4 protein belongs to the basic helix–loop–helix (bHLH) family of proteins. It is known to form a dimer with Ino2p, which regulates phospholipid biosynthetic genes. Mammalian bHLH proteins have been shown to form multiple dimer combinations. However, this flexibility in dimerization had not been documented for yeast bHLH proteins. Using the yeast two-hybrid assay and a biochemical assay we show that Ino4p dimerizes with the Pho4p, Rtg1p, Rtg3p and Sgc1p bHLH proteins. Screening a yeast cDNA library identified three additional proteins that interact with Ino4p: Bck2p, YLR422W and YNR064C. The interaction with Bck2p prompted us to examine if any of the Bck2p-associated functions affect expression of phospholipid biosynthetic genes. We found that hyperosmotic growth conditions altered the growth phase regulation of a phospholipid biosynthetic gene, CHO1. There are two recent reports of initial whole genome yeast two-hybrid interactions. Interestingly, one of these reports identified five proteins that interact with Ino4p: Ino2p, Hcs1p, Apl2p, YMR317W and YNL279W. Ino2p is the only protein in common with the data presented here. Our finding that Ino4p interacts with five bHLH proteins suggests that Ino4p is likely to be a central player in the coordination of multiple biological processes.     

Roles of homeobox and bHLH genes in specification of a retinal cell type

Previous analysis of mutant mice has revealed that the bHLH genes Mash1 and Math3, and the homeobox gene Chx10 are essential for generation of bipolar cells, the interneurons present in the inner nuclear layer of the retina. Thus, a combination of the bHLH and homeobox genes should be important for bipolar cell genesis, but the exact functions of each gene remain largely unknown. We have found that in Mash1-Math3 double-mutant retina, which exhibits a complete loss of bipolar cells, Chx10 expression did not disappear but remained in Müller glial cells, suggesting that Chx10 expression per se is compatible with gliogenesis. In agreement with this, misexpression of Chx10 alone with retrovirus in the retinal explant cultures induced generation of the inner nuclear layer cells, including Müller glia, but few of them were mature bipolar cells. Misexpression of Mash1 or Math3 alone did not promote bipolar cell genesis either, but inhibited Müller gliogenesis. In contrast, misexpression of Mash1 or Math3 together with Chx10 increased the population of mature bipolar cells and decreased that of Müller glia. Thus, the homeobox gene provides the inner nuclear layer-specific identity while the bHLH genes regulate the neuronal versus glial fate determination, and these two classes of genes together specify the bipolar cell fate. Moreover, Mash1 and Math3 promoted the bipolar cell fate, but not the other inner nuclear layer-specific neuronal subtypes in the presence of Chx10, raising the possibility that the bHLH genes may be involved in neuronal subtype specification, in addition to simply making the neuronal versus glial fate choice.