Mauricio X. Zuber

Sodium dodecyl sulfate (SDS)-based whole-mount in situ hybridization of Xenopus laevis embryos.

We describe a streamlined whole-mount in situ hybridization protocol that utilizes high concentrations of the detergent sodium dodecyl sulfate (SDS). Our results suggest that SDS is an effective blocking agent in Xenopus laevis embryos which permeabilizes membranes without disrupting morphology. Consequently, riboprobes appeared to disperse uniformly within the embryo and several arduous and/or laborious steps of conventional procedures could be eliminated without compromising the technique.

Expression of basic-helix-loop-helix transcription factor ME2 during brain development and in the regions of neuronal plasticity in the adult brain☆

We report the isolation of a cDNA encoding the mouse class A bHLH transcription factor ME2 and the analysis of its expression. ME2 is expressed in the cerebral cortex, Purkinje and granule cell layers of the cerebellum, olfactory neuroepithelium, pyramidal cells of hippocampal layers CA1-CA4, and in the granular cells of the dentate gyrus. The specific expression of ME2 during development and in the regions of neuronal plasticity in the adult brain suggest that ME2 may have a regulatory function in developmental processes as well as during neuronal plasticity.

Differential expression and distinct DNA-binding specificity of ME1a and ME2 suggest a unique role during differentiation and neuronal plasticity

Class A basic-helix-loop-helix (bHLH) proteins have been referred to as ubiquitous and are believed to have redundant functions. They are involved in the control of several developmental pathways, such as neurogenesis and myogenesis. To rationalize the existence of multiple class A bHLH proteins, we evaluated the differences and similarities between ME1a and ME2, two class A bHLH proteins, highly expressed in differentiating neuronal cells. In situ hybridization analyses reveal that ME1a and ME2 are characterized by distinguishable patterns of expression in areas of the adult mouse brain where neuronal plasticity occurs. Also, DNA-binding assays show that both proteins bind to E-boxes as homodimers and heterodimers, and show differences in their DNA-binding specificities, which suggest selective interactions with different binding sites of target genes. In addition, in vitro DNA-binding assays demonstrate that Id2 forms heterodimers with ME1a and ME2. As a result of these interactions, their DNA-binding activity is abolished. Furthermore, overexpression of Id2 in neuronal cells suppresses ME1a and ME2 transcriptional activity. Based on our data, we hypothesize that ME1a and ME2 may activate gene expression of different target genes and therefore are likely to be differently involved during neurogenesis.

Cloning and embryonic expression ofXenopus laevis GAP-43 (XGAP-43)

Xenopus laevis GAP-43 (XGAP-43) is highly related to other vertebrate GAP-43 proteins in its N-terminal region which contains a membrane-targeting sequence, serine phosphorylation site, and calmodulin binding domain. Unlike other species examined, however, there appear to be two GAP-43-class genes in X. laevis which resulted from the genome duplication in Xenopus approximately 30 million years ago. During embryogenesis, XGAP-43 is expressed in a complex spatiotemporal pattern that is consistent with its putative role in neuronal growth and development.

Selective conservation of an E-protein gene promoter during vertebrate evolution

The murine E‐protein gene ME1 encodes a non‐tissue‐specific, helix‐loop‐helix transcription factor that is associated with morphological development. ME1 gene expression is regulated by a TATA‐less promoter that contains multiple Sp1 consensus elements, E‐boxes, and a novel transcription initiation site. In this study, we compared DNA homologous to the ME1 promoter from vertebrate species ranging from frog to human. A region of striking sequence similarity was identified in a region corresponding to the ME1 transcription initiation site (ME1 Inr). Within this region, a poly d(A) tract and a 9‐bp inverted repeat (5′‐GTCCGCCTG) were highly conserved in all species that were examined. Protein complexes that recognized these DNA elements were present among distant vertebrates (frog, chick, monkey and human), and were able to bend the ME1 Inr to a similar extent (∼60°) as the previously described murine MBPα and MBPβ proteins. Collectively, these results suggest that an ME1 Inr‐like element and its associated proteins functioned in an ancestral vertebrate more than 350 million years ago.

Identification of non-tissue-specific helix-loop-helix genes in Xenopus laevis ☆

We have isolated four non-tissue-specific helix-loop-helix (HLH) transcription factors from Xenopus laevis (Xl). While some are clearly orthologous to known mammalian HLH proteins, others have dramatic amino-acid differences in otherwise highly conserved protein domains. We propose that these changes arose following the tetraploidization of Xl approx. 30 Myr ago.