A Novel Drosophila, mef2-Regulated Muscle Gene Isolated in a Subtractive Hybridisation-Based Molecular Screen Using Small Amounts of Zygotic Mutant RNA

Abstract

It is unknown how the general patterning mechanisms that subdivide the mesoderm initiate different pathways of cell differentiation. One route to understanding these events is to isolate and analyse genes specifically expressed early in this differentiation process. I have therefore undertaken a novel molecular screen in Drosophila in a systematic search for such genes. The approach utilised subtractive hybridisation coupled to directional cDNA library construction. Libraries were made from as little as 20 μg total RNA isolated from hand-picked embryos of defined stage of development and genotype. In a one-step procedure, the subtraction was 6.5- to 7.25-h wild-type embryos minus 6.5- to 7.25-h twist (twi) zygotic mutant embryos. A two-step procedure in which maternally expressed sequences were subtracted from each of these cDNA libraries, before subtracting twi from wild-type, increased the subtraction efficiency. It resulted in a cDNA population enriched more than 100-fold for mesodermal cDNAs. This was screened by determination of the embryonic expression pattern of each clone in a high throughput procedure and then DNA sequencing. The method, which is comprehensive and does not discriminate against rarer cDNAs, is generally applicable and calculations show that it would work for just 10 embryos. Analysis of one clone, Dmeso18E, that encodes a putative nuclear protein and fulfils the screen's aims is described. It is novel and its expression is mesoderm-specific, twi-dependent, and early during somatic, visceral, and heart muscle differentiation. Two pivotal regulators of mesoderm development and gene expression are Dmef2 and tinman (tin). Analysis of Dmeso18E expression revealed new aspects to their roles: there are effects of Dmef2 on developing muscle much earlier than hitherto believed, and there is tin-independent gene expression in, and invagination of, prospective midgut visceral muscle cells. Dmeso18E expression is regulated by Dmef2, although some expression is Dmef2-independent. The tin-independent and Dmef2-independent expression of Dmeso18E indicates that it either occupies a link between twi and genes like tin and Dmef2 or it lies in a parallel pathway of gene activation.