slouch

DEVELOPMENTAL BIOLOGY

Embryonic

slouch is expressed in several regions: in small subsets of cells in the somatic mesoderm, in specific muscle founder cells, in the CNS, and in a small region of the endoderm. slouch is first expressed at midstage 11, during the third mesodermal mitosis. It is restricted to muscle precursors. At stage 15, after the yolk sac has been surrounded by the fusion of anterior and posterior primordia of the midgut, slouch is expressed in a narrow band of endodermal cells. Expression in the CNS starts during early stage 11, in very specific neural precursors (Dohrmann, 1990).

Terminal divisions of myogenic lineages in the Drosophila embryo generate sibling myoblasts that act as founders for larval muscles or form precursors of adult muscles. The formation of individual muscle fibers is seeded by a special class of founder myoblasts that fuse with neighboring mesodermal cells to form the syncytial precursors of particular muscle. Alternative fates adopted by sibling myoblasts are associated with distinct patterns of gene expression. During normal development (embryonic stage 11), two ventrally located progenitor cells divide once to produce three muscle founders and the precursor of an adult muscle (known as a persistent Twist cell because of its continued expression of twist). The more dorsal of the two progenitors divides, first giving rise to the founders of muscles VA1 and VA2, followed by the more ventral progenitors which produce the VA3 founder and the ventral adult persistent Twist precursor (VaP). As the progenitors divide, Numb is included in one of the two dorsal progenitors and in one of the two ventral progenitors. Thus the division of a muscle progenitor produces an unequal distribution of Numb between the founders: one contains Numb, the other does not. In numb mutants, some muscles are lost and others are transformed. For example VA1 and VaP are duplicated and VA2 and VA3 are lost. Genes expressed in the progenitor cell are maintained in one sibling and repressed in the other. Kruppel, S59 and even skipped expression mark a subset of the developing muscles. In numb mutants the expression of Kruppel, S59 and even skipped is initiated normally but is lost from both founder cells after they are formed. Thus in numb mutants there are no muscles that express Kr, eve or S59. In contrast, when numb is ectopically expressed throughout the mesoderm, Kr, S59 and eve expression are maintained in both founders and in the muscle precursors to which they give rise. In these embryos, Kr, S59 and eve-expressing muscles are duplicated (Gomez, 1997).

Effects of Mutation or Deletion

The expression of the MyoD gene homolog, nautilus (nau), in the Drosophila embryo defines a subset of mesodermal cells known as the muscle 'pioneer' or 'founder' cells. These cells are thought to establish the future muscle pattern in each hemisegment. Founders appear to recruit fusion-competent mesodermal cells to establish a particular muscle fiber type. In support of this concept every somatic muscle in the embryo is associated with one or more nautilus-positive cells. However, because of the lack of known (isolated) nautilus mutations, no direct test of the founder cell hypothesis has been possible. Toxin ablation and genetic interference by double-stranded RNA (RNA interference or RNA-i) have been used to determine both the role of the nautilus-expressing cells and the nautilus gene, respectively, in embryonic muscle formation. In the absence of nautilus-expressing cells muscle formation is severely disrupted or absent. A similar phenotype is observed with the elimination of the nautilus gene product by genetic interference upon injection of nautilus double-stranded RNA (Misquitta, 1999).

The results from the injection of nautilus dsRNA point to a more general approach for the analysis of gene function during Drosophila development and suggest that the RNA interference method essentially would mimic a gene knock-out in the injected generation of Drosophila embryos. To test this idea a variety of cDNA clones were obtained representing a maternal gene expressed in the embryo (daughterless); additional genes involved in myogenesis (S59, DMEF2); homeobox genes (engrailed and S59); a gene important for gastrulation (twist), and a gene expressed in the adult eye (white). This panel of genes covers most stages of Drosophila development. twist was initiatially tested because the mutant has a clear phenotype that is easy to score when compared with wild-type larva. The injection of twist dsRNA (the complete coding region) into embryos produces a twisted larval phenotype that is indistinguishable from the original twist mutation. Similarly, injection of the first 1,200 bp of engrailed dsRNA produces the compressed dentical belt pattern characteristic of an engrailed null mutant. Daughterless mRNA is both maternally loaded and expressed zygotically, and the mutant phenotype produces very characteristic disruptions in the central nervous system (CNS) and peripheral nervous system (PNS). It has been shown previously that mex3, a maternally loaded RNA in C. elegans, can be ablated by dsRNA injection into the gonads. daughterless dsRNA (complete coding region) was injected and the characteristic neuronal phenotypes were sought by using the mAb MAB 22C10. The CNS as well as the PNS were disrupted to varying degrees in the injected embryos. The severity of the phenotype consistently shows a CNS disruption with a variable PNS pattern, possibly reflecting the fact that the CNS is formed before the PNS. This result suggests that maternally loaded as well as zygotically expressed RNA can be affected by RNA-i in Drosophila. The homeobox gene S59 marks a subset of muscle founder cells for 5 of 29 muscles in each hemisegment of the embryo corresponding to muscles 5, 18, 25, 26, and 27. Embryos with an S59 lacZ transgene marking muscles 18 and 25 were injected with S59 dsRNA (complete coding region). In this case, the S59-specific lacZ antibody-staining pattern is abolished. The total muscle pattern for embryos injected with S59 dsRNA, although disrupted, still shows the presence of poorly organized muscle groups in each hemisegment. This is unlike the almost complete absence of muscle observed with the injection of nautilus dsRNA. DMEF2, a member of the MADS domain transcription factor family, is essential for muscle formation in Drosophila. The DMEF2 / embryo has no muscle and is missing the characteristic gut constrictions found in the uninjected embryo. Injection of DMEF2 dsRNA (complete coding region) results in embryos that lack any detectable muscle and an absence of gut morphology (Misquitta, 1999).

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date revised: 20 June 2005

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