Transgenic analysis of the Smad family of TGF-beta signal transducers in Drosophila melanogaster suggests new roles and new interactions between family members.

Smad signal transducers are required for transforming growth factor-beta-mediated developmental events in many organisms including humans. However, the roles of individual human Smad genes (hSmads) in development are largely unknown. Our hypothesis is that an hSmad performs developmental roles analogous to those of the most similar Drosophila Smad gene (dSmad). We expressed six hSmad and four dSmad transgenes in Drosophila melanogaster using the Gal4/UAS system and compared their phenotypes. Phylogenetically related human and Drosophila Smads induced similar phenotypes supporting the hypothesis. In contrast, two nearly identical hSmads generated distinct phenotypes. When expressed in wing imaginal disks, hSmad2 induced oversize wings while hSmad3 induced cell death. This observation suggests that a very small number of amino acid differences, between Smads in the same species, confer distinct developmental roles. Our observations also suggest new roles for the dSmads, Med and Dad, in dActivin signaling and potential interactions between these family members. Overall, the study demonstrates that transgenic methods in Drosophila can provide new information about non-Drosophila members of developmentally important multigene families.

sqv-3, -7, and -8, a set of genes affecting morphogenesis in Caenorhabditis elegans, encode enzymes required for glycosaminoglycan biosynthesis.

sqv (squashed vulva) genes comprise a set of eight independent loci in Caenorhabditis elegans required zygotically for the invagination of vulval epithelial cells and maternally for normal oocyte formation and embryogenesis. Sequencing of sqv-3, sqv-7, and sqv-8 suggested a role for the encoded proteins in glycolipid or glycoprotein biosynthesis. Using a combination of in vitro analysis of SQV enzymatic activities, sqv(+)-mediated rescue of vertebrate cell lines, and biochemical characterization of sqv mutants, we show that sqv-3, -7, and -8 all affect the biosynthesis of glycosaminoglycans and therefore compromise the function of one specific class of glycoconjugates, proteoglycans. These findings establish the importance of proteoglycans and their associated glycosaminoglycans in epithelial morphogenesis and patterning during C. elegans development.

The Drosophila kismet gene is related to chromatin-remodeling factors and is required for both segmentation and segment identity.

The Drosophila kismet gene was identified in a screen for dominant suppressors of Polycomb, a repressor of homeotic genes. Here we show that kismet mutations suppress the Polycomb mutant phenotype by blocking the ectopic transcription of homeotic genes. Loss of zygotic kismet function causes homeotic transformations similar to those associated with loss-of-function mutations in the homeotic genes Sex combs reduced and Abdominal-B. kismet is also required for proper larval body segmentation. Loss of maternal kismet function causes segmentation defects similar to those caused by mutations in the pair-rule gene even-skipped. The kismet gene encodes several large nuclear proteins that are ubiquitously expressed along the anterior-posterior axis. The Kismet proteins contain a domain conserved in the trithorax group protein Brahma and related chromatin-remodeling factors, providing further evidence that alterations in chromatin structure are required to maintain the spatially restricted patterns of homeotic gene transcription.

Smad2 signaling in extraembryonic tissues determines anterior-posterior polarity of the early mouse embryo.

Smad proteins transmit TGFbeta signals from the cell surface to the nucleus. Here we analyze Smad2 mutant embryos created using ES cell technology. Smad2 function is not required for mesoderm production per se, but, rather unexpectedly, in the absence of Smad2 the entire epiblast adopts a mesodermal fate giving rise to a normal yolk sac and fetal blood cells. In contrast, Smad2 mutants entirely lack tissues of the embryonic germ layers. Smad2 signals serve to restrict the site of primitive streak formation and establish anterior-posterior identity within the epiblast. Chimera experiments demonstrate these essential activities are contributed by the extraembryonic tissues. Thus, the extraembryonic tissues play critical roles in establishing the body plan during early mouse development.

Genetic analysis of brahma: the Drosophila homolog of the yeast chromatin remodeling factor SWI2/SNF2.

The Drosophila brahma (brm) gene encodes an activator of homeotic genes related to the yeast chromatin remodeling factor SWI2/SNF2. Here, we report the phenotype of null and dominant-negative brm mutations. Using mosaic analysis, we found that the complete loss of brm function decreases cell viability and causes defects in the peripheral nervous system of the adult. A dominant-negative brm mutation was generated by replacing a conserved lysine in the ATP-binding site of the BRM protein with an arginine. This mutation eliminates brm function in vivo but does not affect assembly of the 2-MD BRM complex. Expression of the dominant-negative BRM protein caused peripheral nervous system defects, homeotic transformations, and decreased viability. Consistent with these findings, the BRM protein is expressed at relatively high levels in nuclei throughout the developing organism. Site-directed mutagenesis was used to investigate the functions of conserved regions of the BRM protein. Domain II is essential for brm function and is required for the assembly or stability of the BRM complex. In spite of its conservation in numerous eukaryotic regulatory proteins, the deletion of the bromodomain of the BRM protein has no discernible phenotype.