An analysis using the hobo genetic system reveals that combinatorial signaling by the Dpp and Wg pathways regulates dpp expression in leading edge cells of the dorsal ectoderm in Drosophila melanogaster.

Our laboratory has contributed to the development of a genetic system based upon the hobo transposable element in Drosophila melanogaster. We recently reported that hobo, like the better-known P element, is capable of local transposition. In that study, we mobilized a hobo enhancer trap vector and generated two unique alleles of decapentaplegic (dpp), a transforming growth factor-beta family member with numerous roles during development. Here we report a detailed study of one of those alleles (dpp(F11)). To our knowledge, this is the first application of the hobo genetic system to understanding developmental processes. First, we demonstrate that lacZ expression from the dpp(F11) enhancer trap accurately reflects dpp mRNA accumulation in leading edge cells of the dorsal ectoderm. Then we show that combinatorial signaling by the Wingless (Wg) pathway, the Dpp pathway, and the transcriptional coactivator Nejire (CBP/p300) regulates dpp(F11) expression in these cells. Our analysis of dpp(F11) suggests a model for the integration of Wg and Dpp signals that may be applicable to other developmental systems. Our analysis also illustrates several new features of the hobo genetic system and highlights the value of hobo, as an alternative to P, in addressing developmental questions.

Combinatorial signaling by an unconventional Wg pathway and the Dpp pathway requires Nejire (CBP/p300) to regulate dpp expression in posterior tracheal branches.

The decapentaplegic (dpp) gene influences many developmental events in Drosophila melanogaster. We have been analyzing dpp expression in two groups of dorsal ectoderm cells at the posterior end of the embryo, in abdominal segment 8 and the telson. These dpp-expressing cells become tracheal cells in the posterior-most branches of the tracheal system (Dorsal Branch10, Spiracular Branch10, and the Posterior Spiracle). These branches are not identified by reagents typically used in analyses of tracheal development, suggesting that dpp expression confers a distinct identity upon posterior tracheal cells. We have determined that dpp posterior ectoderm expression begins during germ band extension and continues throughout development. We have isolated the sequences responsible for these aspects of dpp expression in a reporter gene. We have determined that an unconventional form of Wingless (Wg) signaling, Dpp signaling, and the transcriptional coactivator Nejire (CBP/p300) are required for the initiation and maintenance of dpp expression in the posterior-most branches of the tracheal system. Our data suggest a model for the integration of Wg and Dpp signals that may be applicable to branching morphogenesis in other developmental systems.

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.

Local transposition of a hobo element within the decapentaplegic locus of Drosophila.

We have efficiently mobilized a phenotypically silent hobo transgene inserted within the cis-regulatory heldout region of the decapentaplegic (dpp) locus in Drosophila melanogaster. The goal of our experiment was to identify germline transmission of a local transposition event within the dpp locus that meets two specific criteria. First, excision of the hobo construct does not generate an adult mutant phenotype, suggesting minimal alteration to the original site of insertion. Second, we required a new insertion of the hobo transgene into the Haploinsufficient region of the locus approximately 25 kb away. Genetic and molecular criteria are used to evaluate candidate germlines. In a pilot study, this local transposition event occurred independently in two individuals. Both of the transposition events appear to be new insertions into the dpp transcription unit. One insertion is between the two protein-coding exons, and the other is in the 3'-untranslated region of exon three. Strains carrying these insertions are valuable new reagents for the analysis of dpp function and molecular evolution. These results further support the use of the hobo system as an important tool in Drosophila genetics.

Regulation of BMP7 expression during kidney development.

Members of the Bone Morphogenetic Protein (BMP) family exhibit overlapping and dynamic expression patterns throughout embryogenesis. However, little is known about the upstream regulators of these important signaling molecules. There is some evidence that BMP signaling may be autoregulative as demonstrated for BMP4 during tooth development. Analysis of BMP7 expression during kidney development, in conjunction with studies analyzing the effect of recombinant BMP7 on isolated kidney mesenchyme, suggest that a similar mechanism may operate for BMP7. We have generated a beta-gal-expressing reporter allele at the BMP7 locus to closely monitor expression of BMP7 during embryonic kidney development. In contrast to other studies, our analysis of BMP7/lacZ homozygous mutant embryos, shows that BMP7 expression is not subject to autoregulation in any tissue. In addition, we have used this reporter allele to analyze the expression of BMP7 in response to several known survival factors (EGF, bFGF) and inducers of metanephric mesenchyme, including the ureteric bud, spinal cord and LiCl. These studies show that treatment of isolated mesenchyme with EGF or bFGF allows survival of the mesenchyme but neither factor is sufficient to maintain BMP7 expression in this population of cells. Rather, BMP7 expression in the mesenchyme is contingent on an inductive signal. Thus, the reporter allele provides a convenient marker for the induced mesenchyme. Interestingly LiCl has been shown to activate the Wnt signaling pathway, suggesting that BMP7 expression in the mesenchyme is regulated by a Wnt signal. Treatment of whole kidneys with sodium chlorate to disrupt proteoglycan synthesis results in the loss of BMP7 expression in the mesenchyme whereas expression in the epithelial components of the kidney are unaffected. Heterologous recombinations of ureteric bud with either limb or lung mesenchyme demonstrate that expression of BMP7 is maintained in this epithelial structure. Taken together, these data indicate that BMP7 expression in the epithelial components of the kidney is not dependent on cell-cell or cell-ECM interactions with the metanephric mesenchyme. By contrast, BMP7 expression in the metanephric mesenchyme is dependent on proteoglycans and possibly Wnt signaling.

In vivo functions mediated by the p41 isoform of the MHC class II-associated invariant chain.

We used a "hit and run" gene targeting strategy to generate mice expressing only the p41 isoform of the conserved invariant (Ii) chain associated with MHC class II molecules. In contrast to mutants expressing only p31 Ii chain, a small proportion of A(alpha)b A(beta)b molecules produced by these animals have reduced mobilities in SDS-PAGE and appear incompletely processed. Nonetheless, class II surface expression, peptide occupancy, CD4+ T cell maturation, and proliferative responses toward intact protein Ags are efficiently reconstituted. Moreover, spleen cells exclusively expressing p41 or p31 alone display equivalent dose-response curves in Ag presentation assays. Similar conclusions were reached analyzing mutants expressing two independent MHC haplotypes. Overall, these results demonstrate that Ii chain functional activities as a class II-specific chaperone are largely shared by p31 and p41 isoforms in the intact animal. Mutant mouse strains producing only p31 or p41 under control of endogenous regulatory elements responsible for constitutive and inducible Ii chain expression should prove useful for dissecting the contributions of these isoforms to diverse CD4+ T cell responses in vivo, such as those responsible for Ab production, inflammatory responses, autoimmune diseases, and protection against infectious agents.

Major histocompatibility class II peptide occupancy, antigen presentation, and CD4+ T cell function in mice lacking the p41 isoform of invariant chain.

We used a "hit and run" gene targeting strategy to generate mice expressing only the p31 isoform of the conserved invariant (Ii) chain associated with major histocompatibility complex (MHC) class II molecules. Spleen cells from these mice appear indistinguishable from wild type with respect to class II subunit assembly, transport, peptide acquisition, surface expression, and the ability to present intact protein antigens. Moreover, these mutant mice have normal numbers of thymic and peripheral CD4+ T cells, and intact CD4+ T-dependent proliferative responses towards a soluble antigen. In short, MHC class II expression and function are surprisingly unaffected in mice lacking p41 invariant chain, implying that the p31 and p41 isoforms may be functionally redundant in the intact animal.