This research program focuses on the role of intercellular communication via peptide growth factors in cell differentiation and the formation of tissues. Using the tools of genetics and molecular biology, our goal is to describe biochemical pathways through which growth factors act to modulate morphology, gene expression and cell behavior. The major focus is on the Transforming Growth Factor beta family, with lesser focus on the Wnt and Hedgehog pathways of secreted signaling molecules. Results are relevant to the use of growth factors as cytokines in the manufacture of cellular products as well as therapeutic agents in and of themselves. Furthermore, understanding the stability of differentiated cells and their capacity to be reprogrammed by their environment is a major concern in use of cellular therapies, particularly those using stem cells. Our work uses the fruitfly Drosophila melanogaster as a model system. A completed, annotated genome, and extensive resources for loss of expression and tissue specific over-expression in the context of the intact organism make Drosophila among the most powerful model systems for analysis of cell and tissue interactions. All signal transduction pathways (TGF-beta, Wnt, Hedgehog, FGF, EGF, MAP kinase, JNK kinase) are highly conserved between Drosophila and mammals, therefore results from Drosophila can be extended to higher animals. Major projects: a) Use of genetic interaction screens to identify components of the TGF-beta signal transduction pathway. The product of the decapentaplegic (dpp) gene is the major TGF-beta family member in Drosophila, and the member best characterized by genetic analysis. Mutant phenotypes exist that reflect the many requirements for this growth factor during embryogenesis. We study a class of viable mutations that affects the head of the adult fruitfly. The severity of this class of mutations is closely linked to the functional level of dpp signaling. They therefore serve as a barometer of signal transduction pathway activity and have been used to screen the Drosophila genome for loci that interact with dpp in this pathway. One of the screens used an existing bank of mutations created by P element transposon-tagging to facilitate rapid recovery of identified loci, while a second used an ordered collection of genetic deficiencies covering the majority of the Drosophila genome. Using the completed sequence of the Drosophila genome, we have been able to correlate many interacting loci directly with known genes and putative protein coding regions. Some of the interacting mutations are in previously studied genes, such as transcription factors, cell cycle regulators, cellular adhesion molecules, and chromatin remodeling factors. In the past year we have focused on one of our best-characterized and most strongly interacting genes. This gene is part of a family of mammalian genes thought to play a role in formation of the nervous system, and disruption of human homologues has been implicated in both spina bifida and a structural anomaly of the brain called holoprosencephaly. Loss-of-function alleles of this gene interact dominantly with the dpp head capsule class of mutations to specifically alter the adult Drosophila head, and genetic removal of active protein from this gene using a temperature sensitive mutation also alters adult head formation in a similar manner. No other structures in the adult fly are affected, indicating that this gene and dpp specifically act together in the formation of the adult head. Failure to expresses this gene in the head primordia results in reduction of head specific dpp transcription, as indicated by beta-galactosidase reporter constructs that recapitulate dpp's head-specific expression, introduced into flies transgenically. We cannot yet say whether this influence is direct or indirect. We are examining the post embryonic expression pattern of this gene, both by RNA in situ hybridization, and by creating a series of transgenic flies bearing beta-galactosidase reporter constructs containing regulatory DNA from the gene. These analyses indicate that this gene is expressed in spatially specific regions of the head primordia. We have also begun a study of targeted misexpression of this gene. These analyses use the GAL4/UAS system to misexpress genes of interest in specific tissues in the whole organism. Such studies allow us to assess the cellular effects of protein encoded by this gene and determine its effects on other differentiation markers and signal transduction pathways. Preliminary data suggest that misexpression of this gene in cells determined to take on a retinal fate cause these cells to reprogram to another fate, most likely head cuticle. b) The role of BMP proteins in the formation of the adult head. The phenotype caused by mutations used for the genetic screens described above is limited to the head of the adult fly. Various external structures such as the eye and sensory organs are reduced, eliminated or duplicated. The adult head derives from paired epithelial sacs called imaginal discs that grow during embryogenesis and then undergo final differentiation at the metamorphosis of the pupa. Imaginal discs have become a popular model system for studying signal transduction as they have complex pattern formation, but unlike the embryo, also undergo growth, allowing this aspect of signal transduction to be studied. Our genetic analyses and the expression patterns of our Beta-galactosidase reporter constructs suggest that dpp's head capsule phenotype reflects a requirement for dpp to pattern the ventral surface of the head, and to play a role in aligning and fusing the two epithelial sheets of the paired eye/antennal disks when the adult head is formed from them at metamorphosis. The fusion of imaginal disks at metamorphosis is considered to be a model for the interaction of such epithelial sheets in the construction and regeneration of tissues, particularly wound healing, and the high evolutionary conservation of signaling pathways indicates that these data will be relevant to vertebrates. The dpp hc phenotype results in loss or duplication of structures that arise at the boundary of epithelial sheet fusion, suggesting that misalignment may lead to cell death, and loss or inappropriate regeneration of affected structures. In the past year we have begun to analyze the contribution of the stress-activated protein kinase (SAPK) or JUN kinase pathway. This pathway is activated in response to tissue damage, cell stress, or radiation. Data in Drosophila indicates that discontinuities in growth factor signaling can also activate the SAPK pathway, leading to apoptosis. We have analyzed a surrogate marker of SAPK pathway activation: the expression of an induced phosphatase. Our preliminary results indicate that the SAPK pathway is activated in dpp hc mutations in the area where dpp expression is altered. We are currently analyzing markers of cell death to correlate SAPK pathway induction with apoptosis in our tissue. Our goal is to use the extensive genetic tools available in this model organism to study signal transduction-triggered epithelial sheet fusion and tissue regeneration. c) Identify cis-acting sequences and trans-acting factors required for dpp's role in head-capsule formation. We have identified at the DNA sequence level the lesions associated with our head capsule mutations. We are currently analyzing the region associated with these lesions for the presence of cis-regulatory DNA by the creation of transgenic flies bearing Beta-galactosidase reporter constructs. Transgenic flies bearing eight different constructs have been analyzed in the last year. As reported previously, the expression pattern from these constructs is consistent between the structures altered in the head capsule mutant phenotype and the established developmental fate maps for the adult head. We had previously sequenced one specific mutation, hc1, and found that its lesion is a 17 base pair (bp) deletion that removes putative binding sites for several homeodomain transcription factors. This deletion is within a 600 bp area containing many such putative homeodomain protein binding sites. Constructs bearing only this region of clustered putative binding sites are sufficient to drive beta-galactosidase expression in the eye-antennal imaginal disk, identically to larger constructs containing this region, suggesting this region is sufficient to drive spatially specific gene expression. An equivalent construct containing DNA from the hc1 mutation fails to drive expression of beta-galactosidase, indicating the importance of this region to proper gene expression. In the last year we have systematically altered transcription factor binding sites in and around the region of the 17 bp deletion to assess their individual contribution to gene expression. Results to date indicate that sites critical for the homeodomain transcription factor, extradenticle, are required for the correct level of gene expression. Extradenticle is homologous to the human gene Pbx, mutations in which cause acute lymphoblastic leukemia. We are continuing to assess other sequences in the region for their contribution to gene expression.
