The broad goal of my research program is to understand the molecular mechanisms that underlie cellular differentiation.  We use a combined genetic and molecular approach to examine these mechanisms in the early Drosophila embryo, which provides an excellent system for the study of gene regulation and interaction.  We focus on genes that instruct undetermined cells to differentiate into specific tissue types along the dorsal-ventral (DV) axis of the body.  Initially, the Dorsal transcription factor, which is provided maternally, subdivides the DV axis into three main domains by regulating the expression of downstream zygotic genes that in turn give further instructions to cells.  One important gene is dpp which encodes a secreted molecule that belongs to the TGF-b superfamily of growth factors.  It functions as a ligand in a signal transduction pathway that involves two main steps: binding to a receptor complex that activates intracellular molecules called Smads, and Smads assemble complexes that regulate transcription.  dpp acts in the form of a gradient; high levels of activity in the dorsal-most region instruct cells to differentiate into a tissue called the amnioserosa, a squamous epithelium, and low levels determine dorsal epidermal cells.  In addition, it is crucial to keep Dpp absent from ventral regions to ensure development of the neuroectoderm. 

We are interested in how the activity levels of dpp are tightly controlled along the DV axis, and how these levels are interpreted by downstream target genes.  We have identified several candidate target genes that respond differentially to the Dpp gradient.  They are expressed in nested domains centered along the dorsal midline.  For example, pannier (pnr) is expressed in a broad domain, while u-shaped is a high-level target that is expressed in a 12-14 cell-wide stripe. Race is the most restricted domain comprising only the presumptive amnioserosa (5-6 cell-wide stripe).  With these in hand we have an ideal set of target genes to uncover the molecular mechanisms of how they are differentially regulated by the Dpp gradient.  Thus far we have learned that the broadly expressed genes are regulated by a combination of the Dpp signal mediators, Mad and Medea, and the transcriptional repressor, Brinker which is expressed in regions complementary to dpp with some overlap. It appears that Brinker and Smads compete for DNA binding to the target promoter to define the precise border of target gene expression. However, for the Dpp target genes that respond to the highest levels of Dpp, the highly restricted amnioserosa-specific target genes, like Race, Brinker is not involved but rather a combination of Mad/Medea and the homeodomain protein Zen is required for proper transcriptional activation.  The underlying molecular mechanism involves synergistic interactions between these transcriptional regulators: Mad facilitates the DNA binding of Zen to the Race promoter and this is dependent on their ability to interact with each other at the protein level.  Our studies go on to demonstrate that Dpp and Zen act via a feed forward mechanism to regulate Race, and possibly all amnioserosa-specific target genes, whereby one regulator, Dpp, activates a second regulator, Zen, and then these work together to activate a downstream gene, Race.  We are in the position now to test other high level targets to determine if this mechanism is widespread for the amnioserosa-specific targets.  At this point we do not have evidence to suggest that Dpp utilizes Smad binding site affinity as a means to function as a morphogen as does the Dorsal morphogen.

A second project in the lab addresses the process of tissue differentiation.  Using the amnioserosa as a model embryonic tissue, a long term goal is to understand how cells become determined, differentiate, and function as a specific tissue type.  The amnioserosa is a squamous epithelium that derives from the dorsal-most cells of the early embryo.  It is required for proper germ band extension and other gastrulation movements.  I am interested in regulatory genes that:  (a) determine the fate of embryonic cells to become amniosoersa (dpp and zen for example), (b) direct the differentiation of the amnioserosa (possibly target genes of zen), (c) maintain the differentiated state (the u-shaped group of genes are involved), and (d) are required for proper function of the amnioserosa (what exactly is its function?).