Research

PROJECT 1

Cell Fate Specification in the Developing Heart

During early stages of embryogenesis dpp expression in the medial region of the dorsal ectoderm patterns the underlying mesoderm. These Dpp signals specify both cardial and pericardial cell fates in the developing heart. Subsequently dpp dorsal ectoderm expression becomes restricted to the dorsal-most or leading edge cells. A second round of Dpp signaling then specifies cell shape changes in ectodermal cells leading to dorsal closure. In our Oct. 2003 Developmental Biology paper we showed that a third round of dpp dorsal ectoderm expression initiates during late stages of embryogenesis. This round of dpp expression also is restricted to leading edge cells and these Dpp signals repress the expression of the transcription factor Zfh-1 in a subset of pericardial cells. We found that cis-regulatory sequences that activate this third round of dpp dorsal ectoderm expression are located in the dpp disk region. We also showed that this round of dpp expression requires prior Dpp signals and perhaps release from dTCF-mediated repression.

Since that paper we have made significant progress. We utilized constitutively active and dominant negative components of the Wg pathway to show that Wg signaling represses the third round of dpp expression [Fig.1]. Using site directed mutagenesis of dTCF sites contained in relevant disk region reporter genes we have now shown that Wg repression is direct. We have recently identified a second pericardial cell marker (Odd-skipped) that is repressed by Dpp signals to aid us identifying the pericardial cell lineage responsive to the third round of Dpp signaling. We have identified a cluster of five Mad binding sites in the 3' untranslated region of zfh-1 and generated a reporter gene to test whether Dpp repression of zfh-1 is direct.

PROJECT 2

Branch Formation in the Developing Tracheal System
We have been analyzing dpp expression in the posterior dorsal ectoderm. Here dpp -expressing cells become tracheal cells in the posterior-most branch of the tracheal system (the Posterior Spiracle). In our July 2002 Developmental Biology paper we reported the isolation of the sequences responsible for dpp posterior ectoderm expression in a reporter gene. We also showed that an unconventional form of the Wg pathway, the Dpp pathway and the transcriptional co-activator Nejire (CBP/p300) are required for the initiation and maintenance of dpp posterior dorsal ectoderm expression.

Since that paper we have made considerable progress. We identified a candidate posterior spiracle-combinatorial enhancer (the KX enhancer) containing two sets of overlapping binding sites for Mad and dTCF in our reporter gene. We generated a tracheal-specific dpp mutant strain by effectively deleting the KX enhancer from the genome in vivo. Our analysis of the dpp ΔKX rescue construct in Table1A shows that the KX enhancer is not required to rescue the dorsal-ventral patterning defects engendered by dpp Haploinsufficient alleles. However,
Table 1B shows that the KX enhancer is required to rescue dpp null embryos. Further, as shown in Fig. 2, dpp mutant phenotypes generated in these experiments directly connect the KX enhancer to posterior spiracle development. dpp null embryos bearing the ΔKX rescue construct show normal dorsal-ventral patterning but fail to hatch due to posterior spiracle defects (compare panels 2G and 2K with 2H and 2L). These results also show that the posterior-spiracle defects of dpp Haploinsufficient embryos, long thought to be downstream effects of dorsal-ventral patterning defects, are genetically distinct.

PROJECT 3

Transgenic Analysis of Smad Tumor Suppressor Genes
Based upon clinical studies, oncologists estimate that mutations in human Smad genes (TGF-β signal transducers similar to Drosophila Mad) are involved in roughly 140,000 new cancer cases in the US each year. How the absence of a functional Smad gene leads to a tumor is completely unknown. As a result, the current working hypothesis is that all tumor-associated Smad mutations are loss-of-function mutations and they all lead to tumors via a single mechanism. In our April 2001 Genetics paper we describe a transgenic assay system that allows us to explicitly test this hypothesis. Using our assay, we discovered two gain-of-function mutations in the Smad4 tumor suppressor gene (and one in Drosophila Mad). A phenotypic analysis suggested that the Smad4100T allele is capable of activating Wnt target genes [ Fig. 3]. This suggests that the Smad4100T allele induces tumor formation via ectopic expression of Wnt target genes [Fig. 4].

A mechanism of tumorigenesis associated with the ectopic expression of Wnt target genes was originally identified via mutations in the Wnt signal transducer Adenomatous Polyposis coli (APC). If the Smad4100T mutation causes tumors via an "APC" mechanism rather than a "TGF-β loss-of-function" mechanism there are clinically important implications. There is a 3 year mean life expectancy for people with TGF-β loss-of-function tumors like those found in HNPCC colon cancer but there is a ten year mean life expectancy for APC mutations like those found in FAP colon cancer. If the "APC" hypothesis is confirmed by additional studies we plan to develop a diagnostic test for the Smad4100T mutation.