A major goal of our laboratory is to understand the changes in cell size, shape, and position during embryonic development. The cytoskeletal components and adhesive factors that produce these changes are the same in most organisms; many have been identified using cell biological and biochemical techniques. The genes that directly control transitions from one cytoskeletal or adhesive configuration to another are not known. Embryos provide a useful system to identify such genes because each cell shape change and each restructuring of the cytoskeleton is tightly coupled to the developmental pathways governing cell fate. Over the past 15 years, much of our work has been directed at identifying the genes that control cell fate in Drosophila. We are currently defining the connection between such cell fate genes and the morphological changes that are their immediate consequences. (This work is supported in part by grants from the National Institutes of Health.)
Maternal Patterning Genes and the Accuracy of Cell Fate Determinations
Cells along the anterior-posterior axis of the Drosophila embryo are assigned specific fates based on a gradient of the maternal morphogen Bicoid (BCD). High concentrations of BCD are found in the anterior of the egg and determine anterior cell fate by activating transcription of downstream gap genes such as hunchback (hb). We are investigating how the embryo achieves accurate cell fate decisions in the context of environmental variations in BCD concentration, egg length, and temperature. Our previous work found significant variability of the BCD gradient from one egg to the next. In contrast, HB transcription was almost invariably activated at 48 percent egg length, demonstrating a robust filtering of developmental noise in the BCD gradient. In addition to genetic manipulations of egg length within the D. melanogaster species itself, we are now extending this analysis to other dipteran species (Musca, Calliphora, Lucilia) with average egg lengths 2.5 to 3 times that of Drosophila. We have characterized the BCD distributions in these species and found that they are broader, thus compensating for the increased egg length. These results attest to remarkably robust developmental processes that control cell fate among the insects. By identifying the mechanism that allows diffusion BCD gradients to scale, we hope to identify the underlying processes that control spatial distributions of such developmental morphogens.
Cell Shape Changes and Morphogenetic Movements During Gastrulation
The morphogenetic movements that follow the Drosophila blastoderm stage involve invagination of mesodermal precursor cells into the interior of the embryo and the elongation of the anterior-posterior body axis. The cell shape changes producing these movements are accompanied by a polarized accumulation of myosin on the apical surface of the mesodermal precursors and on the anterior and posterior interfaces of the intercalating ectoderm. In the mesoderm, myosin accumulation is triggered by expression of the Twist (Twi) and Snail (Sna) transcription factors and activation of the fog/cta pathway. The basic outline of this pathway is similar to the LPA/Rho pathway that induces stress fibers in vertebrate tissue culture cells. We are using genetic approaches coupled with microarray analysis and optical visualization strategies to investigate the steps connecting Twi and Sna expression to myosin contractility. The cell polarizations that occur during intercalation of the ectoderm appear to be more complicated in that myosin accumulation does not reflect any single transcription factor but instead depends on the juxtapositions in pair-rule gene expression. The simplest model would require that neighboring cells compare positional identities and polarize themselves along the axes where they confront cells maximally different from themselves. We are investigating the mechanisms that allow cells to make these comparisons, measure differences, and polarize their cytoskeletons accordingly.
The Role of Wingless in Regulation of Armadillo
During epithelial cell development, growth and patterning depend on the stability and accumulation of the β-catenin homolog Armadillo (ARM). This protein functions not only in adhesive junctions but also as a transcriptional cofactor in cell signaling. In the embryonic epidermis, ARM levels are controlled by Wnt signaling; a striped expression of the Wnt homolog Wingless causes a striped accumulation of ARM. In the current model, Wingless signal blocks the normal degradation of cytoplasmic ARM, allowing it to accumulate to high levels in the cell. Degradation of ARM is preceded by its amino-terminal phosphorylation by the zw3/shaggy/GSK3 kinase. We have uncovered a branch of the Wnt pathway in which ARM activity is regulated in a Wnt-dependent manner, independent of zw3 and protein degradation. In this pathway, ARM is anchored in the cytoplasm by Axin and the adenomatous polyposis coli protein, APC. The key step in the activation of Wnt signaling is destabilization of Axin. Axin degradation is controlled by a stabilizing effect of zw3-dependent phosphorylation, and a destabilizing effect of active Arrow. Removing Axin allows ARM to accumulate and relocalize to the nucleus. Nuclear localization of ARM results in transcriptional activation of Wnt target genes.
