Crystal Maki and Melissa Marchal will Present Their Research June 27, 2013
Identification and characterization of developmental enhancers in two model systems of epithelial morphogenesis
Conditional gene expression is the crux of growth regulation and development within complex eukaryotes. Regulatory elements such as enhancers play critical roles in determining the morphologies of various cells, tissues and entire organisms by integrating inputs from diverse signaling pathways. Enhancers themselves are modular in nature and act on their target genes in a position- and orientation-independent manner. Interestingly, genomic regulatory regions are under differential selective constraints relative to their protein-coding counterparts and as such are more likely to experience compositional change over time. Unfortunately, very little is known about the evolutionary diversification of enhancer composition within the context of morphological change. The approach here utilizes a post-genomic evo-devo perspective to take a look at the phenotypic divergence of the dorsal respiratory appendage between Drosophila subgenera, with specific attention to alterations in enhancer sequences. Drosophila is an ideal model organism with which to pursue this aim due to its experimental amenability and availability of multiple species with available sequence and a well-characterized phylogeny. Furthermore, the number of respiratory appendages present in the follicular egg chamber of Drosophila is one of most basal morphological divisions between the Drosophila and Sophophora subgenera. Virtually all identified species within the Drosophila subgenera exhibit two pairs of respiratory appendages and all those within Sophophora have a single pair. We postulate that a series of developmental enhancers for the dorsal respiratory appendage existed in the latest common ancestor of the Drosophila genus (2 pairs) and that subsets of enhancers underwent loss or gain of function within the early Sophophora stem-lineage. An alternate evolutionary hypothesis is that enhancers evolved de novo in the Sophophoran stem-lineage. For our purposes, we focus on transcription factors with known roles during oogenesis: Pointed (Pnt) and Suppressor of Hairless [Su(H)], which participate in EGFR and Notch signaling, respectively.
The Role of Maternal Wnts and Frizzleds in Dorso-Ventral Axis Specification
Elucidating the molecular mechanisms that transform a symmetric egg into a more complex embryonic body has interested developmental biologists for some time. The asymmetric distribution of determinants within the oocyte is critical to normal developmental processes such as dorsal axis determination and cell migration. In Xenopus laevis in particular, maternal mRNAs involved in the patterning of the dorsal body axis are localized to the oocyte vegetal cortex, and upon sperm entry, are subsequently translocated to the future dorsal side. This process, called cortical rotation, is required for the asymmetric activation of the Wnt/Beta-Catenin signaling pathway on the dorsal side of the blastula. Beta-catenin then regulates the transcriptional activation of dorsal-specific genes at the midblastula transition. Maternal mRNA depletion studies in Xenopus first established the role for Beta-Catenin signaling in the specification of the dorso-ventral axis, although the upstream mechanisms activating Beta-catenin have remained elusive. Recent evidence has suggested that secreted Wnt ligands Wnt11 and Wnt5a may act together in axis formation. However, the function of other maternally expressed Wnts and their cognate receptors (frizzleds) remains uncharacterized. We have examined the expression of several wnts and frizzleds (fzds) in the oocyte and throughout early development. Through this analysis, we have found that fzds 1, 4, 6, and 7 are expressed abundantly in the oocyte, with fzd7 maintaining high expression levels until the tailbud stages. We have begun to identify the roles of these maternal factors in dorso-ventral axis patterning via maternal mRNA depletion/host transfer studies. We present evidence that maternal fzd1 -depleted embryos show partial defects in dorsal-specific gene expression. Additionally, we have begun testing the efficacy of several anti-sense oligos against additional fzds for use in future maternal loss-of-function studies.