Abstracts - Society for Developmental Biology

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Program/Abstract # 242
Mutational and biochemical analysis of a UBX-responsive regulatory element
Hersh, Brad; Biery, Amy; Sharpnack, William, Allegheny College, Meadville, United States
Identification of direct Hox target genes is crucial to understand how the Hox family establishes anterior-posterior pattern
in developing animals. The Hox protein Ultrabithorax (UBX), which is necessary for specification of the Drosophila
haltere, binds the DNA sequence TAAT in vitro. Co-factors, such as Extradenticle, can increase the DNA sequence
specificity of UBX at some target sites, but in many instances the mechanisms for discriminating between target and nontarget
sequences are poorly characterized. A single conserved UBX binding site is necessary for the function of a cisregulatory
element (CRE) for the Cpr47Ee gene in the developing haltere. We identified additional sequence important for
activation of GFP reporter gene expression in the haltere by mutagenesis of conserved sequences flanking the UBX
binding site. In addition, we purified full-length UBXIa protein to test its ability to bind mutated CRE sequence, and to
determine whether the conserved regions are necessary for binding of UBX. We have also assessed the evolution of this
regulatory element by testing homologous CRE sequences from several Drosophila species for function in the
D.melanogaster haltere. To identify specific evolved sequence changes that alter CRE function we generated chimeric
elements combining melanogaster and ananassae sequence. These combined mutational, evolutionary, and biochemical
analyses contribute to a more complete understanding of the mechanisms of Hox protein function.
Program/Abstract # 243
Transcriptional repression of Fgf8 by retinoic acid signaling during early mouse embryogenesis
Kumar, Sandeep; Duester, Gregg, Sanford-Burnham Med Research Institute, La Jolla, United States
Retinoic acid (RA) is a signaling molecule required for vertebrate embryogenesis. RA serves as a ligand for RA receptors
(RARs) that directly control transcription via RA response elements (RAREs) located near genes. During early
embryogenesis, fibroblast growth factor-8 (Fgf8) plays important roles in the caudal progenitor zone, heart, and
somitogenesis as the body axis extends from head to tail. RA represses Fgf8 at the anterior end of the caudal progenitor
zone thus preventing FGF signaling from extending into the developing trunk. During early heart organogenesis, RA
mediates anteroposterior patterning of the heart by repressing Fgf8 in mesoderm lying posterior to the heartfield. These
opposing gradients of RA and Fgf8 operate in concert as a switch that controls mesodermal development. A direct role of
RA in regulating Fgf8 was suggested by previous studies that identified a conserved RARE upstream of Fgf8. Here, we
investigate the mechanism of RA-Fgf8 antagonism. Chromatin immunoprecipitation (ChIP) performed on E8.25 mouse
embryos shows that the Fgf8 RARE binds all three RARs in vivo, and mutation of this RARE abolishes the binding of
RARs in gel shift assays. To test a potential repressive role of RA, we analyzed the ChIP patterns of repressive and
activating histone marks (H3K27me3 and H3K4me3, respectively) near the Fgf8 RARE in different regions of
E8.25embryos. Chromatin obtained from head/heart and caudal progenitor zone showed enrichment of H3K4me3
compared to H3K27me3 correlating with transcriptionally active Fgf8. In contrast, the trunk showed enrichment of
H3K27me3 versus H3K4me3 suggesting Fgf8 repression. Our findings thus provide evidence that RA may directly repress
Fgf8 via its RARE.
Program/Abstract # 244
The promoter regulates the dynamics of gene activation in development
Lagha, Mounia; Bothma, Jacques P.;Esposito, Emilia (UC Berkeley, United States); Stefanik, Laura (Penn State
University, University Park, United States); Tsui, Chiahao (UC Berkeley, United States); Johnston, Jeffrey; Chen, Kai
(Stowers Institute, Kansas City, United States); Gilmour, David S. (Penn State University, University Park, United States);
Zeitlinger, Julia (Stowers Institute, Kansas City, United States); Levine Michael (UC Berkeley, United States)
BMP/Dpp signaling gradients have been implicated in a variety of metazoan developmental processes, and much is known
about how these gradients produce different spatial patterns of gene expression. However, there is considerably less
information about the mechanisms underlying the temporal control of gene expression. Here we employ a novel
quantitative imaging assay to measure the dynamics of gene activation during Dpp signaling in the Drosophila embryo.
Genes containing paused RNA polymerase (Pol II) exhibit more synchronous and rapid induction of gene expression than
those lacking Pol II, but not all paused promoters are equivalent. Some mediate faster activation dynamics than others.
This differential timing correlates with Pol II pausing stability, and mutations that destabilize paused Pol II cause a delay in
gene expression. We conclude that the promoter is a prime determinant of developmental timing.
Program/Abstract # 245
Fhl1 promotes myogenesis of C2C12 in response to Wnt signaling