Abstracts - Society for Developmental Biology

81
Lee, Hu-Hui, National Chiayi ersityChiayi City ,, Taiwan; Lee, Jing-Yu; Chien, I-Chun; Lin, Win-Yu; Wu, Shao-min; Wei,
Bo-Huei; Lee, Yu-En, Univ Chiayi City, Taiwan
Previous studies have shown that Wnt signaling involves in postnatal mammalian myogenesis, in which for example, Wnt4
can activate the canonical beta-catenin pathway to stimulate myogenesis, and R-spondin, a Wnt signaling activator,
promotes skeletal myogenesis. On the other hand, soluble freezle inhibits myoblast differentiation. However, despite the
influence of Wnt is evident, the downstream mechanism ofWnt signaling in muscle cell differentiation is limited. Here, we
report that the Four and a half LIM domains 1 (Fhl1), which induces muscle hypertrophic, can be stimulated by betacatenin
and Licl treatment. On the other hand, knock-down of Fhl1 gene expression in C2C12 cells caused a reduction of
myotube formation. By reporter gene expression assays, we demonstrate that either beta-catenin or LiCl significantly
activated Fhl1 promoter, which contains 4 conserved Tcf/Lef binding sites. Mutations of 2 of these sites caused a
significant decrease in promoter activity by luciferase reporter assay. Thus, we suggest that Wnt signaling induces muscle
cell differentiation, at least partly, through Fhl1 activation.
Program/Abstract # 246
Novel enhancers regulate patched in Drosophila embryos
Lorberbaum, David S.; Ramos, Andrea; Barolo, Scott, University of Michigan, Ann Arbor, United States
The Hedgehog (Hh) signaling pathway is one of the most conserved pathways in development, and proper Hh signaling is
essential for the formation and function of nearly all animals. All targets of the pathway are regulated by Cubitus
interruptus (Ci), the Drosophila effector of Hh signaling. This transcription factor, conserved invertebrates as the Gli
family, functions through binding to DNA in cis-regulatory regions of the genome known as enhancers. These elements are
the key to the spatial and temporal regulation of target genes. Relatively few Hh target genes have been identified, but
among those that have is patched (ptc), which encodes the Hh receptor and is activated in all Hh-responding cells in both
flies and vertebrates. The only known ptc enhancer requires three consensus Ci binding sites to promote expression in the
developing wing, but does not respond to Hh signaling in embryos, underscoring that we do not know how Hh signaling
directly contributes to embryonic ptc expression. Clarification of this context specific regulation is essential to understand
how the Hh signaling pathway promotes normal, healthy development in all animals. Our recent work identifies novel ptc
enhancers that suggest a role for non-consensus Ci bindingsites in the proper regulation of ptc inthe embryo.
Program/Abstract # 247
Tlx3 modulates Prrxl1 promoter activity via two distinct mechanisms
Regadas, Isabel; Soares-dos-Reis, Ricardo; Matos, Mariana; Pessoa, Ana; Falcão, Miguel; A. Monteiro, Filipe; Lima,
Deolinda; Reguenga, Carlos, University of Porto, Portugal
The establishment of the nervous nociceptive circuitry depends on a group of transcription factors, including Prrxl1
(Drg11) and Tlx3, that controls the differentiation and further specification of neuronal cells. It was recently suggested that
Tlx3, which highly co-localizes with Prrxl1, is required to maintain Prrxl1 expression. In order to dissect the Tlx3-
dependent transcriptional mechanisms that drive Prrxl1 expression, luciferase reporter assays were performed using two
Prrxl1 regulatory regions containing different alternative promoters. Tlx3 overexpression induced the activity of the
TATA-containing promoter by directly binding to a bipartite DNA motif, as it was demonstrated by DNA-pull down
experiments followed by western-blotting. Regarding the other promoter region (lacking a TATA box), Tlx3
overexpression prompted a higher induction of luciferase expression, an effect that was significantly enhanced by Prrxl1.
Prrxl1 silencing reduced the inductive effect caused by Tlx3 overexpression, suggesting that Prrxl1/Tlx3 interaction is
required to activate the Prrxl1 TATA-less promoter. This hypothesis was confirmed by co-immuno precipitation assays.
Moreover, pull-down assays showed that this Prrxl1/Tlx3 induction on Prrxl1 promoter is indirect. The Tlx3-interaction
domain on Prrxl1 protein was mapped using different N- and C-terminal truncated Prrxl1 versions. Luciferase reporter
assays and co-immuno precipitation experiments demonstrated that the Prrxl1 C-terminal region is important for
transcriptional activity while the domain encompassing the 143-180 residues is implicated in Tlx3 interaction. Altogether,
our results suggest that Tlx3 strongly modulates the Prrxl1 promoter activity using two distinct mechanisms.
Program/Abstract # 248
Meis gene regulation during embryonic development
Zerucha, Ted; Barrett, Cody; Nelson, Kyle; Wellington, Allen, Appalachian State University, Boone, United States
The Meis genes (named for myeloid ecotropic leukemia virus integration site) are an evolutionarily conserved family of
genes and homologues of this family have been found in all animals. Meis proteins function as cofactors, interacting
directly with other transcription factors as well as DNA to facilitate transcriptional regulation. Most notably, they appear to
act as co-factors of Hox proteins as well as of other homeodomain proteins. The Meis genes are expressed in similar