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Poster Full Abstracts - Cell Biology and Signal Transduction
Poster board number is above title. The first author is the presenter
183
185B
A BMP-dependent feedback loop regulates
dpp
expression by direct and indirect mechanisms in the
Drosophila
wing imaginal disc.
Maryanna M.
Aldrich, Lorena Soares, Kristi Wharton. MCB Department, Brown University, Providence, RI.
Positional information across a field of unspecified cells is often established by a gradient of morphogen activity that results in threshold-specific
transcriptional responses. As the tissue develops, regulatory mechanisms likely exist to ensure robustness within signal transduction pathways that generate
such morphogen activity gradients. In the
Drosophila
wing disc, the graded distribution of the phosphorylated BMP signal transducer, phospho-Mad
(pMad), directs the transcriptional response of distinct sets of target genes critical for patterning along the anterior-posterior axis. BMP signaling is thought
to maintain robustness as a result of feedback mechanisms involving regulation of the pathway modulators,
dad
and
pent
. The work presented here
characterizes an additional feedback mechanism that directly impacts transcriptional regulation of the BMP ligand,
dpp
. We show that a negative feedback
loop mediated through canonical Mad-mediated BMP signaling regulates
dpp
transcription within its endogenous wing pouch expression domain. Through a
combined analysis of binding sites within the
dpp cis
-regulatory sequences and of requirements for candidate transcription factors, we determined that Shn is
required for negative feedback but Ci is not essential. Furthermore, we find that BMP signaling exhibits a domain-specific effect on transcription of
dpp
. In
the lateral region of the wing disc, BMP signaling positively regulates
dpp
expression. Curiously, this effect is blocked by action at a putative Mad binding
site within the control regions of
dpp
. Within the medial region of the wing pouch, the endogenous expression domain of
dpp, dpp
is regulated in a pMad
dose-dependent manner. Taken together, we propose that this complex regulation of
dpp
serves to fine tune BMP signaling should fluctuations in signaling
output be encountered, thus, ensuring robustness during patterning of the wing pouch.
186C
A
Drosophila
cell culture model for Dpp-induced epithelial plasticity.
David J. Casso
1
, Björn Gärtner
2
, J. Alex Rondon
1,4
, Aiguo Tian
1,3
, Rik Derynck
1
,
Julia Zeitlinger
2
, Katja Brückner
1
. 1) University of California San Francisco, San Francisco, CA; 2) Stowers Institute for Medical Research, Kansas City,
MO; 3) Present Address: Univ Texas Southwestern Medial Center, Dallas, TX; 4) Present Address: Genentech, South San Francisco, CA.
Epithelial plasticity, which reflects changes of epithelial cells regarding their morphology and differentiation state, is an essential program in normal
development, and underlies life-threatening pathologies such as fibrosis and tumor metastasis. In vertebrates, members of the TGF-β/BMP family are potent
inducers of epithelial plasticity.
Drosophila
has been an excellent model to study epithelial architecture and epithelial plasticity in vivo, yet no cell-based
system has been available to take advantage of
Drosophila
in the molecular dissection of epithelial plasticity. We now introduce KaBrü1D, a
Drosophila
epithelial cell line closely related to wing imaginal disc cells, that undergoes BMP/decapentaplegic (dpp) induced epithelial plasticity, similar to the
elongation of wing imaginal cells during thorax closure of the developing adult fly. Based on an RNAi screen comprising all
Drosophila
kinases and
phosphatases, expression profiling, and ChIP analyses, we identified Mad (mothers against Dpp) transcriptional targets and genes functionally involved in
Dpp/BMP-induced epithelial plasticity. Akt/Tor signaling is essential in this process, and activity of this pathway is enhanced over the course of several days
of BMP stimulation, consistent with a secondary transcriptional wave leading to elevated receptor tyrosine kinase signaling. We now focus on the
mechanism of cooperation between the Dpp and Akt/Tor pathways, and investigate the role of these pathways and their transcriptional targets during thorax
closure in
Drosophila
in vivo.
187A
A Molecular Competition between Wingless and BMP Signaling Controlled by Mad Phosphorylations.
Edward V Eivers
1
, Hadrien Degmany
2
, Edward
DeRobertis
2
. 1) Department of Biological Sciences, California State University, Los Angeles, CA 90032-8201; 2) Howard Hughes Medical Institute,
University of California, Los Angeles, CA 90095-1662.
Bone morphogenetic proteins (BMPs) and Wnts are growth factors that provide essential patterning signals for cell proliferation and differentiation. Here
we describe a novel mechanism of action for the transcription factor Mad in Wingless signal transduction. Traditionally, Mad has been shown to transmit
BMP signals in response to phosphorylation of its C-terminal domain. We now propose a novel role for Mad in Wg signaling, independent of its C-terminal
phosphorylation by BMP receptors. By applying both genetic and biochemical approaches we demonstrate that Mad binds to the Wg transcriptional complex
and is required for signaling in the wing imaginal disc and cell culture assays. Wg signaling is inhibited by phosphorylation of Mad by the BMP receptor
resulting from activation of the BMP pathway. The results presented here show that Mad has distinct signal transduction roles in the BMP and Wg signaling
pathways, with the outcome depending on its phosphorylation state.
188B
JNK Signaling Antagonism: The role of Raw during Drosophila dorsal closure.
Molly C. Jud, Melissa Ratcliffe, Matt Higley, Gregory B. Humphreys,
Anthea Letsou. Department of Human Genetics, University of Utah, Salt Lake City, UT.
Signaling pathways are important to several life processes including development, differentiation, growth, homeostasis, and apoptosis. One major family
of signaling pathways is the Mitogen-activated protein kinase (MAPK) cascade. The MAPK family includes ERKs, JNKs/SAPKs, and p38/HOG; these
kinases activate transcription factors in response to cell growth and/or stress signals. The positive regulation of the MAPK pathways is well characterized;
however, less is known about their negative regulation. Our lab studies the novel gene
raw
, an antagonist of JNK signaling, using embryonic dorsal closure
as a model.
raw
’s embryonic loss of function phenotype includes dorsal closure defects, hypotrophy of ventral denticle belts, and ectopic expression of the
JNK target gene,
dpp
, in cells beyond the leading edge (LE) epidermis. Using genetics, we have previously shown that
raw
is widely expressed during
embryogenesis and is required to suppress Basket (JNK)-independent AP-1 activity in the lateral epidermis of embryos undergoing dorsal closure.
Therefore, Raw functions to silence basal levels of the epidermal AP-1 transcription factor. Furthermore, we show biochemical data that activated phospho-
Jun accumulates to high levels in
raw
and
raw basket
mutant embryos, about 2.5 fold higher than wild type. Since Jun is active in
raw
mutants even in the
absence of
basket
, this indicates that another kinase is responsible for activating the AP-1 transcription factor. As Raw does not act through the JNK
signaling negative feedback loop involving the MKP, Puckered, our findings indicate Raw functions in a previously unrecognized JNK/AP-1 regulatory
system. To better understand this regulatory system, here we test: (1) whether the ectopic
dpp
expression in
raw
mutants is due to Jun mislocalization, and
(2) that a MAPK other than the
basket
-encoded JNK activates Jun in the epidermis of
raw
mutants.