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Poster Full Abstracts - Cell Biology and Signal Transduction
Poster board number is above title. The first author is the presenter
205
factors that are required for Wg secretion in polarized cells of
Drosophila
wing imaginal discs. We are addressing this using
Drosophila in vivo
RNAi
screening approaches. A focused RNAi library subset was chosen, containing genes with functions in trafficking, such as Rab-protein family member,
Exocyst complex genes, retromer and others. Approximately 451 genes from this subset were knocked down in Wg producing polarized cells of the wing
imaginal disc. From this subset screen we have identified various candidates, which show reduced secretion of Wg. We will discuss the current findings and
their implication in polarized Wg/Wnt transport.
274A
Pebble RhoGEF acts as a negative regulator of Wg/Wnt signaling.
Elisabeth R Greer, Kieran R Hendricksen, Anna T Chao, Amy Bejsovec. Dept of
Biology, Duke University, Durham, NC.
Wingless/Wnt (Wg/Wnt) signaling directs cell fate decisions in developing flies and other animal species. In humans, deregulated Wnt signaling is
associated with cancer; this highlights the importance of understanding how this developmental pathway is regulated. In previous work, we found that the
cytokinesis proteins Tumbleweed/RacGAP50C (Tum) and Pavarotti (Pav) act as negative regulators of Wg/Wnt signaling in Drosophila embryos and in
mammalian cell lines. Because Tum binds to the RhoGEF Pebble (Pbl) to organize the contractile ring during cell division, we tested whether Pbl also
interacts with Tum in regulating Wg/Wnt signaling. We find that Pbl does play a role in the negative regulation of Wg/Wnt signaling, but it does so
independently of its interaction with Tum.
pbl
loss of function mutant embryos show expanded expression of the Wg target gene,
engrailed
, and produce an
epidermal pattern with excess naked cuticle, which correlates with Wg signaling activity. Conversely,
pbl
overexpression in the embryonic epidermis
diminishes the specification of naked cuticle. Thus the
pbl
gene functions to restrict the activity of the Wg pathway. Indeed, overexpressing
pbl
or its human
homolog,
Ect2
, represses Wg/Wnt target gene expression in either Drosophila or mammalian cells, indicating that this property is highly conserved. We
show that the guanine nucleotide exchange factor (GEF) activity of Pbl or Ect2 is required for this regulation, while other protein domains important for
cytokinesis, such as the Tum binding domain, are not. In contrast to Tum and Pav regulation of the Wnt pathway, Pbl and Ect2 do not require nuclear
localization to exert their effect. Both Pbl and Ect2 function downstream of Armadillo/beta-catenin stabilization to control Wnt target gene expression, and
we find evidence that Pbl acts through a Rho family GTPase to modulate Wnt signaling. These results provide new insight into possible connections between
G protein regulation and Wnt pathway modulation.
275B
A developmental function of dWNK kinase in the regulation of canonical Wnt/β-catenin signaling.
Andreas Jenny
1
, Ekatherina Serysheva
2
, Hebist
Berhane
1
, Kubilay Demir
3
, Michael Boutros
3
, Marek Mlodzik
2
. 1) Dept Molec & Dev Biol, Albert Einstein Col Med, New York, NY; 2) Dept. of
Developmental and Regenerative Biology, Mount Sinai School of Medicine, New York, NY; 3) Signaling and Functional Genomics, German Cancer
Research Center, Heidelberg, Germany.
During vertebrate development major inductive and morphogenetic events pattern and shape the embryo. For example, canonical Wnt signaling is
necessary to specify the embryonic dorsoventral axis. Since D/V axis formation is intrinsically linked to establishment of the anteroposterior axis, Wnt
signaling is essential for the three-dimensional vertebrate body plan including the formation and positioning of organs. Furthermore, aberrant Wnt signaling
can lead to severe developmental disabilities such as heart abnormalities, ablation of the forebrain, and a variety of cancers. The canonical Wnt/β-Catenin
pathway is highly conserved from invertebrates to humans. In
Drosophila
, canonical Wnt signaling is required for segmentation and for the formation of
adult structures such as wings. A conserved and relatively upstream readout for Wnt pathway activation is phosphorylation of Dishevelled (Dsh), the major
adapter protein of Wnt signaling. We performed a systematic RNAi screen to knock-down all
Drosophila
kinases in cell culture and identified previously
known and novel kinases affecting Dsh phosphorylation. In particular, our data show that the single fly ortholog of the conserved Wnk (With No Lysine [K])
kinase family, dWNK (CG7177) modulates peak levels of canonical Wnt/β-catenin signaling. A reduction of
dwnk
activity suppresses overactivation of Wnt
signaling and dwnk mutations cause wing margin defects due to a requirement for dWnk for the activation of high threshold targets of canonical Wnt
signaling
in vivo
. Human Wnks regulate ion transport and thus cell volume in the kidney and brain, and mutations of Wnk1/4 are associated with Gordon's
syndrome (PHAII). Our studies identified an unappreciated and novel role for Wnk in early development and suggest that Wnks potentially have functions
beyond the regulation of ion homeostasis.
276C
Role of Wingless in pigment rim formation.
Sudha R Kumar, Hinaben Patel, Andrew Tomlinson. Department of Genetics & Development, Columbia
University, NY.
The eye of the fly is an ideal tissue to understand how a morphogen gradient can direct cells to various fates. Here, Wingless (Wg) diffusing into the
developing eye from the circumscribing head capsule directs the formation of three peripheral retinal specializations, each with a specific threshold response
to Wg concentration. The lowest threshold response denudes the ommatidia by removing the bristles. The intermediate response directs the formation of the
dorsal rim ommatidia (plane polarized light detectors). The highest threshold response directs the formation of the pigment rim, a thick band of pigment cells
that circumscribes the eye and optically insulates it from extraneous light rays. We are focused on how the pigment rim is formed. The outermost ommatidial
row dies, leaving the surrounding pigment cells to coalesce to form the pigment rim. In the moribund ommatidia the cone cells respond to the high levels of
Wg signaling by activating expression of Wg itself, along with Snail family transcription factors. Shortly afterwards, these ommatidia collapse and undergo
apoptosis. Here, we ask whether the expression of these proteins in the cone cells is sufficient for the collapse and death of the ommatidia, and the nature of
the death signal that directs this apoptosis. Activation of high levels of Wg throughout the developing eye converts the whole eye into pigment rim. Timed
developmental analysis showed that the death of the ommatidia occurred throughout the eye in a similar manner to that which occurs at the periphery. Thus
we can use normal ommatidia found throughout the eye as models for testing which gene expressions, in which cells, can mimic high levels of Wg signaling
and induce the collapse and apoptosis. We are conducting experiments that express Snail family transcription factors, with and without the activation of the
Wg pathway, in the cone cells to determine which aspects of pigment rim formation are regulated by these expressions. We are also investigating other
signaling pathways to gain a better insight into the death cascade triggered in response to Wg.
277A