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Poster Full Abstracts - Gametogenesis and Organogenesis
Poster board number is above title. The first author is the presenter
285
Tubulogenesis forms the neural tube, gut, and other organs. Errors in tubulogenesis lead to birth defects such as spina bifida. A Drosophila model that
resembles neural tube formation is dorsal appendage (DA) formation in the egg chamber. The DAs are made by patches of follicle cells that reorganize into
tubes, extend anteriorly over the squamous stretch cells (SCs), and secrete chorion into the tube lumens. In
bullwinkle
(
bwk
) mutants, the egg chamber forms
moose-antler shaped DAs. Mutations in
shark
, which encodes a tyrosine kinase required in the SCs, strongly enhance the
bwk
phenotype. Other components
of this pathway are unknown. Intriguingly,
shark
mRNA localizes in distinct foci at stage 10. We hypothesize that this pattern, which is transient and is
lacking in
bwk
mutants, localizes Shark protein and is important for its function. To identify differences in protein expression and phosphorylation
specifically in SCs from wild-type vs.
bwk
egg chambers, we optimized a magnetic bead cell-purification protocol for use with mass spectrometry (MS). An
initial MS trial using this method revealed a number of potential differences in expression between wild-type and bwk SCs. We will choose candidates based
on potential relevance and statistical significance over multiple MS runs, and test their function using phenotypic analysis of egg chambers in which
candidate protein expression or phosphorylation state is altered. To investigate localization of endogenous or fluorescently-tagged Shark and Shark
phosphorylation mutants relative to
shark
mRNA, we developed a method for simultaneous fluorescent
in situ
hybridization (FISH) and protein
immunostaining for the ovary. Methods that work well in other tissues do not work in ovaries. Success of our protocol in ovaries depends critically on signal
amplification, choice of blocking reagent, tissue permeabilization method, and RNase inactivation, factors that also influence FISH alone. These studies will
increase our understanding of mechanisms governing tubulogenesis and may provide insight into the basis of human developmental defects.
575B
Patterns of molecular evolution of germ line specification genes in
Drosophila
.
Abha Ahuja, Victor Zeng, Cassandra Extavour. Department of
Organismic and Evolutionary Biology, Harvard University, Cambridge, MA.
Specification of germ cells during embryogenesis is critical for reproductive success and survival of sexually reproducing animals. In
Drosophila
molecular interactions between mRNA and protein products of different germ cell specification genes are required for the proper differentiation of germ
cells. To gain insight into the molecular mechanisms underlying this process, here we assess the patterns of evolution of known germ line specification
genes. For these genes, we estimate and compare the rates of synonymous (
dS
) and non-synonymous (
dN
) substitutions between sequenced
Drosophila
species, and identify genes that show signatures of positive selection. Next, we identify groups of genes that exhibit correlated rates of evolution. Co-
evolving genes are expected to be functionally linked, and we discuss our findings in the context of known genetic and molecular interactions between these
genes. Our goal is to identify conserved gene regulatory networks involved in germ cell specification. These results provide important insight into the
modular nature of developmental processes underlying germ line specification, and will also help guide future biochemical and physical association studies
of germ line genes.
576C
A surprising role for the Anaphase Promoting Complex in sex determination.
Osamah Batiha, Varsha Padmanaban, Eric Fifield, Rami Mechael, Alison
Petrie, Andrew Swan. Biological sciences, University of Windsor, Windsor.
Anaphase Promoting Complex (APC) is an E3 ubiquitin ligase that plays important roles in the progression from metaphase to anaphase in mitosis and
meiosis by targeting cyclins and other proteins for destruction. In female meiosis, the APC requires a meiosis specific activator, Cort, in addition to the
canonical mitotic activator, Fzy. Cort expression and degradation is highly regulated as Cort is expressed in late oogenesis and is degraded in early
embryogenesis. To determine the significance of the tight control of Cort expression we mis-expressed Cort zygotically. Surprisingly, Cort mis- expression
caused a female to male sex transformation. This sex transformation is APC dependent as it is suppressed by mutations in cort that disrupt APC interaction
or by knockdown of APC genes. Based on these results we hypothesized that APC/Cort targets a protein in the sex determination pathway. Sex
determination in Drosophila depends on a cascade of differential splicing starting with Sxl which is expressed initially only in females. Sxl acts to splice its
pre-mRNA and the downstream target, tra. Tra is also a splicing factor and works with Tra-2 to splice dsx in the female mode. We used RT-PCR to show
that Cort acts downstream of tra splicing and upstream of dsx splicing. Consistent with this conclusion our genetic experiments point toward Cort acting
specifically on Tra protein. Interestingly, we found that Cort has the ability to affect Tra activity from a distant Diptera relative, which suggests that Cort
plays a conserved role in sex determination. The main question we are trying to answer now is: how a germline- specific protein like Cort can affect sex
determination, a process that does not happen until zygotic proteins are expressed in the embryo. An interesting paper by the Cline lab recently discovered a
reciprocal activation loop between Tra and Sxl; in which maternal Tra can promote Sxl splicing. Currently, we are investigating the potential germline role
of APC/Cort in keeping Tra levels low until sex determination is initiated zygotically.
577A
Hrp48 functions as a moderator of Sxl expression to allow for proper Notch expression and signaling in monomorphic organ development.
Dvora
Burshtein
1
, Yaron Suissa
1
, Yossi Kalifa
2
, Tama Dinur
1
, Patricia Graham
3
, Girish Deshpande
3
, Paul Schedl
3
, Offer Gerlitz
1
. 1) Developmental Biology and
Cancer Research, IMRIC, The Hebrew University-Hadassah Medical School, Jerusalem; 2) Department of Molecular Genetics, Weizmann Institute of
Science, Rehovot Israel; 3) Department of Molecular Biology, Princeton University, Princeton, NJ 08540, USA.
Sex-determination genes specify the sexual identity of tissues, mainly by deploying the major signaling pathways that direct cells and organ primordia. The
Notch (N) signaling pathway is central to a wide variety of contexts throughout development because of its ability to specify alternative cell fates. Recently,
Sex-lethal (Sxl), the female determinant in Drosophila melanogaster was shown to downregulate N signaling to accomplish sex-specific patterning. With the
exception of genital discs, most organ primordia follow identical developmental routes and develop in similar fashion in both sexes (monomorphic organs).
Moreover, irrespective of the sex, N signaling plays a central role during organ development. Paradoxically however, Sxl activity is essential in every female
cell to ensure the proper level of X-linked gene expression. This raises a key question as to how, during female development, N signaling escapes the
negative impact of Sxl in monomorphic tissues. We have uncovered a novel mechanism where Hrp48, an abundant essential hnRNP, functions to restrict Sxl
expression in monomorphic tissues and thus allows for proper development. Phenotypic consequences of the partial loss of hrp48 resemble that of N but are
more pronounced in females than in males. Likewise, N levels are drastically diminished only in females. Interestingly, monomorphic female tissues
including wing, eye and antennal discs display considerable increase in Sxl amounts. Lastly, female-specific attenuation of N signaling is rescued upon
simultaneous removal of Sxl. Our findings bring into focus the critical role played by general homeostatic factors in specification of diverse cell fates and