Bicaudal-C (Bic-C) encodes a KH-type RNA binding protein required maternally for anterior patterning of the Drosophila oocyte and correct migration of the centripetal follicle cells. In Drosophila, premature translation of the germ-plasm determinant Oskar in Bic-C mutant oocytes suggests a function for Bic-C in post-transcriptional gene regulation. / Purification and microarray analysis of Bic-C containing ribonucleoprotein complexes revealed that Bic-C associates with multiple transcripts encoding functionally-related components of the Wnt/Frizzled/Dishevelled signaling pathway that regulate actin dynamics, in addition to its own mRNA. Using transgenic reporter constructs, Bic-C was demonstrated to destabilize its own mRNA via cis-acting 5' UTR elements. When auto-regulation was bypassed and Bic-C was over-expressed in the female germline, premature cytoplasmic streaming was induced, disrupting axial patterning through displacement of both Gurken (Grk) and oskar. These phenotypes can also be induced by disruption of the actin cytoskeleton with pharmacological agents and are similar to those described for hypomorphic mutant alleles of orb, which encodes a CPEB-like protein that promotes polyadenylation of target mRNAs. The Bic-C overexpression phenotypes require its RNA binding activity, are substantially enhanced by mutations affecting orb and poly(A) polymerase, and are suppressed by mutations affecting the deadenylase CCR4 and its accessory protein NOT3. Co-immunoprecipitation experiments demonstrate that Bic-C associates with components of the deadenylase complex and with components of an ER-associated RNP complex that includes Me31B, PABP and Trailer-hitch. The latter complex is involved in Grk exocytosis. Accordingly, Grk secretion is defective in Bic-C mutants. / Taken together, these results support a model whereby Bic-C antagonizes Orb function by negatively regulating the expression of Orb target mRNAs, through recruitment of the deadenylase machinery, that are involved in coordinating cytoplasmic movements. Furthermore, this work identifies a novel function of Bic-C in dorsal/ventral patterning by promoting Grk secretion.

I have investigated the mechanisms by which developmental asymmetry arises, using oocyte determination in Drosophila melanogaster as a model system. The Bicaudal-D (Bic-D) gene is required early in oogenesis for the asymmetric localization of specific mRNAs and proteins and for the differentiation of an oocyte from one of a cluster of 16 interconnected germarial cells. To better understand how Bic-D functions in creating this asymmetry, I took two approaches. First, I examined the role of Bic-D in the asymmetric localization of mRNA and other cellular components during later oogenesis. Second, I molecularly and genetically characterized a gene that interacts with Bic-D in oocyte determination. To determine the role of Bic-D in later oogenesis, I used an inducible source of Bic-D activity to selectively rescue the block at oocyte determination in Bic-D null mutants. Using this system, I find that Bic-D is indeed required in the later stages of oogenesis for the localization of specific mRNAs at both the anterior and posterior of the oocyte. Bic-D is also required for oocyte growth and nuclear positioning, processes which also depend on microtubules. / In the second part of this thesis, I describe the characterization of a Bic-D interacting gene which I have identified as the Drosophila homologue of the human Lissencephaly-1 (Lis-1) gene, DLis-1. Human Lis-1 is the causative gene for Miller-Dieker Syndrome and is required for neuronal migration in the developing brain, while fungal homologues have been implicated in dynein dependent nuclear migration. Like Bic-D , DLis-1 is essential for oocyte determination and for intracellular localization throughout oogenesis. DLis-1 is required for correct positioning of the oocyte nucleus, and appears to function upstream of dynein in this process. Immunolocalization studies suggest that DLis-1 functions as part of a cortical anchor that links microtubules and the oocyte nucleus, via dynein and microtubules, to the cell cortex. DLis-1 and Bic-D are also required for nuclear positioning during neural development in Drosophila, supporting a model in which the neuronal migration defects in Miller-Dieker Syndrome are due to a disruption of dynein/Lis-1 dependent nuclear migration.

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The goal of my doctoral work is to understand how proteins involved in vesicle trafficking contribute to proper animal development. To understand aspects of this process, I studied how two vesicle trafficking proteins, Liquid facets(Lqf)/epsin1 and D-Epsin-Related, affect Drosophila eye development. I determined that Lqf, an endocytosis protein, together with Fat facets (Faf), a deubiquitinating enzyme, regulate the Notch and Delta signaling in the developing Drosophila eye. Notch signaling pathway is used in most developmental processes and is dependent on its ligand Delta. Faf deubiquitinates Lqf in the signaling cells, thereby increasing Lqf protein levels and also levels of Delta endocytosis. This event is necessary for Notch activation in neighboring cells. Lqf probably works in concert with the E3 ubiquitin ligase Neuralized (Neur), which ubiquitinates Delta. These conclusions are consistent with a relatively new model describing an obligate role for endocytosis in the signaling cells to effect activation in neighboring cells. To understand how Lqf functions mechanistically in this process, I performed a structure/function analysis of the Lqf protein. Lqf proteins with strategic deletions of certain functional domains were tested for their ability to function in vivo. The major result of these experiments is that the N-terminal ENTH domain of Lqf and a protein without the ENTH domain each retain significant activity. This suggests that Lqf has two functions: the ENTH domain function and the ENTH-less function. These data are in contrast with the most popular model suggesting that ENTH-less epsins are non-functional proteins. I present possible models for how ENTH-less epsins may retain function. The final part of my thesis focuses on D-Epsin-Related (D-Epsin-R) protein. I showed that D-Epsin-R is a Golgi protein, like its homologs. Surprisingly, D-Epsin-R ENTH domain is not required for function because an ENTH-less D-Epsin-R can substitute for endogenous D-Epsin-R. Also, D-Epsin-R has essential and probably specific developmental roles in the eye as D-Epsin-R mutants exhibit impaired cell growth. This work suggests that epsins are specific components of certain developmental pathways. / text

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Cell cycle checkpoints are signal transduction pathways that control the order and timing of cell cycle transitions, ensuring that critical events are completed before the cell cycle proceeds. The Chk2 family of kinases plays a central role in mediating responses to DNA damage or DNA replication blocks in various organisms. My functional analysis of the Drosophila serine/threonine kinase Loki/Chk2 shows that fly chk2 monitors double-strand breaks caused by irradiation during S and G2 phases and induces cell cycle arrest in embryonic cells around cellularization. / loki is also required for the normal number of germ line cells to form in the embryo, and for normal modification of Vasa, a crucial factor in germ cell formation. However, during normal oogenesis loki expression is suppressed by orb. Another group described the involvement of Drosophila loki/chk2 in the meiotic pachytene checkpoint. Using our loki·null mutant, I obtained the opposite result: loki/chk2 does not have an essential function in this process. / The second part of my thesis deals with the question of how cells are instructed about their identity in a developing organism. (Abstract shortened by UMI.)

Drosophila Lasp contains a LIM domain, two nebulin repeats, and a SH3 domain, and exhibits high homology with mammalian Lasp family proteins. Vertebrate Lasp localizes to focal adhesions and to the leading edge of migrating cells and binds filamentous actin. To investigate Drosophila Lasp in vivo, we generated a Lasp null mutant, named Laspl, and showed that Laspl is male sterile. We observed two major functions of Lasp during Drosophila spermatogenesis. First, in the stem cell niche, hub cells fail to localize to the apical end of Drosophila testis in Laspl mutant. Hub cell anchoring is dependent on cell adhesion between cells and extracellular matrix (ECM), which is mediated by integrins. Lasp genetically interacts with betaPS integrin showing complete hub cell mislocalization. This indicates that Lasp is involved in an integrin-dependent process. However, hub cell anchoring is not required for fertility or stem cell maintenance. Secondly, we observe that actin cones, a unique actin structure during spermatid individualization, are perturbed in Laspl. Our data for Lasp expression in actin cones and incomplete individualization indicate that Lasp may play a role in tethering actin to the plasma membrane.

We report the cloning and characterization of valois (vls), a posterior group gene of Drosophila melanogaster , which was initially identified in a screen for female steriles. Three EMS alleles of vls contain premature stop codons in the open reading frame. Sequence analyses show the presence of WD domains in Vls and find significant similarity with the human MEP50 protein which is involved in the assembly of the splicing machinery. We did not find evidence that this function is conserved in flies yet. / We created a null mutant for vls, which shows a maternal effect lethal phenotype accompanied by posterior polarity defects in the embryos. Hemizygous vlsEMS females show a weaker, partially maternal-effect lethal and a fully penetrant grandchildless phenotype. The posterior localization of Vasa is disrupted in vlsnull ovaries, but the initial distribution of Oskar protein and mRNA appear normal. However, levels of the Short Oskar isoform responsible for pole plasm assembly are greatly reduced and Vasa appears to be differently modified post-translationally. Furthermore, a Vls::GFP fusion protein is detected all throughout oogenesis in the nurse cell and oocyte cytoplasm. Taken together, these data suggest that Vls is a cytoplasmic protein involved in the transport or activation of Vasa at the posterior of the oocyte essential for the accumulation of Short Osk.

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