A critical step in animal development is the specification of primordial germ cells (PGCs), the precursors of the germline. Two seemingly mutually exclusive mechanisms are implemented across the animal kingdom: epigenesis and preformation. In epigenesis, PGC specification is non-autonomous and depends on extrinsic signaling pathways. The BMP pathway provides the key PGC specification signals in mammals. Preformation is autonomous and mediated by determinants localized within PGCs. In Drosophila, a classic example of preformation, constituents of the germ plasm localized at the embryonic posterior are thought to be both necessary and sufficient for proper determination of PGCs. Contrary to this longstanding model, here we show that these localized determinants are insufficient by themselves to direct PGC specification in blastoderm stage embryos. Instead, we find that the BMP signaling pathway is required at multiple steps during the specification process and functions in conjunction with components of the germ plasm to orchestrate PGC fate.

CPEB proteins are conserved translation regulators involved in multiple biological processes. One of these proteins in Drosophila, Orb2, is a principal player in spermatogenesis. It is required for meiosis and spermatid differentiation. During the later process, orb2 mRNA and protein are localized within the developing spermatid. To evaluate the role of the orb2 mRNA 3'UTR in spermatogenesis, we used the CRISPR/Cas9 system to generate a deletion of the orb2 3'UTR, orb2R. This deletion disrupts the process of spermatid differentiation but has no apparent effect on meiosis. Differentiation abnormalities include defects in the initial polarization of the 64-cell spermatid cysts, mislocalization of mRNAs and proteins in the elongating spermatid tails, altered morphology of the elongating spermatid tails, and defects in the assembly of the individualization complex. These disruptions in differentiation appear to arise because orb2 mRNA and protein are not properly localized within the 64-cell spermatid cyst.

BACKGROUND: Epigenetic memory plays a critical role in the establishment and maintenance of cell identities in multicellular organisms. Polycomb and trithorax group (PcG and TrxG) proteins are responsible for epigenetic memory, and in flies, they are recruited to specialized DNA regulatory elements termed polycomb response elements (PREs). Previous transgene studies have shown that PREs can silence reporter genes outside of their normal context, often by pairing sensitive (PSS) mechanism; however, their silencing activity is non-autonomous and depends upon the surrounding chromatin context. It is not known why PRE activity depends on the local environment or what outside factors can induce silencing. RESULTS: Using an attP system in Drosophila, we find that the so-called neutral chromatin environments vary substantially in their ability to support the silencing activity of the well-characterized bxdPRE. In refractory chromosomal contexts, factors required for PcG-silencing are unable to gain access to the PRE. Silencing activity can be rescued by linking the bxdPRE to a boundary element (insulator). When placed next to the PRE, the boundaries induce an alteration in chromatin structure enabling factors critical for PcG silencing to gain access to the bxdPRE. When placed at a distance from the bxdPRE, boundaries induce PSS by bringing the bxdPREs on each homolog in close proximity. CONCLUSION: This proof-of-concept study demonstrates that the repressing activity of PREs can be induced or enhanced by nearby boundary elements.

The Drosophila GAGA factor (GAF) is a multifunctional protein implicated in nucleosome organization and remodeling, activation and repression of gene expression, long distance enhancer-promoter communication, higher order chromosome structure, and mitosis. This broad range of activities poses questions about how a single protein can perform so many seemingly different and unrelated functions. Current studies argue that GAF acts as a "pioneer" factor, generating nucleosome-free regions of chromatin for different classes of regulatory elements. The removal of nucleosomes from regulatory elements in turn enables other factors to bind to these elements and carry out their specialized functions. Consistent with this view, GAF associates with a collection of chromatin remodelers and also interacts with proteins implicated in different regulatory functions. In this review, we summarize the known activities of GAF and the functions of its protein partners.

Posttranscriptional gene regulation includes mRNA transport, localization, translation, and regulation of mRNA stability. CPEB (cytoplasmic polyadenylation element binding) family proteins bind to specific sites within the 3'-untranslated region and mediate poly- and deadenylation of transcripts, activating or repressing protein synthesis. As part of ribonucleoprotein complexes, the CPEB proteins participate in mRNA transport and localization to different sub-cellular compartments. The CPEB proteins are evolutionarily conserved and have similar functions in vertebrates and invertebrates. In the nervous system, the CPEB proteins are involved in cell division, neural development, learning, and memory. Here we consider the functional features of these proteins in the nervous system of phylogenetically distant organisms: Drosophila, a well-studied model, and mammals. Disruption of the CPEB proteins functioning is associated with various pathologies, such as autism spectrum disorder and brain cancer. At the same time, CPEB gene regulation can provide for a recovery of the brain function in patients with fragile X syndrome and Huntington's disease, making the CPEB genes promising targets for gene therapy.

The chromatin remodeler SWI/SNF is an important participant in gene activation, functioning predominantly by opening the chromatin structure on promoters and enhancers. Here, we describe its novel mode of action in which SWI/SNF factors mediate the targeted action of an enhancer. We studied the functions of two signature subunits of PBAP subfamily, BAP170 and SAYP, in . These subunits were stably tethered to a transgene reporter carrying the core promoter. The tethered subunits mediate transcription of the reporter in a pattern that is generated by enhancers close to the insertion site in multiple loci throughout the genome. Both tethered SAYP and BAP170 recruit the whole PBAP complex to the reporter promoter. However, we found that BAP170-dependent transcription is more resistant to the depletion of other PBAP subunits, suggesting that BAP170 may play a more critical role in establishing enhancer-dependent transcription.

The autonomy of segment-specific regulatory domains in the Bithorax complex is conferred by boundary elements and associated Polycomb response elements (PREs). The Fab-6 boundary is located at the junction of the iab-5 and iab-6 domains. Previous studies mapped it to a nuclease hypersensitive region 1 (HS1), while the iab-6 PRE was mapped to a second hypersensitive region HS2 nearly 3 kb away. To analyze the role of HS1 and HS2 in boundary we generated deletions of HS1 or HS1 + HS2 that have attP site for boundary replacement experiments. The 1389 bp HS1 deletion can be rescued by a 529 bp core Fab-6 sequence that includes two CTCF sites. However, Fab-6 HS1 cannot rescue the HS1 + HS2 deletion or substitute for another BX-C boundary - Fab-7. For this it must be combined with a PRE, either Fab-7 HS3, or Fab-6 HS2. These findings suggest that the boundary function of Fab-6 HS1 must be bolstered by a second element that has PRE activity.

Since the seminal 1961 paper of Monod and Jacob, mathematical models of biomolecular circuits have guided our understanding of cell regulation. Model-based exploration of the functional capabilities of any given circuit requires systematic mapping of multidimensional spaces of model parameters. Despite significant advances in computational dynamical systems approaches, this analysis remains a nontrivial task. Here, we use a nonlinear system of ordinary differential equations to model oocyte selection in Drosophila, a robust symmetry-breaking event that relies on autoregulatory localization of oocyte-specification factors. By applying an algorithmic approach that implements symbolic computation and topological methods, we enumerate all phase portraits of stable steady states in the limit when nonlinear regulatory interactions become discrete switches. Leveraging this initial exact partitioning and further using numerical exploration, we locate parameter regions that are dense in purely asymmetric steady states when the nonlinearities are not infinitely sharp, enabling systematic identification of parameter regions that correspond to robust oocyte selection. This framework can be generalized to map the full parameter spaces in a broad class of models involving biological switches.

Transcriptional quiescence, an evolutionarily conserved trait, distinguishes the embryonic primordial germ cells (PGCs) from their somatic neighbors. In , PGCs from embryos maternally compromised for () misexpress somatic genes, possibly resulting in PGC loss. Recent studies documented a requirement for Gcl during proteolytic degradation of the terminal patterning determinant, Torso receptor. Here we demonstrate that the somatic determinant of female fate, (), is a biologically relevant transcriptional target of Gcl. Underscoring the significance of transcriptional silencing mediated by Gcl, ectopic expression of a degradation-resistant form of Torso () can activate transcription in PGCs, whereas simultaneous loss of () reinstates the quiescent status of PGCs. Intriguingly, like mutants, embryos derived from mothers expressing in the germline display aberrant spreading of pole plasm RNAs, suggesting that mutual antagonism between Gcl and Torso ensures the controlled release of germ-plasm underlying the germline/soma distinction.

Embryonic patterning is critically dependent on zygotic genome activation (ZGA). In Drosophila melanogaster embryos, the pioneer factor Zelda directs ZGA, possibly in conjunction with other factors. Here, we have explored the novel involvement of Chromatin-Linked Adapter for MSL Proteins (CLAMP) during ZGA. CLAMP binds thousands of sites genome-wide throughout early embryogenesis. Interestingly, CLAMP relocates to target promoter sequences across the genome when ZGA is initiated. Although there is a considerable overlap between CLAMP and Zelda binding sites, the proteins display distinct temporal dynamics. To assess whether CLAMP occupancy affects gene expression, we analyzed transcriptomes of embryos zygotically compromised for either clamp or zelda and found that transcript levels of many zygotically activated genes are similarly affected. Importantly, compromising either clamp or zelda disrupted the expression of critical segmentation and sex determination genes bound by CLAMP (and Zelda). Furthermore, clamp knockdown embryos recapitulate other phenotypes observed in Zelda-depleted embryos, including nuclear division defects, centrosome aberrations, and a disorganized actomyosin network. Based on these data, we propose that CLAMP acts in concert with Zelda to regulate early zygotic transcription.

The Abdominal-B (Abd-B) gene belongs to the bithorax complex and its expression is controlled by four regulatory domains, iab-5, iab-6, iab-7 and iab-8, each of which is thought to be responsible for directing the expression of Abd-B in one of the abdominal segments from A5 to A8. A variety of experiments have supported the idea that BX-C regulatory domains are functionally autonomous and that each domain is both necessary and sufficient to orchestrate the development of the segment they specify. Unexpectedly, we discovered that this model does not always hold. Instead, we find that tissue-specific enhancers located in the iab-5 domain are required for the proper activation of Abd-B not only in A5 but also in A6. Our findings indicate that the functioning of the iab-5 and iab-6 domains in development of the adult cuticle A5 and A6 in males fit better with an additive model, much like that first envisioned by Ed Lewis.

REDOX mechanisms that induce biosynthesis of the reactive oxygen species (ROS) have attracted considerable attention due to both the deleterious and beneficial responses elicited by the reactive radical. In several organisms including Drosophila melanogaster, modulation of ROS activity is thought to be crucial for the maintenance of cell fates in developmental contexts. Interestingly, REDOX mechanisms have been shown to be involved in maintaining progenitor fate of stem cells as well as their proliferation and differentiation. Here, we have explored the possible functions of ROS during proper specification and developmental progression of embryonic primordial germ cells (PGCs). Indicating its potential involvement in these processes, ROS can be detected in the embryonic PGCs and the surrounding somatic cells from very early stages of embryogenesis. Using both "loss" and "gain" of function mutations in two different components of the REDOX pathway, we show that ROS levels are likely to be critical in maintaining germ cell behavior, including their directed migration. Altering the activity of a putative regulator of ROS also adversely influences the ability of PGCs to adhere to one another in cellular blastoderm embryos, suggesting potential involvement of this pathway in orchestrating different phases of germ cell migration.

In mammals, a C2H2 zinc finger (C2H2) protein, CTCF, acts as the master regulator of chromosomal architecture and of the expression of Hox gene clusters. Like mammalian CTCF, the homolog, dCTCF, localizes to boundaries in the bithorax complex (BX-C). Here, we have determined the minimal requirements for the assembly of a functional boundary by dCTCF and two other C2H2 zinc finger proteins, Pita and Su(Hw). Although binding sites for these proteins are essential for the insulator activity of BX-C boundaries, these binding sites alone are insufficient to create a functional boundary. dCTCF cannot effectively bind to a single recognition sequence in chromatin or generate a functional insulator without the help of additional proteins. In addition, for boundary elements in BX-C at least four binding sites for dCTCF or the presence of additional DNA binding factors is required to generate a functional insulator.

encodes one of the two fly CPEB proteins. These widely conserved proteins bind to the 3'UTRs of target messenger RNAs (mRNAs) and activate or repress their translation. We show here that a positive autoregulatory loop driven by the gene propels the specification of oocyte identity in egg chambers. Oocyte fate specification is mediated by a 3'UTR-dependent mechanism that concentrates mRNAs and proteins in one of the two pro-oocytes in the 16-cell germline cyst. When the 3'UTR is deleted, mRNA and protein fail to localize and all 16 cells become nurse cells. In wild type, the oocyte is specified when and other gene products concentrate in a single cell in region 2b of the germarium. A partially functional 3'UTR replacement delays oocyte specification until the egg chambers reach stage 2 of oogenesis. Before this point, mRNA and protein are unlocalized, as are other markers of oocyte identity, and the oocyte is not specified. After stage 2, ∼50% of the chambers successfully localize in a single cell, and this cell assumes oocyte identity. In the remaining chambers, the autoregulatory loop is not activated and no oocyte is formed. Finally, maintenance of oocyte identity requires continuous activity.

Boundaries in the bithorax complex (BX-C) enable the regulatory domains that drive parasegment-specific expression of the three genes to function autonomously. The four regulatory domains (, , , and ) that control the expression of the () gene are located downstream of the transcription unit, and are delimited by the , , , and boundaries. These boundaries function to block cross talk between neighboring regulatory domains. In addition, three of the boundaries (, , and ) must also have bypass activity so that regulatory domains distal to the boundaries can contact the promoter. In the studies reported here, we have undertaken a functional dissection of the boundary using a boundary-replacement strategy. Our studies indicate that the boundary has two separable subelements. The distal subelement blocks cross talk, but cannot support bypass. The proximal subelement has only minimal blocking activity but is able to mediate bypass. A large multiprotein complex, the LBC (large boundary complex), binds to sequences in the proximal subelement and contributes to its bypass activity. The same LBC complex has been implicated in the bypass activity of the boundary.

Boundaries in the complex (BX-C) delimit autonomous regulatory domains that drive parasegment-specific expression of the genes , and The boundary is located between the and domains and has two key functions: blocking cross-talk between these domains and at the same time promoting communication (boundary bypass) between and the promoter. Using a replacement strategy, we found that multimerized binding sites for the architectural proteins Pita, Su(Hw), and dCTCF function as conventional insulators and block cross-talk between the and domains; however, they lack bypass activity, and is unable to regulate Here we show that an ∼200-bp sequence of dHS1 from the boundary rescues the bypass defects of these multimerized binding sites. The dHS1 sequence is bound in embryos by a large multiprotein complex, Late Boundary Complex (LBC), that contains the zinc finger proteins CLAMP and GAF. Using deletions and mutations in critical GAGAG motifs, we show that bypass activity correlates with the efficiency of recruitment of LBC components CLAMP and GAF to the artificial boundary. These results indicate that LBC orchestrates long-distance communication between the regulatory domain and the gene, while the Pita, Su(Hw), and dCTCF proteins function to block local cross-talk between the neighboring regulatory domains and .

During embryonic gonad coalescence, primordial germ cells (PGCs) follow a carefully choreographed migratory route circumscribed by guidance signals towards somatic gonadal precursor cells (SGPs). In , SGP-derived Hedgehog (Hh), which serves as a guidance cue for the PGCs, is potentiated by mesodermally restricted HMGCoA-reductase (Hmgcr) and the ABC transporter Multi-drug-resistant-49 (Mdr49). Given the importance of cholesterol modification in the processing and long-distance transmission of the Hh ligand, we have analyzed the involvement of the Niemann-Pick disease type C-1a (NPC1a) protein, a cholesterol transporter, in germ cell migration and Hedgehog signaling. We show that mesoderm-specific inactivation of results in germ cell migration defects. Similar to , PGC migration defects in the embryos are ameliorated by a cholesterol-rich diet. Consistently, reduction in weakens the ability of ectopic HMG Coenzyme A reductase () to induce germ cell migration defects. Moreover, compromising levels influences Hh signaling adversely during wing development, a process that relies upon long-range Hh signaling. Last, doubly heterozygous embryos () display enhanced germ cell migration defects when compared with single mutants ( or ), supporting cooperative interaction between the two.

The Paip2 protein is a factor regulating mRNA translation and stability in the cytoplasm. It has also been found in the nuclei of several cell types in Drosophila. Here, we aim to elucidate the functions of Paip2 in the cell nucleus. We find that nuclear Paip2 is a component of an ~300-kDa protein complex. Paip2 interacts with mRNA capping factor and factors of RNA polymerase II (Pol II) transcription initiation and early elongation. Paip2 functionally cooperates with the Cbp80 subunit of the cap-binding complex, with both proteins ensuring proper Pol II C-terminal domain (CTD) Ser5 phosphorylation at the promoter. Thus, Paip2 is a novel player at the stage of mRNA capping and early Pol II elongation.

BACKGROUND: Boundaries in the Drosophila bithorax complex delimit autonomous regulatory domains that activate the parasegment (PS)-specific expression of homeotic genes. The Fab-7 boundary separates the iab-6 and iab-7 regulatory domains that control Abd-B expression in PS11 and PS12. This boundary is composed of multiple functionally redundant elements and has two key activities: it blocks crosstalk between iab-6 and iab-7 and facilitates boundary bypass. RESULTS: Here, we have used a structure-function approach to elucidate the biochemical properties and the in vivo activities of a conserved BEN domain protein, Insensitive, that is associated with Fab-7. Our biochemical studies indicate that in addition to the C-terminal BEN DNA-binding domain, Insv has two domains that mediate multimerization: one is a coiled-coil domain in the N-terminus, and the other is next to the BEN domain. These multimerization domains enable Insv to bind simultaneously to two canonical 8-bp recognition motifs, as well as to a ~ 100-bp non-canonical recognition sequence. They also mediate the assembly of higher-order multimers in the presence of DNA. Transgenic proteins lacking the N-terminal coiled-coil domain are compromised for boundary function in vivo. We also show that Insv interacts directly with CP190, a protein previously implicated in the boundary functions of several DNA-binding proteins, including Su(Hw) and dCTCF. While CP190 interaction is required for Insv binding to a subset of sites on polytene chromosomes, it has only a minor role in the boundary activity of Insv in the context of Fab-7. CONCLUSIONS: The subdivision of eukaryotic chromosomes into discrete topological domains depends upon the pairing of boundary elements. In flies, pairing interactions are specific and typically orientation dependent. They occur in cis between neighboring heterologous boundaries, and in trans between homologous boundaries. One potential mechanism for ensuring pairing-interaction specificity is the use of sequence-specific DNA-binding proteins that can bind simultaneously with two or more recognition sequences. Our studies indicate that Insv can assemble into a multivalent DNA-binding complex and that the N-terminal Insv multimerization domain is critical for boundary function.

Drosophila bithorax complex (BX-C) is one of the best model systems for studying the role of boundaries (insulators) in gene regulation. Expression of three homeotic genes, Ubx, abd-A, and Abd-B, is orchestrated by nine parasegment-specific regulatory domains. These domains are flanked by boundary elements, which function to block crosstalk between adjacent domains, ensuring that they can act autonomously. Paradoxically, seven of the BX-C regulatory domains are separated from their gene target by at least one boundary, and must "jump over" the intervening boundaries. To understand the jumping mechanism, the Mcp boundary was replaced with Fab-7 and Fab-8. Mcp is located between the iab-4 and iab-5 domains, and defines the border between the set of regulatory domains controlling abd-A and Abd-B. When Mcp is replaced by Fab-7 or Fab-8, they direct the iab-4 domain (which regulates abd-A) to inappropriately activate Abd-B in abdominal segment A4. For the Fab-8 replacement, ectopic induction was only observed when it was inserted in the same orientation as the endogenous Fab-8 boundary. A similar orientation dependence for bypass activity was observed when Fab-7 was replaced by Fab-8. Thus, boundaries perform two opposite functions in the context of BX-C-they block crosstalk between neighboring regulatory domains, but at the same time actively facilitate long distance communication between the regulatory domains and their respective target genes.

In most animal species, newly formed primordial germ cells (PGCs) acquire the special characteristics that distinguish them from the surrounding somatic cells. Proper fate specification of the PGCs is coupled with transcriptional quiescence, whether they are segregated by determinative or inductive mechanisms. Inappropriate differentiation of PGCs into somatic cells is thought to be prevented due to repression of RNA polymerase (Pol) II-dependent transcription. In the case of a determinative mode of PGC formation (Drosophila, Caenorhabditis elegans, etc.), there is a broad downregulation of Pol II activity. By contrast, PGCs display only gene-specific repression in organisms that rely on inductive signaling-based mechanism (e.g., mice). In addition to the global block of Pol II activity in PGCs, gene expression can be suppressed in other ways, such as chromatin remodeling and Piwi-mediated RNAi. Here, we discuss the mechanisms responsible for the transcriptionally silent state of PGCs in common experimental animals, such as Drosophila, C. elegans, Danio rerio, Xenopus, and mouse. While a PGC-specific downregulation of transcription is a common feature among these organisms, the diverse nature of underlying mechanisms suggests that this functional trait likely evolved independently on several instances. We discuss the possible biological relevance of these silencing mechanisms vis-a-vis fate determination of PGCs.

Boundaries (insulators) in the bithorax complex (BX-C) delimit autonomous regulatory domains that orchestrate the parasegment (PS)-specific expression of the BX-C homeotic genes. The boundary separates the and regulatory domains, which control Abd-B expression in PS11 and PS12, respectively. This boundary is composed of multiple functionally redundant elements and has two key functions: it blocks cross talk between and and facilitates boundary bypass. Here, we show that two BEN domain protein complexes, Insensitive and Elba, bind to multiple sequences located in the nuclease hypersensitive regions. Two of these sequences are recognized by both Insv and Elba and correspond to a CCAATTGG palindrome. Elba also binds to a related CCAATAAG sequence, while Insv does not. However, the third Insv recognition sequences is ∼100 bp in length and contains the CCAATAAG sequence at one end. Both Insv and Elba are assembled into large complexes (∼420 and ∼265-290 kDa, respectively) in nuclear extracts. Using a sensitized genetic background, we show that the Insv protein is required for boundary function and that PS11 identity is not properly established in mutants. This is the first demonstration that a BEN domain protein is important for the functioning of an endogenous fly boundary.

Expression of the three bithorax complex homeotic genes is orchestrated by nine parasegment-specific regulatory domains. Autonomy of each domain is conferred by boundary elements (insulators). Here, we have used an in situ replacement strategy to reanalyze the sequences required for the functioning of one of the best-characterized fly boundaries, Fab-7. It was initially identified by a deletion, Fab-71, that transformed parasegment (PS) 11 into a duplicate copy of PS12. Fab-71 deleted four nuclease hypersensitive sites, HS*, HS1, HS2, and HS3, located between the iab-6 and iab-7 regulatory domains. Transgenic and P-element excision experiments mapped the boundary to HS*+HS1+HS2, while HS3 was shown to be the iab-7 Polycomb response element (PRE). Recent replacement experiments showed that HS1 is both necessary and sufficient for boundary activity when HS3 is also present in the replacement construct. Surprisingly, while HS1+HS3 combination has full boundary activity, we discovered that HS1 alone has only minimal function. Moreover, when combined with HS3, only the distal half of HS1, dHS1, is needed. A ~1,000 kD multiprotein complex containing the GAF protein, called the LBC, binds to the dHS1 sequence and we show that mutations in dHS1, that disrupt LBC binding in nuclear extracts, eliminate boundary activity and GAF binding in vivo. HS3 has binding sites for GAF and Pho proteins that are required for PRE silencing. In contrast, HS3 boundary activity only requires the GAF binding sites. LBC binding with HS3 in nuclear extracts, and GAF association in vivo, depend upon the HS3 GAF sites, but not the Pho sites. Consistent with a role for the LBC in HS3 boundary activity, the boundary function of the dHS1+HS3mPho combination is lost when the flies are heterozygous for a mutation in the GAF gene. Taken together, these results reveal a novel function for the iab-7 PREs in chromosome architecture.

Paip2 (Poly(A)-binding protein - interacting protein 2) is a conserved metazoan-specific protein that has been implicated in regulating the translation and stability of mRNAs. However, we have found that Paip2 is not restricted to the cytoplasm but is also found in the nucleus in Drosophila embryos, salivary glands, testes, and tissue culture cells. Nuclear Paip2 is associated with chromatin, and in chromatin immunoprecipitation experiments it maps to the promoter regions of active genes. However, this chromatin association is indirect, as it is RNA-dependent. Thus, Paip2 is one more item in the growing list of translation factors that are recruited to mRNAs co-transcriptionally.

As collective cell migration is intimately involved in different aspects of metazoan development, molecular mechanisms underlying this process are being explored in a variety of developmental contexts. Border cell (BC) migration during oogenesis has emerged as an excellent genetic model for studying collective cell migration. BCs are of epithelial origin but acquire partial mesenchymal characteristics before migrating as a group towards the oocyte. Here, we report that insulin signaling modulates collective BC movement during oogenesis. Supporting the involvement of Insulin pathway, we demonstrate that compromising Insulin-like Receptor (InR) levels in BCs, inhibits their migration. Furthermore, we show that canonical Insulin signaling pathway components participate in this process. Interestingly, visualization of -depleted BC clusters, using time-lapse imaging, revealed a delay in detachment of BC clusters from the surrounding anterior follicle cells and altered protrusion dynamics. Lastly, based on genetic interactions between , the polarity determinant, and a regulatory subunit of Myosin (), we propose that Insulin signaling likely influences activity to engineer border cell detachment and subsequent movement via Myosin.

Proper specification of germline stem cells (GSCs) in ovaries depends on niche derived non-autonomous signaling and cell autonomous components of transcriptional machinery. Stonewall (Stwl), a MADF-BESS family protein, is one of the cell intrinsic transcriptional regulators involved in the establishment and/or maintenance of GSC fate in ovaries. Here we report identification and functional characterization of another member of the same protein family, CG3838/ Brickwall (Brwl) with analogous functions. Loss of function alleles of exhibit age dependent progressive degeneration of the developing ovarioles and loss of GSCs. Supporting the conclusion that the structural deterioration of mutant egg chambers is a result of apoptotic cell death, activated caspase levels are considerably elevated in ovaries. Moreover, as in the case of mutants, on several instances, loss of activity results in fusion of egg chambers and misspecification of the oocyte. Importantly, phenotypes can be partially rescued by germline specific over-expression of arguing for overlapping yet distinct functional capabilities of the two proteins. Taken together with our phylogenetic analysis, these data suggest that and likely share a common MADF-BESS ancestor and they are expressed in overlapping spatiotemporal domains to ensure robust development of the female germline.

Chromatin entry sites (CES) are 100- to 1,500-bp elements that recruit male-specific lethal (MSL) complexes to the X chromosome to upregulate expression of X-linked genes in male flies. CES contain one or more ∼20-bp GA-rich sequences called MSL recognition elements (MREs) that are critical for dosage compensation. Recent studies indicate that CES also correspond to boundaries of X-chromosomal topologically associated domains (TADs). Here, we show that an ∼1,000-kDa complex called the late boundary complex (LBC), which is required for the functioning of the Bithorax complex boundary , interacts specifically with a special class of CES that contain multiple MREs. Mutations in the MRE sequences of three of these CES that disrupt function abrogate interactions with the LBC. Moreover, reducing the levels of two LBC components compromises MSL recruitment. Finally, we show that several of the CES that are physically linked to each other are LBC interactors.

Boundaries in the Bithorax complex (BX-C) of delimit autonomous regulatory domains that drive parasegment-specific expression of homeotic genes. BX-C boundaries have two crucial functions: they must block crosstalk between adjacent regulatory domains and at the same time facilitate boundary bypass. The C2H2 zinc-finger protein Pita binds to several BX-C boundaries, including and To study Pita functions, we have used a boundary replacement strategy by substituting modified DNAs for the boundary, which is located between the and regulatory domains. Multimerized Pita sites block crosstalk but fail to support regulation of (bypass). In the case of , we used a novel sensitized background to show that the two Pita-binding sites contribute to its boundary function. Although is from BX-C, it does not function appropriately when substituted for : it blocks crosstalk but does not support bypass. Mutation of the Pita site disrupts blocking activity and also eliminates dCTCF binding. In contrast, mutation of the dCTCF site does not affect Pita binding, and this mutant boundary retains partial function.

In Drosophila melanogaster the progenitors of the germ-line stem cells, the primordial germ cells (PGCs) are formed on the outside surface of the early embryo, while the somatic gonadal precursor cells (SGPs) are specified during mid-embryogenesis. To form the primitive embryonic gonad, the PGCs travel from outside of the embryo, across the mid-gut and then migrate through the mesoderm to the SGPs. The migratory path of PGCs is dictated by a series of attractive and repulsive cues. Studies in our laboratory have shown that one of the key chemoattractants is the Hedgehog (Hh) ligand. Although, Hh is expressed in other cell types, the long-distance transmission of this ligand is specifically potentiated in the SGPs by the hmgcr isoprenoid biosynthetic pathway. The distant transmission of the Hh ligand is gated by restricting expression of hmgcr to the SGPs. This is particularly relevant in light of the recent findings that an ABC transporter, mdr49 also acts in a mesoderm specific manner to release the germ cell attractant. Our studies have demonstrated that mdr49 functions in hh signaling likely via its role in the transport of cholesterol. Given the importance of cholesterol in the processing and long distance transmission of the Hh ligand, this observation has opened up an exciting avenue concerning the possible role of components of the sterol transport machinery in PGC migration.

The Drosophila GAGA factor (GAF) has an extraordinarily diverse set of functions that include the activation and silencing of gene expression, nucleosome organization and remodeling, higher order chromosome architecture and mitosis. One hypothesis that could account for these diverse activities is that GAF is able to interact with partners that have specific and dedicated functions. To test this possibility we used affinity purification coupled with high throughput mass spectrometry to identify GAF associated partners. Consistent with this hypothesis the GAF interacting network includes a large collection of factors and complexes that have been implicated in many different aspects of gene activity, chromosome structure and function. Moreover, we show that GAF interactions with a small subset of partners is direct; however for many others the interactions could be indirect, and depend upon intermediates that serve to diversify the functional capabilities of the GAF protein.

Chromosomes in multicellular animals are subdivided into a series of looped domains. In addition to being the underlying principle for organizing the chromatin fiber, looping is critical for processes ranging from gene regulation to recombination and repair. The subdivision of chromosomes into looped domains depends upon a special class of architectural elements called boundaries or insulators. These elements are distributed throughout the genome and are ubiquitous building blocks of chromosomes. In this review, we focus on features of boundaries that are critical in determining the topology of the looped domains and their genetic properties. We highlight the properties of fly boundaries that are likely to have an important bearing on the organization of looped domains in vertebrates, and discuss the functional consequences of the observed similarities and differences.

The primordial germ cells (PGCs) specified during embryogenesis serve as progenitors to the adult germline stem cells. In Drosophila, the proper specification and formation of PGCs require both centrosomes and germ plasm, which contains the germline determinants. Centrosomes are microtubule (MT)-organizing centers that ensure the faithful segregation of germ plasm into PGCs. To date, mechanisms that modulate centrosome behavior to engineer PGC development have remained elusive. Only one germ plasm component, Germ cell-less (Gcl), is known to play a role in PGC formation. Here, we show that Gcl engineers PGC formation by regulating centrosome dynamics. Loss of gcl leads to aberrant centrosome separation and elaboration of the astral MT network, resulting in inefficient germ plasm segregation and aborted PGC cellularization. Importantly, compromising centrosome separation alone is sufficient to mimic the gcl loss-of-function phenotypes. We conclude Gcl functions as a key regulator of centrosome separation required for proper PGC development.

Chromatin boundary elements subdivide chromosomes in multicellular organisms into physically independent domains. In addition to this architectural function, these elements also play a critical role in gene regulation. Here we investigated the evolution of a Drosophila Bithorax complex boundary element called Fab-7, which is required for the proper parasegment specific expression of the homeotic Abd-B gene. Using a "gene" replacement strategy, we show that Fab-7 boundaries from two closely related species, D. erecta and D. yakuba, and a more distant species, D. pseudoobscura, are able to substitute for the melanogaster boundary. Consistent with this functional conservation, the two known Fab-7 boundary factors, Elba and LBC, have recognition sequences in the boundaries from all species. However, the strategies used for maintaining binding and function in the face of sequence divergence is different. The first is conventional, and depends upon conservation of the 8 bp Elba recognition sequence. The second is unconventional, and takes advantage of the unusually large and flexible sequence recognition properties of the LBC boundary factor, and the deployment of multiple LBC recognition elements in each boundary. In the former case, binding is lost when the recognition sequence is altered. In the latter case, sequence divergence is accompanied by changes in the number, relative affinity, and location of the LBC recognition elements.

The chromosomes of multicellular animals are organized into a series of topologically independent looped domains. This domain organization is critical for the proper utilization and propagation of the genetic information encoded by the chromosome. A special set of architectural elements, called boundaries or insulators, are responsible both for subdividing the chromatin into discrete domains and for determining the topological organization of these domains. Central to the architectural functions of insulators are homologous and heterologous insulator:insulator pairing interactions. The former (pairing between copies of the same insulator) dictates the process of homolog alignment and pairing in trans, while the latter (pairing between different insulators) defines the topology of looped domains in cis. To elucidate the principles governing these architectural functions, we use two insulators, Homie and Nhomie, that flank the Drosophila even skipped locus. We show that homologous insulator interactions in trans, between Homie on one homolog and Homie on the other, or between Nhomie on one homolog and Nhomie on the other, mediate transvection. Critically, these homologous insulator:insulator interactions are orientation-dependent. Consistent with a role in the alignment and pairing of homologs, self-pairing in trans is head-to-head. Head-to-head self-interactions in cis have been reported for other fly insulators, suggesting that this is a general principle of self-pairing. Homie and Nhomie not only pair with themselves, but with each other. Heterologous Homie-Nhomie interactions occur in cis, and we show that they serve to delimit a looped chromosomal domain that contains the even skipped transcription unit and its associated enhancers. The topology of this loop is defined by the heterologous pairing properties of Homie and Nhomie. Instead of being head-to-head, which would generate a circular loop, Homie-Nhomie pairing is head-to-tail. Head-to-tail pairing in cis generates a stem-loop, a configuration much like that observed in classical lampbrush chromosomes. These pairing principles provide a mechanistic underpinning for the observed topologies within and between chromosomes.

How cell polarity is established and maintained is an important question in diverse biological contexts. Molecular mechanisms used to localize polarity proteins to distinct domains are likely context-dependent and provide a feedback loop in order to maintain polarity. One such mechanism is the localized translation of mRNAs encoding polarity proteins, which will be the focus of this review and may play a more important role in the establishment and maintenance of polarity than is currently known. Localized translation of mRNAs encoding polarity proteins can be used to establish polarity in response to an external signal, and to maintain polarity by local production of polarity determinants. The importance of this mechanism is illustrated by recent findings, including orb2-dependent localized translation of aPKC mRNA at the apical end of elongating spermatid tails in the Drosophila testis, and the apical localization of stardust A mRNA in Drosophila follicle and embryonic epithelia.

The embryonic gonad of Drosophila melanogaster begins to display sexually dimorphic traits soon after its formation. Here we demonstrate the involvement of a wnt family ligand, wnt-2, in the induction of these sex-specific differences. We show that wnt-2 contributes to the survival of a male-specific population of somatic gonadal precursor cells (SGPs), the male-specific SGPs that are located at the posterior of the male gonad. We also show that the Wnt-2 ligand synergizes with the JAK-STAT ligand Upd, which is produced by SGPs at the anterior of the gonad to activate the STAT pathway in male germ cells. We suggest that the use of two spatially separated signaling systems to initiate the JAK-STAT stem cell maintenance pathway in germ cells provides a mechanism for increasing the pool of potential progenitors of the germline stem cells in the adult testes. Finally, we present evidence indicating that, like the JAK-STAT pathway, wnt-2 stimulates germ cells in male embryos to re-enter the cell cycle.

Functionally autonomous regulatory domains direct the parasegment-specific expression of the Drosophila Bithorax complex (BX-C) homeotic genes. Autonomy is conferred by boundary/insulator elements that separate each regulatory domain from its neighbors. For six of the nine parasegment (PS) regulatory domains in the complex, at least one boundary is located between the domain and its target homeotic gene. Consequently, BX-C boundaries must not only block adventitious interactions between neighboring regulatory domains, but also be permissive (bypass) for regulatory interactions between the domains and their gene targets. To elucidate how the BX-C boundaries combine these two contradictory activities, we have used a boundary replacement strategy. We show that a 337 bp fragment spanning the Fab-8 boundary nuclease hypersensitive site and lacking all but 83 bp of the 625 bp Fab-8 PTS (promoter targeting sequence) fully rescues a Fab-7 deletion. It blocks crosstalk between the iab-6 and iab-7 regulatory domains, and has bypass activity that enables the two downstream domains, iab-5 and iab-6, to regulate Abdominal-B (Abd-B) transcription in spite of two intervening boundary elements. Fab-8 has two dCTCF sites and we show that they are necessary both for blocking and bypass activity. However, CTCF sites on their own are not sufficient for bypass. While multimerized dCTCF (or Su(Hw)) sites have blocking activity, they fail to support bypass. Moreover, this bypass defect is not rescued by the full length PTS. Finally, we show that orientation is critical for the proper functioning the Fab-8 replacement. Though the inverted Fab-8 boundary still blocks crosstalk, it disrupts the topology of the Abd-B regulatory domains and does not support bypass. Importantly, altering the orientation of the Fab-8 dCTCF sites is not sufficient to disrupt bypass, indicating that orientation dependence is conferred by other factors.

Coalescence of the embryonic gonad in Drosophila melanogaster requires directed migration of primordial germ cells (PGCs) towards somatic gonadal precursor cells (SGPs). It was recently proposed that the ATP-binding cassette (ABC) transporter Mdr49 functions in the embryonic mesoderm to facilitate the transmission of the PGC attractant from the SGPs; however, the precise molecular identity of the Mdr49-dependent guidance signal remained elusive. Employing the loss- and gain-of-function strategies, we show that Mdr49 is a component of the Hedgehog (hh) pathway and it potentiates the signaling activity. This function is direct because in Mdr49 mutant embryos the Hh ligand is inappropriately sequestered in the hh-expressing cells. Our data also suggest that the role of Mdr49 is to provide cholesterol for the correct processing of the Hh precursor protein. Supporting this conclusion, PGC migration defects in Mdr49 embryos are substantially ameliorated by a cholesterol-rich diet.

In Drosophila, Polycomb (PcG) and Trithorax (TrxG) group proteins are assembled on Polycomb response elements (PREs) to maintain tissue and stage-specific patterns of gene expression. Critical to coordinating gene expression with the process of differentiation, the activity of PREs can be switched "on" and "off." When on, the PRE imposes a silenced state on the genes in the same domain that is stably inherited through multiple rounds of cell division. When the PRE is switched off, the domain is in a state permissive for gene expression that can be stably inherited. Previous studies have suggested that a burst of transcription through a PRE sequence displaces PcG proteins and provides a universal mechanism for inducing a heritable switch in PRE activity from on to off; however, the evidence favoring this model is indirect. Here, we have directly tested the transcriptional read-through mechanism. Contrary to previous suggestions, we show that transcription through the PRE is not sufficient for inducing an epigenetic switch in PRE activity. In fact, even high levels of continuous transcription through a PRE fails to dislodge the PcG proteins, nor does it remove repressive histone marks. Our results indicate that other mechanisms involving adjacent DNA regulatory elements must be implicated in heritable switch of PRE activity.

The subdivision of cell populations in compartments is a key event during animal development. In Drosophila, the gene apterous (ap) divides the wing imaginal disc in dorsal vs ventral cell lineages and is required for wing formation. ap function as a dorsal selector gene has been extensively studied. However, the regulation of its expression during wing development is poorly understood. In this study, we analyzed ap transcriptional regulation at the endogenous locus and identified three cis-regulatory modules (CRMs) essential for wing development. Only when the three CRMs are combined, robust ap expression is obtained. In addition, we genetically and molecularly analyzed the trans-factors that regulate these CRMs. Our results propose a three-step mechanism for the cell lineage compartment expression of ap that includes initial activation, positive autoregulation and Trithorax-mediated maintenance through separable CRMs.

Chromatin boundaries are architectural elements that determine the three-dimensional folding of the chromatin fiber and organize the chromosome into independent units of genetic activity. The Fab-7 boundary from the Drosophila bithorax complex (BX-C) is required for the parasegment-specific expression of the Abd-B gene. We have used a replacement strategy to identify sequences that are necessary and sufficient for Fab-7 boundary function in the BX-C. Fab-7 boundary activity is known to depend on factors that are stage specific, and we describe a novel ∼700-kDa complex, the late boundary complex (LBC), that binds to Fab-7 sequences that have insulator functions in late embryos and adults. We show that the LBC is enriched in nuclear extracts from late, but not early, embryos and that it contains three insulator proteins, GAF, Mod(mdg4), and E(y)2. Its DNA binding properties are unusual in that it requires a minimal sequence of >65 bp; however, other than a GAGA motif, the three Fab-7 LBC recognition elements display few sequence similarities. Finally, we show that mutations which abrogate LBC binding in vitro inactivate the Fab-7 boundary in the BX-C.

BACKGROUND: Insulators play a central role in gene regulation, chromosomal architecture and genome function in higher eukaryotes. To learn more about how insulators carry out their diverse functions, we have begun an analysis of the Drosophila CTCF (dCTCF). CTCF is one of the few insulator proteins known to be conserved from flies to man. RESULTS: In the studies reported here we have focused on the identification and characterization of two dCTCF protein interaction modules. The first mediates dCTCF multimerization, while the second mediates dCTCF-CP190 interactions. The multimerization domain maps in the N-terminus of the dCTCF protein and likely mediates the formation of tetrameric complexes. The CP190 interaction module encompasses a sequence ~200 amino acids long that spans the C-terminal and mediates interactions with the N-terminal BTB domain of the CP190 protein. Transgene rescue experiments showed that a dCTCF protein lacking sequences critical for CP190 interactions was almost as effective as wild type in rescuing the phenotypic effects of a dCTCF null allele. The mutation did, however, affect CP190 recruitment to specific Drosophila insulator elements and had a modest effect on dCTCF chromatin association. A protein lacking the N-terminal dCTCF multimerization domain incompletely rescued the zygotic and maternal effect lethality of the null and did not rescue the defects in Abd-B regulation evident in surviving adult dCTCF mutant flies. Finally, we show that elimination of maternally contributed dCTCF at the onset of embryogenesis has quite different effects on development and Abd-B regulation than is observed when the homozygous mutant animals develop in the presence of maternally derived dCTCF activity. CONCLUSIONS: Our results indicate that dCTCF-CP190 interactions are less critical for the in vivo functions of the dCTCF protein than the N-terminal dCTCF-dCTCF interaction domain. We also show that the phenotypic consequences of dCTCF mutations differ depending upon when and how dCTCF activity is lost.

The parasegment-specific expression of the three Drosophila Bithorax complex homeotic genes is orchestrated by nine functionally autonomous regulatory domains. Functional autonomy depends upon special elements called boundaries or insulators that are located between each domain. The boundaries ensure the independent activity of each domain by blocking adventitious interactions with initiators, enhancers and silencers in the neighboring domains. However, this blocking activity poses a regulatory paradox--the Bithorax boundaries are also able to insulate promoters from regulatory interactions with enhancers and silencers and six of the nine Bithorax regulatory domains are separated from their target genes by at least one boundary element. Here we consider several mechanisms that have been suggested for how the Bithorax regulatory domains are able to bypass intervening boundary elements and direct the appropriate parasegment-specific temporal and spatial expression of their target gene.

The selector gene apterous (ap) plays a key role during the development of the Drosophila melanogaster wing because it governs the establishment of the dorsal-ventral (D-V) compartment boundary. The D-V compartment boundary is known to serve as an important signaling center that is essential for the growth of the wing. The role of Ap and its downstream effectors have been studied extensively. However, very little is known about the transcriptional regulation of ap during wing disc development. In this study, we present a first characterization of an essential wing-specific ap enhancer. First, we defined an 874-bp fragment about 10 kb upstream of the ap transcription start that faithfully recapitulates the expression pattern of ap in the wing imaginal disc. Analysis of deletions in the ap locus covering this element demonstrated that it is essential for proper regulation of ap and formation of the wing. Moreover, we showed that the mutations ap(blot) and ap(Xasta) directly affect the integrity of this enhancer, leading to characteristic wing phenotypes. Furthermore, we engineered an in situ rescue system at the endogenous ap gene locus, allowing us to investigate the role of enhancer fragments in their native environment. Using this system, we were able to demonstrate that the essential wing enhancer alone is not sufficient for normal wing development. The in situ rescue system will allow us to characterize the ap regulatory sequences in great detail at the endogenous locus.

Ramathal et al. have employed an elegant xenotransplantation technique to study the fate of human induced pluripotent stem cells (hiPSCs) from fertile males and from males carrying Y chromosome deletions of the azoospermia factor (AZF) region. When placed in a mouse testis niche, hiPSCs from fertile males differentiate into germ cell-like cells (GCLCs). Highlighting the crucial role of cell autonomous factors in male sterility, hiPSCs derived from azoospermic males prove to be less successful under similar circumstances. Their studies argue that the agametic "Sertoli cell only" phenotype of two of the AZF deletions likely arises from a defect in the maintenance of germline stem cells (GSCs) rather than from a defect in their specification. These observations underscore the importance of the dialogue between the somatic niche and its inhabitant stem cells, and open up interesting questions concerning the functioning of the somatic niche and how it communicates to the GSCs.

Mature Drosophila sperm are highly polarized cells--on one side is a nearly 2 mm long flagellar tail that comprises most of the cell, while on the other is the sperm head, which carries the gamete's genetic information. The polarization of the sperm cells commences after meiosis is complete and the 64-cell spermatid cyst begins the process of differentiation. The spermatid nuclei cluster to one side of the cyst, while the flagellar axonemes grows from the other. The elongating spermatid bundles are also polarized with respect to the main axis of the testis; the sperm heads are always oriented basally, while the growing tails extend apically. This orientation within the testes is important for transferring the mature sperm into the seminal vesicles. We show here that orienting cyst polarization with respect to the main axis of the testis depends upon atypical Protein Kinase C (aPKC), a factor implicated in polarity decisions in many different biological contexts. When apkc activity is compromised in the male germline, the direction of cyst polarization within this organ is randomized. Significantly, the mechanisms used to spatially restrict apkc activity to the apical side of the spermatid cyst are different from the canonical cross-regulatory interactions between this kinase and other cell polarity proteins that normally orchestrate polarization. We show that the asymmetric accumulation of aPKC protein in the cyst depends on an mRNA localization pathway that is regulated by the Drosophila CPEB protein Orb2. orb2 is required to properly localize and activate the translation of apkc mRNAs in polarizing spermatid cysts. We also show that orb2 functions not only in orienting cyst polarization with respect to the apical-basal axis of the testis, but also in the process of polarization itself. One of the orb2 targets in this process is its own mRNA. Moreover, the proper execution of this orb2 autoregulatory pathway depends upon apkc.

The specification of primordial germ cells (PGCs) and subsequent maintenance of germ-line identity in Drosophila embryos has long been thought to occur solely under the control of cell-autonomous factors deposited in the posterior pole plasm during oogenesis. However, here we document a novel role for somatic BMP signaling in the maintenance of PGC fate during the period leading up to embryonic gonad coalescence. We find that PGCs fail to maintain their germline identity when BMP signaling is compromised. They initiate but are unable to properly assemble the germline stem cell-specific organelle, the spectrosome, and they lose expression of the germline-specific gene Vasa. BMP signaling must, however, be finely tuned as there are deleterious consequences to PGCs when the pathway is excessively active. We show that one mechanism used to calibrate the effects of BMP signals is dependent on the Ubc9 homolog Lesswright (Lwr).

Chromatin immunoprecipitation (ChIP) is widely used for mapping DNA-protein interactions across eukaryotic genomes in cells, tissues or even whole organisms. Critical to this procedure is the efficient cross-linking of chromatin-associated proteins to DNA sequences that are in close proximity. Since the mid-nineties formaldehyde fixation has been the method of choice. However, some protein-DNA complexes cannot be successfully captured for ChIP using formaldehyde. One such formaldehyde refractory complex is the developmentally regulated insulator factor, Elba. Here we describe a new embryo fixation procedure using the bi-functional cross-linking reagents DSG (disuccinimidyl glutarate) and DSP (dithiobis[succinimidyl propionate). We show that unlike standard formaldehyde fixation protocols, it is possible to capture Elba association with insulator elements in 2-5 h embryos using this new cross-linking procedure. We show that this new cross-linking procedure can also be applied to localize nuclear proteins that are amenable to ChIP using standard formaldehyde cross-linking protocols, and that in the cases tested the enrichment was generally superior to that achieved using formaldehyde cross-linking.

The determination of cell fate and the establishment of polarity axes during Drosophila oogenesis depend upon pathways that localize mRNAs within the egg chamber and control their on-site translation. One factor that plays a central role in regulating on-site translation of mRNAs is Orb. Orb is a founding member of the conserved CPEB family of RNA-binding proteins. These proteins bind to target sequences in 3' UTRs and regulate mRNA translation by modulating poly(A) tail length. In addition to controlling the translation of axis-determining mRNAs like grk, fs(1)K10, and osk, Orb protein autoregulates its own synthesis by binding to orb mRNA and activating its translation. We have previously shown that Rasputin (Rin), the Drosophila homologue of Ras-GAP SH3 Binding Protein (G3BP), associates with Orb in a messenger ribonucleoprotein (mRNP) complex. Rin is an evolutionarily conserved RNA-binding protein believed to function as a link between Ras signaling and RNA metabolism. Here we show that Orb and Rin form a complex in the female germline. Characterization of a new rin allele shows that rin is essential for oogenesis. Co-localization studies suggest that Orb and Rin form a complex in the oocyte at different stages of oogenesis. This is supported by genetic and biochemical analyses showing that rin functions as a positive regulator in the orb autoregulatory pathway by increasing Orb protein expression. Tandem Mass Spectrometry analysis shows that several canonical stress granule proteins are associated with the Orb-Rin complex suggesting that a conserved mRNP complex regulates localized translation during oogenesis in Drosophila.

The Drosophila embryonic gonad is assembled from two distinct cell types, the Primordial Germ Cells (PGCs) and the Somatic Gonadal Precursor cells (SGPs). The PGCs form at the posterior of blastoderm stage embryos and are subsequently carried inside the embryo during gastrulation. To reach the SGPs, the PGCs must traverse the midgut wall and then migrate through the mesoderm. A combination of local repulsive cues and attractive signals emanating from the SGPs guide migration. We have investigated the role of the hedgehog (hh) pathway gene shifted (shf) in directing PGC migration. shf encodes a secreted protein that facilitates the long distance transmission of Hh through the proteoglycan matrix after it is released from basolateral membranes of Hh expressing cells in the wing imaginal disc. shf is expressed in the gonadal mesoderm, and loss- and gain-of-function experiments demonstrate that it is required for PGC migration. Previous studies have established that the hmgcr-dependent isoprenoid biosynthetic pathway plays a pivotal role in generating the PGC attractant both by the SGPs and by other tissues when hmgcr is ectopically expressed. We show that production of this PGC attractant depends upon shf as well as a second hh pathway gene gγ1. Further linking the PGC attractant to Hh, we present evidence indicating that ectopic expression of hmgcr in the nervous system promotes the release/transmission of the Hh ligand from these cells into and through the underlying mesodermal cell layer, where Hh can contact migrating PGCs. Finally, potentiation of Hh by hmgcr appears to depend upon cholesterol modification.