Top PDF Uncoupling Dorsal mediated activation from Dorsal mediated repression in the Drosophila embryo

Uncoupling Dorsal mediated activation from Dorsal mediated repression in
the Drosophila embryo

Uncoupling Dorsal mediated activation from Dorsal mediated repression in the Drosophila embryo

As expected, expression of the activation targets twi, sna, sog and brk, as determined by in situ hybridization, was nearly normal in embryos produced by females bearing the wild-type dl transgene under the control of a maternal Gal4 driver (Fig. 2, compare A with C). Expression of DL-VP16 (Fig. 2D) and DL-p65 (Fig. 2E) also resulted in ventral activation of sna, twi, sog and brk. Consistent with the expanded nuclear localization of these variants, the domain of ventral activation was also expanded relative to wild type. By contrast, DL-WRPW failed to activate twi, sna, sog or brk (Fig. 2F). The repression targets zen and dpp are both expressed in the dorsal 40% of the precellular wild-type embryo (Fig. 2A), and show uniform expression along the DV axis in dl null embryos (Fig. 2B). Expression of transgenic DL restores a near wild-type pattern of zen and dpp expression (Fig. 2C). By contrast, early zen and dpp expression are greatly expanded in DL-VP16 and DL-p65 embryos – expression was frequently almost uniform along the DV axis (Fig. 2D,E). This is consistent with the function of DL-VP16 and DL-p65 as dedicated activators. By contrast, like wild-type DL, DL-WRPW repressed zen and dpp (Fig. 2F).
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Multiple RTK pathways downregulate Groucho mediated repression in
Drosophila embryogenesis

Multiple RTK pathways downregulate Groucho mediated repression in Drosophila embryogenesis

In early Drosophila embryogenesis, signals mediated by different receptor tyrosine kinases (RTKs) establish cell fates in a wide range of developmental processes. All RTK pathways transduce signals via the canonical Ras/Raf/MEK/MAPK cascade, yet they clearly elicit diverse outcomes: the Torso RTK pathway defines the embryonic termini (Furriols and Casanova, 2003); two FGF receptors (FGFR) are required for the patterning of the mesoderm and trachea (Huang and Stern, 2005); and the EGF receptor (EGFR) pathway controls various processes such as the formation of the ventral neuroectoderm, the specification of muscle precursors and the invagination of tracheal branches (Shilo, 2003). These differential transcriptional and morphological responses to RTK activation are context specific, and probably depend on the strength, range and duration of the signal. Additional specificity of the response is conferred by crosstalk between RTK and other signalling pathways (Culi et al., 2001), as well as by the combinatorial activity of nuclear pathway effectors together with distinct tissue-specific factors, at the level of specific DNA enhancers (Flores et al., 2000; Simon, 2000).
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The promoter activity of long terminal repeats of the HERV-H family of human retrovirus-like elements is critically dependent on Sp1 family proteins interacting with a GC/GT box located immediately 3' to the TATA box.

The promoter activity of long terminal repeats of the HERV-H family of human retrovirus-like elements is critically dependent on Sp1 family proteins interacting with a GC/GT box located immediately 3' to the TATA box.

The results presented in this paper show that members of the Sp1 protein family are critically involved in the regulation of the transcriptional activity of the HERV-H LTR promoters. We have found that transcriptionally active LTRs contain a GC/GT box immediately downstream of the TATA box and that three different members of the Sp1 protein family can bind to this site and modulate the promoter activity. First, Sp1, which is a well-known activator of transcription of both cellular and viral genes, binds to this GC/GT box and stimulates the promoter activity in all cell lines tested. Second, Sp3, which has been reported to act as a repressor of Sp1-mediated transcrip- tional activation and of the HIV-1 LTR promoter (20, 21, 33, 46), binds to the GC/GT box. As expected, we found that Sp3 repressed the Sp1-mediated transcriptional activation of the HERV-H LTRs in Drosophila SL-2 cells and had no stimula- tory effect on the transcription from the LTR promoters and the TK promoter in HeLa cells and Drosophila SL-2 cells. However, in the embryonal carcinoma cell line NTera2-D1, Sp3 stimulated the transcriptional activities of both the TK and the HERV-H LTR promoters. These results suggest that Sp3 may be dependent on cell-specific cofactors to act as a tran- scriptional activator and thus may be involved in cell-specific regulation of both the HERV-H LTRs and other GC/GT box- containing promoters. Interestingly, a B-cell-specific coactiva- tor converting the ubiquitously expressed transcription factor Oct-1 to a cell-type-specific activator has recently been char- acterized (19). The Sp3-mediated repression of Sp1-directed transcriptional activation is most probably due to competitive binding between these two proteins. Thus, various levels of the ubiquitously expressed Sp1 and Sp3 proteins in different cell lines may result in significant variation of the transcriptional activity from the LTR promoters, which together with the cell-specific activation potential of Sp3 may contribute to the different expression levels of HERV-H transcripts in different cell lines and tissues (22, 59). Third, competition with the Sp1 oligonucleotide showed that a third protein also bound to the GC/GT box of the LTRs. This protein could be the Sp1-related Sp4 or another, not-yet-identified, member of the Sp1 protein family or an unrelated GC/GT box-binding protein. Fourth, the more distantly Sp1-related GC/GT box-binding protein BTEB also bound to and stimulated the promoter activities of the HERV-H LTRs. However, gel shift assays indicated that BTEB is not expressed in NTera2-D1 and HeLa cells, which is in agreement with the fact that BTEB is specifically translated in brain and testis (26). Thus, tissue-specific members of the Sp1 protein family also can bind to and activate the LTR
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The AF-6 Homolog Canoe Acts as a Rap1 Effector During Dorsal Closure of the Drosophila Embryo

The AF-6 Homolog Canoe Acts as a Rap1 Effector During Dorsal Closure of the Drosophila Embryo

R ap1 belongs to the Ras superfamily of small GTP- of Rap1 are emerging from studies in Drosophila. Loss- ases, which cycle between an inactive GDP-bound of-function (lof) mutations in Drosophila Rap1 cause and an active GTP-bound state, eliciting distinct down- severe morphogenetic abnormalities during embryonic stream responses in the active state. Rap proteins were development, while cell proliferation and cell fate deter- originally identified as antagonists of oncogenic Ras mination, processes that rely heavily on regulation by (Kitayama et al. 1989; Cook et al. 1993; Okada et al. Ras, appear to be unaffected. Specifically, the ventral 1998; Mochizuki et al. 1999), but more recent studies invagination and migration of mesodermal precursors suggest that the function of Rap1 is largely independent in the embryo are severely impaired, as are head involu- of Ras (reviewed in Zwartkruis and Bos 1999; Bos et tion, dorsal closure, and the migration of gonadal pre- al. 2001; Caron 2003). While Ras is mainly localized at cursors (Asha et al. 1999). More recently, Rap1 has been the plasma membrane, Rap1 has been found in differ- shown to play a role in cell adhesion, specifically in ent membrane compartments, depending on the cell the positioning of adherens junctions in proliferating type. Further, Rap1 activation appears to be stimulated epithelial cells (Knox and Brown 2002). These findings by numerous exchange factors that do not act on the strongly suggest that Rap1 plays a largely Ras-indepen- prototypic Ras GTPases. Rap1 has been shown to act in dent role in cell migration and morphogenesis. a Ras-independent manner in the production of super- Little is currently known about the signaling pathways oxide (Bokoch et al. 1991; Maly et al. 1994), in cAMP- mediating the downstream effects of Rap1 in vertebrates induced neurite outgrowth (York et al. 1998), and, most or Drosophila. A number of molecules that were origi- recently, in the regulation of integrin-mediated cell ad- nally identified in vertebrates as Ras-interacting pro- hesion and AMPA receptor trafficking during synaptic teins, including B-Raf, members of the RalGEF family, plasticity (Tsukamoto et al. 1999; Caron et al. 2000; and AF-6, were subsequently shown to associate with Reedquist et al. 2000; Arai et al. 2001; Zhu et al. 2002; Rap1 as well (Linnemann et al. 1999; Quilliam et al. Rangarajan et al. 2003). 1999; Zwartkruis and Bos 1999; Boettner et al. 2000; Perhaps the most important insights into the function Bos et al. 2001). However, the relevance of these interac- tions for Rap1 function in vivo remains largely unknown; to date, none of these molecules have been shown to act as Rap1 targets in an in vivo context.
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Dorsal Ventral Patterning and Gene Regulation in the Early Embryo of Drosophila melanogaster

Dorsal Ventral Patterning and Gene Regulation in the Early Embryo of Drosophila melanogaster

Additional evidence also suggests that TGF-ß signaling may also regulate the ind expression domains, but whether or not this signaling pathway functions through the A- box element was not known. Decapentaplegic (Dpp) is a TGFß/BMP homolog that is limited in its expression to dorsal regions of the embryo and functions as a morphogen to support patterning of the amnioserosa, at higher levels in dorsal-most regions of the embryo, and the non-neurogenic ectoderm, at lower levels in dorsal-lateral regions of the embryo (Ferguson and Anderson, 1992). A previous study found that in mutants in which Dpp signaling is expanded into lateral regions of the embryo, ind expression is lost (Von Ohlen and Doe, 2000). Likewise, ectopic expression of dpp in lateralized embryos that exhibit expanded ind expression throughout the embryo was able to repress ind in the domain where Dpp signaling was presented (Mizutani et al., 2006). Also, the ind CRM contains a 15 bp DNA sequence implicated in TGF- β signaling-mediated repression (Stathopoulos and Levine, 2005). Similar sites have been shown to mediate repression by recruiting a Dpp-dependent Schnurri/Mad/Medea (SMM) protein complex, but SMM dependent repression of ind has never been shown and in fact this mechanism of repression has only been shown to act at later stages of development (Dai et al., 2000; Pyrowolakis et al., 2004).
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Drosophila Dok is required for embryonic dorsal closure

Drosophila Dok is required for embryonic dorsal closure

Tyrosine phosphorylated residues mediate interaction with SH2 domains (Bradshaw and Waksman, 2002) and there is little tyrosine phosphorylation in Saccharomyces cerevisiae. To increase the probability of obtaining proteins whose interactions with Shark rely on the SH2 domains, we modified the yeast two- hybrid system by co-expressing the activated Src-kinase domain, which has been found to tyrosine phosphorylate library proteins in yeast (Keegan and Cooper, 1996; Lioubin et al., 1996). The bait plasmid was based on pGilda (Gimeno et al., 1996) and contained the LexA DNA-binding domain fused to the shark sequences, together with the DNA sequences encoding an activated Src- kinase domain (Lioubin et al., 1996), placed under the control of a constitutive promoter. The bait plasmid was co-transformed into yeast together with a Drosophila ‘prey’ library, in which cDNAs from 0- 21-hour-old embryos were fused to the B42 activation domain AD (Gyuris et al., 1993) (pB42AD; Clontech). Two out of the 17 clones whose interaction with the bait protein mediated LexA-dependent lacZ and Leu2 reporter gene expression were shown to encode overlapping portions of a protein similar to the Downstream of kinase (Dok) family of proteins (Carpino et al., 1997; Di Cristofano et al., 1998; Ellis et al., 1990; Lemay et al., 2000; Yamanashi and Baltimore, 1997). The Drosophila Dok gene (Ddok) has been annotated in the Drosophila genome
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Cranial kinesis in palaeognathous birds

Cranial kinesis in palaeognathous birds

bending zones found in the bending experiments of the upper bill (see arrows in Figs·7, 8) nor the location suggested by other authors (Hofer, 1954; Simonetta, 1960; Bock, 1963), nor with the behavioural observations (Gussekloo and Bout, 2005). In Dromaius (Fig.·9), the dorsal bar is relatively thick near the rostrum maxillae and becomes thinner to caudal. This suggests that the bar would bend far more caudal than is expected from the observed position of the bending zone. In fact, bending of the caudal part is impossible due to the presence of a thick rostrum parasphenoidale. The position of the thinnest zones in the ventral bars is for all palaeognathous species also situated caudal of, or at the most rostral point of, the rostrum parasphenoidale. This again indicates that these thin zones cannot be bending zones. The difference in position of the areas of low thickness is also remarkable. While in the Calidris species thin areas in the dorsal and ventral bar are located at an almost equal distance from the bill tip, such a relationship is absent in the Palaeognathae. In palaeognaths no discrete zone is seen where both the ventral and dorsal bar show locally reduced thickness, indicating that no clear bending zone is present in the upper bills of this taxon.
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Parallels between tissue repair and embryo morphogenesis

Parallels between tissue repair and embryo morphogenesis

Epithelial fusion is the climax of many morphogenetic episodes and of wound healing. During dorsal closure in flies, the leading edge epithelial cells extend filopodial protrusions that appear to play a key role in bonding the two epithelial sheets together (Fig. 5A,B,E). Filopodia from confronting epithelial cells interdigitate at the zipper front and in the fusion seam, several cell diameters back from the zipper front, these interdigitations resolve to leave mature adherens junctions linking opposing cells (Jacinto et al., 2000). In vitro studies of keratinocytes adhering to one another to form confluent sheets show that these cells too use filopodial interdigitation to prime the formation of adherens junctions (Vasioukhin et al., 2000). Live studies of equivalent filopodial interactions between opposing epithelial leader cells during C. elegans ventral enclosure show that α- catenin is pre-localised to filopodial tips, which may aid in assembly of rapid, transient adhesions between filopodia that go on to nucleate the formation of mature junctions (Raich et al., 1999). Evidence that filopodia are pivotal for epithelial fusion during dorsal closure comes from experiments where their assembly is blocked by expressing a dominant-negative mutant form of CDC42 in Engrailed stripes of the embryonic fly epithelium. In such cases, fusion of the opposing epithelial sheets in these regions fails (Jacinto et al., 2000). Moreover, these experiments reveal one further role for the filopodia in closure of the dorsal hole. Live imaging of the leading edge in the minutes preceding fusion shows how filopodia scan the opposing leading edge, rather like filopodia from a growth cone sensing for axon guidance cues. If assembly of filopodia is blocked, then the opposing epithelial fronts fail to align properly at the midline, much like a poorly buttoned up waistcoat, indicating that filopodia are, in part, responsible for the cell:cell matching needed to align segments across the midline seam (Jacinto et al., 2000).
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The Anatomy of Dorsal Ramus Nerves and Its Implications in Lower Back Pain

The Anatomy of Dorsal Ramus Nerves and Its Implications in Lower Back Pain

Spinal dorsal ramus mediated back pain can occur at any level of the human spine [18,21,22,44,52,53]. For low back pain mediated by dorsal ramus, the primary pain is commonly at the thoracolumbar junction [19-22, 38,44]. Within the thoracic region, the coronal orienta- tion of the zygapophysial joints grants spine free rotation. However, this rotation is limited by a rigid rib cage, ex- cept at the T10-12 levels because of floating ribs. The upper lumbar facets also have a relative coronal orienta- tion. Therefore, spine rotation is relatively free at the thoracolumbar junction and the greatest shear force oc- curs at the more mobile upper lumbar segments. This normal spinal movement can cause zygapophysial joint separation or rotation. If these movements occur rapidly or overcome the body’s physiological limit, they can cause stretching tension and irritation to the dorsal ramus, resulting in low back pain [20,40,44,54]. Shao and his colleagues reported that seventy four percent (74%) of the 1263 patients with spinal dorsal ramus mediated low back pain had the pain originating from L1 and/or L2 dorsal ramus [36].
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Effector CD4+ T-Cell Involvement in Clearance of Infectious Herpes Simplex Virus Type 1 from Sensory Ganglia and Spinal Cords

Effector CD4+ T-Cell Involvement in Clearance of Infectious Herpes Simplex Virus Type 1 from Sensory Ganglia and Spinal Cords

In primary infection, CD8 ⴙ T cells are important for clearance of infectious herpes simplex virus (HSV) from sensory ganglia. In this study, evidence of CD4 ⴙ T-cell-mediated clearance of infectious HSV type 1 (HSV-1) from neural tissues was also detected. In immunocompetent mice, HSV-specific CD4 ⴙ T cells were present in sensory ganglia and spinal cords coincident with HSV-1 clearance from these sites and remained detectable at least 8 months postinfection. Neural CD4 ⴙ T cells isolated at the peak of neural infection secreted gamma interferon, tumor necrosis factor alpha, interleukin-2 (IL-2), or IL-4 after stimulation with HSV antigen. HSV-1 titers in neural tissues were greatly reduced over time in CD8 ⴙ T-cell-deficient and CD8 ⴙ T-cell-depleted mice, suggesting that CD4 ⴙ T cells could mediate clearance of HSV-1 from neural tissue. To examine possible mechanisms by which CD4 ⴙ T cells resolved neural infection, CD8 ⴙ T cells were depleted from perforin-deficient or FasL-defective mice. Clearance of infectious virus from neural tissues was not significantly different in perforin-deficient or FasL-defective mice compared to wild-type mice. Further, in spinal cords and brains after vaginal HSV-1 challenge of chimeric mice expressing both perforin and Fas or neither perforin nor Fas, virus titers were significantly lower than in control mice. Thus, perforin and Fas were not required for clearance of infectious virus from neural tissues. These results suggest that HSV-specific CD4 ⴙ T cells are one component of a long-term immune cell presence in neural tissues following genital HSV-1 infection and play a role in clearance of infectious HSV-1 at neural sites, possibly via a nonlytic mechanism.
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Opiate mediated inhibition of calcium signaling is decreased in dorsal root ganglion neurons from the diabetic BB/W rat

Opiate mediated inhibition of calcium signaling is decreased in dorsal root ganglion neurons from the diabetic BB/W rat

dissociated, capsaicin-sensitive dorsal root ganglion (DRG) neurons from the spontaneously diabetic BioBreeding/ Worcester (BB/W)-rat model of type I human diabetes melli- tus (9). The magnitude of the current enhancement increased with the duration of diabetes and nerve conduction velocity slowed. Both abnormalities were prevented by long-term oral treatment of diabetic animals with an aldose reductase inhibitor. Endogenous opiates, such as dynorphin A (Dyn A), have been detected in dorsal root ganglia from adult rats (12), and local release of opiate transmitters may play an important role in modulating neurotransmission in primary afferent path- ways. Decreased sensitivity to opiates at the spinal level, as ev- idenced by decreased inhibition of tail flick response to nox- ious stimuli (13) and impaired vagal transport of opiate receptors (14) have been previously demonstrated in diabetic animal models. However, the cellular mechanism(s) underly- ing the decreased neuronal response to opiates in diabetes are unknown. Because previous studies suggested that calcium channels were affected in diabetes mellitus (9), we hypothe- sized that altered opiate-mediated regulation of neuronal cal- cium channels might contribute to the changes in calcium sig- naling observed in diabetes. Activation of k and m opiate receptors on DRG neurons inhibits calcium influx through voltage-activated calcium channels via a pertussis-sensitive, in- hibitory (G i/o ) G protein–coupled pathway (15–17). We com-
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Dorsal ventral midline signaling in the developing Drosophila
eye

Dorsal ventral midline signaling in the developing Drosophila eye

Eye discs were double-stained for transcriptional activity of the iro complex (dorsally expressed) and the slp complex (ventrally expressed) to determine the spatial relationship of the two expression domains. In second-instar eye discs, mirror (mirr, one of the three iro genes) transcription was exclusively dorsal (Fig. 1D,E) whereas slp was exclusively ventral (Fig. 1C,E), and the two expression domains directly abutted (with no overlap) in the medial region of the eye disc (Fig. 1E). In the third larval instar the morphogenetic furrow (MF) begins to progress across the presumptive retina, and slp and mirr transcription was extinguished in post-furrow regions as evidenced by in situ hybridization (not shown). However, lacZ reporters from both genes showed persistent expression posterior to the MF, which is likely to result from perdurance of the long-lived lacZ gene product (Fig. 1F). Ahead of the furrow mirr was still exclusively dorsal, and slp ventral, but now the two expression domains did not abut; instead, a gap opened that appeared to be generated by loss of slp expression in the region close to the D-V midline (white arrowhead in Fig. 1F).
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A facilitated diffusion mechanism establishes the Drosophila Dorsal gradient

A facilitated diffusion mechanism establishes the Drosophila Dorsal gradient

Further analysis of our model showed that the mechanism of facilitated diffusion can be tested by slowing diffusion of the Dl/Cact complex. The model predicts that different instances in a hallmark series of phenotypes will be observed, depending on the severity of the perturbation. In order of increasing strength of perturbation: the Dl gradient widens, becomes flat on top, or splits into two peaks (Fig. 2C, D). These predictions and the shuttling phenomenon itself arise naturally out of biophysical processes known to occur in the embryo: global movement, binding of Dl and Cact, Toll-mediated destruction of the Dl/Cact complex on the ventral side, and nuclear capture of free Dl. Indeed, previous models of the Dl gradient, which included these processes, also exhibit shuttling, even when it was not considered in the models (Fig. S2, Supplementary Materials and Methods) (Kanodia et al., 2009; Ambrosi et al., 2014; O ’ Connell and Reeves, 2015). In particular, in previous work using a model of the Dl/Cact system, the authors found that increasing the diffusion rate caused the gradient to sharpen (Ambrosi et al., 2014), which is the same prediction detailed here (Fig. 2C,D). We analyzed the model presented in that study, and show that this result arose because shuttling was indeed occurring in that model, even though the authors did not intend it to be (Fig. S2).
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Herpes simplex virus-based nerve targeting gene therapy in pain management

Herpes simplex virus-based nerve targeting gene therapy in pain management

Another set of potential therapeutic targets accessible to HSV vectors are the receptors that transduce noxious stimuli to electrical potentials within the PANs. These targets are only now being examined, but could provide a more fine-tuned approach to pain control, ie, decrease pain from a specific stimuli such as heat. For example, the transient receptor potential vanilloid type 1 (TRPV1) receptor is an ion channel activated by high temperature and involved in painful heat sensation. Transduction of rodent dorsal root ganglia by footpad inoculation with an HSV vector expressing a mutant TRPV1 gene that does not form the channel results in a sig- nificantly attenuated thermal response, 76 but does not affect
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Superoxide mediated activation of uncoupling protein 2 causes pancreatic β cell dysfunction

Superoxide mediated activation of uncoupling protein 2 causes pancreatic β cell dysfunction

Failure to secrete adequate amounts of insulin in response to increasing concentrations of glucose is an important feature of type 2 diabetes. The mechanism for loss of glucose responsiveness is unknown. Uncoupling protein 2 (UCP2), by virtue of its mitochondrial proton leak activity and con- sequent negative effect on ATP production, impairs glucose-stimulated insulin secretion. Of inter- est, it has recently been shown that superoxide, when added to isolated mitochondria, activates UCP2-mediated proton leak. Since obesity and chronic hyperglycemia increase mitochondrial super- oxide production, as well as UCP2 expression in pancreatic β cells, a superoxide-UCP2 pathway could contribute importantly to obesity- and hyperglycemia-induced β cell dysfunction. This study demonstrates that endogenously produced mitochondrial superoxide activates UCP2-mediated pro- ton leak, thus lowering ATP levels and impairing glucose-stimulated insulin secretion. Furthermore, hyperglycemia- and obesity-induced loss of glucose responsiveness is prevented by reduction of mitochondrial superoxide production or gene knockout of UCP2. Importantly, reduction of super- oxide has no beneficial effect in the absence of UCP2, and superoxide levels are increased further in the absence of UCP2, demonstrating that the adverse effects of superoxide on β cell glucose sensing are caused by activation of UCP2. Therefore, superoxide-mediated activation of UCP2 could play an important role in the pathogenesis of β cell dysfunction and type 2 diabetes.
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Vulva morphogenesis involves attraction of plexin 1 expressing primordial vulva cells to semaphorin 1a sequentially expressed at the vulva midline

Vulva morphogenesis involves attraction of plexin 1 expressing primordial vulva cells to semaphorin 1a sequentially expressed at the vulva midline

migration during vulva morphogenesis. (A) Lateral perspective; D, dorsal; P, posterior; unlabelled arrow indicates left-right depth. A series of short-range migrations is repeated for the formation of vulva rings. (B) Ventral perspective of the boxed area in A. Intermediate steps of the proposed model for vulva cell migration showing the PLX-1-expressing leading edge as a blue band on the future lumen side of cells poised to enter the forming vulva. The leading edge spreads ventrally under forming vulva rings expressing SMP-1 on their lumen surface (green spiked ring). SMP-1 expression increases dramatically in the newly formed ring, while the next external vulva half ring (not yet expressing SMP-1) initiates its migration by extending processes underneath it. (C) A genetically derived model of the molecular cascade regulating vulva morphogenesis. SMP-1, PLX-1 and CED-10 function in the same pathway when guiding vulva cells (attractive guidance to the vulva midline). UNC-73 and MIG-2 act in one or more parallel pathways whose function is similar to that of the SMP-1/PLX-1 signaling pathway. It is possible that MIG-2 and CED-10 also function downstream of PLX-1 (broken arrows from UNC-73 and MIG-2), as suggested by the literature, indicating
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Epstein-Barr virus EBNA3A and EBNA3C proteins both repress RBP-J kappa-EBNA2-activated transcription by inhibiting the binding of RBP-J kappa to DNA.

Epstein-Barr virus EBNA3A and EBNA3C proteins both repress RBP-J kappa-EBNA2-activated transcription by inhibiting the binding of RBP-J kappa to DNA.

Both EBNA3A and EBNA3C repress EBNA2-mediated ac- tivation from a TK promoter carrying binding sites for RBP- J k . Since the EBNA2 transcriptional activation is mediated by RBP-J k , it was necessary to determine if EBNA3A and EBNA3C would downregulate EBNA2-mediated activation from a thymidine kinase (TK) promoter carrying binding sites for RBP-J k . The reporter plasmid pTK-CAT-Cp4x is shown in Fig. 1A. Upon transfection of pTK-CAT-Cp4x in HeLa cells, transcription of the CAT gene was very inefficient (Fig. 1B, lane 1) but was strongly activated by EBNA2 (lane 4), indicat- ing that endogenous RBP-J k binds to the reporter plasmid and recruits EBNA2 to the TK promoter. The EBNA2-mediated activation of CAT transcription was strongly repressed by EBNA3A (Fig. 1B, lanes 5 to 7) and EBNA3C (lanes 8 to 10), the repression being proportional to the amounts of EBNA3A- and EBNA3C-expressing plasmids transfected. These results suggest that EBNA3A and EBNA3C either directly contact RBP-J k or prevent the recruitment of EBNA2 by RBP-J k or, alternatively, impair the binding of the RBP-J k –EBNA2 com- plex to DNA. Another possibility is that EBNA3A and EBNA3C could titrate a factor required by EBNA2 to activate transcription (squelching), or they could interfere with the activation function of EBNA2.
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Quantitative Single-Embryo Profile of Drosophila Genome Activation and the Dorsal–Ventral Patterning Network

Quantitative Single-Embryo Profile of Drosophila Genome Activation and the Dorsal–Ventral Patterning Network

activator of AP transcription is maternally deposited bicoid, which is transported to the anterior pole and forms a concen- tration gradient. The nuclear concentration of Bicoid during the final five nuclear cycles remains mostly constant during each nuclear cycle, indicating that Bicoid itself activates transcrip- tion of AP genes at a constant rate through these nuclear cycles (Gregor et al. 2007). In contrast, the protein product of the maternal gene dorsal, found in a DV gradient, increases in concentration within nuclei during each of the final five nuclear cycles (Reeves et al. 2012). This increase in nuclear Dorsal concentration suggests that the DV network is activated differ- ently at each nuclear cycle, both by Dorsal itself, and by a network of transcription factors that respond to different levels of Dorsal. The combination of the rapidly changing transcrip- tional landscape during the MZT, the increasing nuclear con- centration of Dorsal on the DV axis, and the small number of studies that have examined embryogenesis at the single nuclear cycle level present an opportunity to use emerging technologies to provide additional insight into this gene patterning network. In this study, we examine the MZT and gene expression dynamics of the DV network at 10 time points during Drosophila embryonic development between NC10 and gastrulation at a 10- to 15-min resolution. We utilize the NanoString nCounter instrument to directly detect and quantify 68 early embryonic genes from single embryos, and we calculate the absolute num- ber of transcripts per embryo for every gene at every time point in the study. The NanoString system is able to precisely quantify transcripts across five orders of magnitude from a single em- bryo without the need to fragment, amplify, or reverse tran- scribe the RNA (Geiss et al. 2008). The direct detection of messenger RNA (mRNA) molecules minimizes steps between sample collection and data acquisition, reducing error, sample loss, or contamination. RNA-seq has been used in past studies of the Drosophila MZT to quantify the number of transcripts for a gene in the early embryo, and while these studies provide an abundance of data for all genes transcribed, the methods used have been shown to introduce bias in transcript count and read coverage that can hamper absolute quantification of transcripts (Hansen et al. 2010; Lott et al. 2011; Roberts et al. 2011; Ali- Murthy et al. 2013; Petkova et al. 2014).
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Dorsal activity of maternal squint is mediated by a non coding function of the RNA

Dorsal activity of maternal squint is mediated by a non coding function of the RNA

functions that are independent of Oskar protein in early oogenesis (Jenny et al., 2006). Similarly, Xenopus veg-T RNA has a scaffolding function that is independent of the later functions of Veg-T protein in germ layer specification (Zhang and King, 1996; Kloc et al., 2005). Since sqt RNA is normally present in limiting amounts in early embryos (Rebagliati et al., 1998; Gore et al., 2005), even small increases in the amount of sqt 3 ⬘ UTR could lead to an amplified effect of any UTR-bound factors (see model in Fig. 9) in the future embryonic dorsal side. These factor(s) might function via the canonical Wnt/-catenin pathway, as the sqt 3 ⬘ UTR by itself is unable to expand the dorsal domain in the context of ich embryos, which are deficient in  -catenin signaling. It is conceivable that the factor(s) binding to the sqt 3 ⬘ UTR are components of the Wnt/  -catenin pathway. In Xenopus, RNA encoding Xwnt11, which is required for dorsal specification and functions via -catenin (Tao et al., 2005), is localized to dorsal vegetal cells of early embryos. The factors that bind and localize Xwnt11 RNA are not known. In zebrafish, embryos from ich mutant mothers show that maternal -catenin function is required for dorsal specification, and maternal wnt8a RNA is likely to activate Wnt/-catenin signaling (Lu et al., 2011). Asymmetric localization of sqt RNA in the blastoderm by the 4-cell stage precedes dorsal accumulation of nuclear  -catenin at the 128-cell stage (Dougan et al., 2003; Gore et al., 2005).
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Drosophila WntD is a target and an inhibitor of the
Dorsal/Twist/Snail network in the gastrulating embryo

Drosophila WntD is a target and an inhibitor of the Dorsal/Twist/Snail network in the gastrulating embryo

embryos that lacked maternal and zygotic functions of both Frizzled 1 and Frizzled 2 but did not observe any obvious defects in Dorsal or snail expression. A similar experiment using a dishevelled null mutant also did not reveal any such defects (data not shown). Furthermore, overexpression of Dishevelled or dominant-negative Gsk3 did not cause a detectable change of dorsoventral patterning (data not shown). These results suggest that Drosophila WntD may use other components for signaling. Wnt molecules employ multiple receptors and pathways to regulate various processes (Nelson and Nusse, 2004; Veeman et al., 2003). For example, Drosophila Wnt5 interacts with the receptor tyrosine kinase Derailed to regulate axon guidance (Yoshikawa et al., 2003). There are seven Wnt proteins and five Frizzled receptors in Drosophila, and WntD showed detectable affinity towards Frizzled 4 in cell culture assays (Wu and Nusse, 2002), but the in vivo relevance of this interaction is not clear. It is important to elucidate how Drosophila WntD transmits its signal. Equally important is to find out whether WntD interacts with the Toll pathway, and whether the interaction also occurs in processes such as the immune response and cancer progression in other organisms.
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