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Ihog and Boi are essential for Hedgehog signaling in Drosophila
Neural Development volume 5, Article number: 28 (2010)
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The Hedgehog (Hh) signaling pathway is important for the development of a variety of tissues in both vertebrates and invertebrates. For example, in developing nervous systems Hh signaling is required for the normal differentiation of neural progenitors into mature neurons. The molecular signaling mechanism underlying the function of Hh is not fully understood. In Drosophila, Ihog (Interference hedgehog) and Boi (Brother of Ihog) are related transmembrane proteins of the immunoglobulin superfamily (IgSF) with orthologs in vertebrates. Members of this IgSF subfamily have been shown to bind Hh and promote pathway activation but their exact role in the Hh signaling pathway has remained elusive. To better understand this role in vivo, we generated loss-of-function mutations of the ihog and boi genes, and investigated their effects in developing eye and wing imaginal discs.
While mutation of either ihog or boi alone had no discernible effect on imaginal tissues, cells in the developing eye disc that were mutant for both ihog and boi failed to activate the Hh pathway, causing severe disruption of photoreceptor differentiation in the retina. In the anterior compartment of the developing wing disc, where different concentrations of the Hh morphogen elicit distinct cellular responses, cells mutant for both ihog and boi failed to activate responses at either high or low thresholds of Hh signaling. They also lost their affinity for neighboring cells and aberrantly sorted out from the anterior compartment of the wing disc into posterior territory. We found that ihog and boi are required for the accumulation of the essential Hh signaling mediator Smoothened (Smo) in Hh-responsive cells, providing evidence that Ihog and Boi act upstream of Smo in the Hh signaling pathway.
The consequences of boi;ihog mutations for eye development, neural differentiation and wing patterning phenocopy those of smo mutations and uncover an essential role for Ihog and Boi in the Hh signaling pathway.
The Hedgehog (Hh) signaling pathway is essential for proper embryonic development, and aberrant Hh pathway activity is the cause of several human congenital defects and cancers [1–3]. In developing nervous systems, Hh is an important factor governing neural fate specification, neural precursor proliferation, and axon guidance [3–5]. For example, Hh signaling specifies neural cell fate identity in the developing neural tube of vertebrates [4], and is essential for the normal differentiation of photoreceptors in the Drosophila compound eye [6]. Despite its importance in these and many other developmental events in diverse species, the molecular signaling mechanism underlying the function of Hh is still not fully elucidated.
Hh is a secreted protein that elicits concentration-dependent effects [3, 4]. Genetic and biochemical experiments have led to a model where Patched (Ptc), a 12-pass transmembrane protein, is involved in sensing the extracellular Hh concentration [2]. In the absence of Hh, Ptc maintains the 7-pass transmembrane protein Smoothened (Smo) in a repressed state. Under these conditions, the Cubitus interruptus (Ci) transcription factor is proteolytically cleaved and acts as a transcriptional repressor. Conversely, in the presence of Hh, Ptc-mediated repression of Smo is relieved and this leads to the stabilization of full-length Ci (Ci155), which activates transcription of Hh target genes.
Ihog (Interference hedgehog; CG9211) and Boi (Brother of Ihog; CG13756) are two related immunoglobulin superfamily (IgSF) members composed of four immunoglobulin-like (Ig) domains, two fibronectin type III (FN3) repeats, a transmembrane domain, and a cytoplasmic tail [7–9]. The first FN3 domain of Ihog is required and sufficient for direct binding to Hh [9, 10]. In transcription reporter assays in cultured cells, modulation of Ihog and Boi levels affected the strength of responses to Hh [8, 9]. Although Ptc plays a critical role in sensing the Hh morphogenic gradient, the identification of Ihog and Boi as proteins that bind to Hh and promote pathway activation raised questions about their exact role in the Hh signaling pathway. To better understand this role in vivo, we generated loss-of-function mutations of the ihog and boi genes in Drosophila. We have investigated the effects of these mutations in developing eye and wing imaginal discs, and found that Ihog and Boi are functionally redundant and are required for Hh signaling in these tissues. This work uncovers an essential role for Ihog and Boi in the Hh signaling pathway.
Generation of ihog and boi mutants in Drosophila
We generated an ihog null mutant (ihogDC1) that completely lacks the ihog coding sequence plus a portion of the 5' untranslated region of the adjacent gene CG10158 (Figure 1A; see Materials and methods). The absence of ihog mRNA transcripts in ihogDC1/DC1mutants was confirmed by in situ hybridization (Figure 1F) and by reverse transcriptase PCR (RT-PCR; Figure 2D). ihogDC1/DC1homozygotes or ihogDC1/Dfhemizygotes were viable and fertile (Table 1). However, we observed semi-lethality when mutant progeny (ihogDC1/DC1or ihogDC1/Df) but not heterozygous progeny (ihogDC1/+) were derived from ihogDC1/DC1homozygous mothers (Table 1).
We characterized a UAS-ihog:myc transgenic line with basal levels of expression in the absence of a Gal4 driver (Figure 2D). Eclosion and adult viability were fully rescued in the presence of UAS-ihog::myc (Table 1), demonstrating that death at pupal stages was caused by loss of Ihog function and not CG10158. Together, the data indicate that zygotic Ihog is important for adult viability only in the absence of maternal Ihog.
The viability and lack of overt Hh-like phenotypes in ihogDC1mutants prompted us to also investigate Boi, since it behaves similarly to Ihog in Hh transcription reporter assays in vitro [9]. We used chemical mutagenesis to generate a boi mutation (boiC1) in a genetic screen (Figure 1B; Additional file 1; see Materials and methods). boiC1causes a premature stop codon at amino acid Trp626 (Figure 1B). This mutation is predicted to truncate the Boi protein within the second FN3 domain, and fails to encode the transmembrane domain and cytoplasmic tail (Figure 1C). Therefore, the boiC1mutation is predicted to severely disrupt the properties of Boi. The boi gene is situated on the X chromosome and therefore males are boiC1/-hemizygotes. Similar to ihog, boiC1/C1female flies and boiC1/-male flies were viable and fertile, but unlike ihog there was no maternal effect.
Thus, zygotic mutants for either ihogDC1or boiC1were viable and exhibited no overt phenotype reminiscent of mutations of components of the Hh signaling pathway. However, larvae that were double mutants for ihogDC1and boiC1died 24 to 48 hours after hatching, and never reached third instar (L3), suggesting that ihog and boi might act redundantly. In support of this idea, viability of double mutants was rescued in the presence of UAS-ihog::myc, suggesting that Ihog could supplant Boi function in these flies (Figure 2C).
Effects of ihog and boi mutations in the developing visual system
In the developing Drosophila retina, neuronal differentiation within the eye imaginal disc proceeds stepwise in the wake of a constriction in the disc epithelium called the morphogenetic furrow (MF) [11]. Hh signaling is required for the initiation of the MF at the posterior margin of the disc, and for its wave-like progression across the disc that marks the boundary between undifferentiated anterior cells and differentiating posterior cells [6]. Previous studies have demonstrated that clones of cells in the developing eye disc that lack the essential Hh mediator Smo retard MF progression and exhibit a cell-autonomous delay of differentiation into photoreceptors (R-cells) [12, 13].
To begin to investigate whether ihog and boi could play a role in the developing visual system, we studied their expression in wild-type L3 larvae using in situ hybridization (Figure 1D, E). Transcripts for both genes were observed throughout the developing eye disc, but were enriched in anterior regions characterized by undifferentiated cells compared to the posterior regions where there is ongoing differentiation of retinal cell types. Transcripts for neither ihog nor boi were detected in the optic lobe and other regions of the brain at this stage. We confirmed the complete loss of ihog mRNA in ihogDC1mutants (Figure 1F) and thereby demonstrated the specificity of the ihog probe. We could not do likewise with the boi probe, since it was predicted to hybridize with transcripts from the boiC1nonsense mutation. However, the boi probe is unlikely to cross-hybridize with ihog transcripts as the ihog probe does not detect boi transcripts in ihogDC1/DC1mutants. Together, these data revealed overlapping expression of ihog and boi in the developing eye disc, supporting the idea that they might act redundantly in this context.
To test this directly, we needed to generate mosaic animals because, as noted above, double mutant larvae died prior to L3. In boiC1/-hemizygotes, we used FLP-mediated mitotic recombination to render the majority of cells in the developing eye discs also mutant for ihogDC1/DC1[14]. Eye development was severely compromised in these mosaic animals (Figure 2B), leading to structural defects reminiscent of those found in smo and hh mosaics [15–17]. Control mosaics in which ihogDC1/DC1eye discs were generated in boiC1/+heterozygotes had normal eyes (Figure 2A). Additional controls with normal eyes were boiC1/-; ihogDC1/+males (not shown) and rescued double mutants (genotype: boiC1/-; ihogDC1/DC1; UAS-ihog::myc/+; Figure 2C). These results indicated that Ihog and Boi are together required for the proper development of the Drosophila eye.
Ihog and Boi are required for Hh signaling and neuronal differentiation in the eye imaginal disc
To determine whether the disruption of eye development that we observed was due to perturbation of the Hh signaling pathway, we first examined the distribution of Ci155, a Gli-related zinc-finger protein that regulates transcription of Hh-responsive genes. Hh signaling inhibits proteolytic processing of Ci155 to the truncated repressor form Ci75 [3, 18]. Using an antibody that detects Ci155 but not Ci75, one can monitor Hh signaling in cells that accumulate high levels of Ci155 [19]. In eye discs of L3 larvae, Ci155 normally accumulates to high levels just anterior to the advancing MF, marking Hh signaling that is required for MF progression [12, 20, 21]. In control experiments, we generated ihogDC1/DC1clones in boiC1/+heterozygotes and found no effect on Ci155 accumulation just anterior to the MF (eight of eight clones; Figure 3A-B''). In contrast, ihogDC1/DC1clones in boiC1/-mutants had no detectable Ci155 expression at the MF (11 of 11 clones; Figure 3C-D''), indicating a loss of Hh signal transduction in double mutant cells.
Like smo mutant clones [12, 15], we found that boi;ihog double mutant clones failed to express Elav, a neuron-specific marker for R-cells in the eye disc (green fluorescent protein (GFP)-negative in Figure 3E-E''). In controls, cells posterior to the MF that were mutant for only ihog (GFP-negative in Figure 3A, B) or only boi (GFP-positive in Figure 3E) expressed Elav normally, indicating that simultaneous loss of both family members is required to affect R-cell differentiation.
Elevated Ci155 levels drop sharply in cells posterior to the MF in a Cullin-3-dependent proteolytic process associated with the onset of R-cell differentiation [12, 15]. As in the case of smo mutant clones [12, 15], the absence of R-cells in clones lacking ihog and boi is accompanied by ectopic accumulation of Ci155 in clones located posterior to the MF (Figure 3E-E''). Taken together, our data suggest that Ihog and Boi are functionally redundant and, like Smo, are essential for the differentiation of R-cells in response to Hh.
Ihog and Boi are required cell-autonomously for both high- and low-threshold responses to Hh pathway activation
To determine whether Ihog and Boi also function in Hh signaling elsewhere, and to explore whether they mediate high-threshold and low-threshold responses to a Hh morphogen gradient in vivo, we examined their role in wing development. Subdivision of the developing imaginal wing disc into anterior and posterior compartments involves the posterior-specific expression of the selector gene Engrailed [22], which programs posterior cells to produce and secrete Hh, and simultaneously prevents their response to Hh by blocking expression of Ci [23, 24]. Therefore, only anterior cells limited to a broad stripe along the anterior/posterior compartment boundary can respond to Hh, and they do so by upregulating the expression of Hh target genes in a manner that depends on their proximity to the boundary and therefore the concentration of Hh to which they are exposed [25]. Cells immediately adjacent to the compartment boundary respond to high levels of Hh by expressing Ptc, a direct transcriptional target of Hh signaling [26]. Cells positioned more anteriorly respond to lower levels of Hh marked by high Ci155 accumulation, though all anterior cells have low baseline levels of Ci155. In control experiments, levels of Ptc and Ci155 were unaffected in ihogDC1/DC1mutants (not shown), in boiC1/-hemizygous cells (GFP-positive in Figure 4C, D) and in ihogDC1/DC1clones in boiC1/+heterozygotes (12 of 12 clones; GFP-negative in Figure 4A, B).
In contrast, cells lacking both ihog and boi in anterior clones situated near the compartment boundary did not express Ptc and did not accumulate high levels of Ci155 (12 of 12 clones; Figure 4C-D'''), similar to smo clones [27]. Ci155 normally accumulates to high levels even at low concentrations of Hh [28, 29], and so the failure to elicit this low-threshold response indicates that removal of ihog and boi results in complete loss of Hh pathway activation. Importantly, while cells lacking both ihog and boi lost the ability to respond to Hh, cells immediately adjacent to these clones expressed Ptc and accumulated Ci155 normally (arrowheads in Figure 4D'''), indicating that Ihog and Boi are required cell-autonomously in Hh responding cells.
Ihog and Boi sequester Hh activity
In response to Hh, upregulation of Ptc levels in anterior cells near the compartment boundary reduces the anterior range of Hh activity [27]. This finding has been fundamental to the prevailing model that Ptc is a Hh receptor that can directly bind and sequester Hh protein. An alternative hypothesis is that Ptc interacts indirectly with Hh, perhaps by tethering another protein that directly binds Hh [27]. Since Ihog and Boi bind directly to Hh in vitro [9], and since Ptc and Ihog have been shown to synergize to increase binding of Hh to cultured cells [9], we wondered whether Ihog and Boi could likewise limit the range of Hh activity within the anterior compartment of the wing disc. Therefore, we studied large anterior clones that spanned the usual Ptc expression domain, and examined Ptc and Ci levels in cells immediately anterior to these clones. In cells that were immediately anterior to control ihogDC1/DC1clones in boiC1/+heterozygotes, there was no evidence for Hh pathway activation. In contrast, high levels of both Ptc and Ci155 were observed in cells immediately anterior to clones lacking Ihog and Boi (Figure 4C-D'''), indicating that the loss of both Ihog and Boi fails to restrict the movement of Hh through the clone.
Ihog and Boi act upstream of Smo
Activation of the Hh signaling pathway leads to increased accumulation of Smo protein at the surface of cells near the compartment boundary of the wing disc [30]. This accumulation of Smo at the cell surface is required for its signaling activity, and therefore marks Smo activation by Hh [30–33]. Thus, to test whether Ihog and Boi could function upstream of Smo, we examined Smo accumulation in ihog;boi mutant clones. While control clones showed normal Smo accumulation (eight of eight clones; Figure 5A-B''), cells within ihog;boi mutant clones had Smo protein levels that were markedly reduced (seven of seven clones; Figure 5C-D''). Thus, Ihog and Boi are required for the Hh-dependent stabilization of Smo, and these results provide evidence that Ihog and Boi act upstream of Smo in the Hh signaling pathway.
Ihog and Boi are required for compartment-specific cell affinity
Cells of the wing imaginal disc normally do not cross the boundary between the anterior and posterior compartments, and implementation of this boundary is thought to involve mechanisms that control compartment-specific cell affinity and adhesion [34]. Clonal analysis has demonstrated that Hh signaling is required to maintain cell affinities in the anterior compartment. For example, if anterior cells that are adjacent to the boundary are mutant for smo or ci [35–37], they sort out from other anterior cells and segregate into posterior territory. To determine if Ihog and Boi are involved in maintaining compartment-specific cell affinities, we examined the segregation behavior of clones near the anterior-posterior compartment boundary. To do this, clones of anterior origin were unambiguously identified if they expressed baseline levels of Ci, and if an adjacent wild-type sister clone, the twin-spot, was situated in the anterior compartment since, by definition, the mutant clone and twin-spot must arise from the same compartment. In controls (ihogDC1/DC1clones in boiC1/+heterozygotes), anterior-derived clones exclusively occupied anterior territory and defined a straight border with posterior cells in the normal position of the boundary (14 of 14 clones; Figure 4A-B'''). In contrast, double mutant clones (ihogDC1/DC1clones in boiC1/-hemizygotes) often straddled the boundary and formed tight borders with both anterior and posterior cells (8 of 16 clones; Figure 6). This modified segregation behavior indicates that anterior cells lacking both Ihog and Boi sort out from the anterior compartment into posterior territory. These clones do not readily integrate into posterior territory because they do not possess affinity for posterior cells conferred by expression of Engrailed [35]. Consistent with the lack of a known role for Hh signaling in the posterior compartment, double mutant posterior clones respected the compartment boundary and did not sort into anterior territory (11 of 11 clones; Figure 6A'). Together, these results indicate that, like Smo or Ci, Ihog and Boi are required to maintain cell affinities in the anterior compartment.
Ihog and Boi are essential components of the Hh pathway
Using genetic approaches in Drosophila, we show that ihog and boi act redundantly and are required for viability. Mutation of ihog and boi in the developing eye disc prevents Hh signaling and causes severe disruption of photoreceptor differentiation. Similarly, mutation of ihog and boi in the developing wing disc completely abrogates Hh signaling in a cell-autonomous manner. All these phenotypic effects are identical to mutations of the essential Hh signaling mediator smo and thus indicate that the transmembrane proteins Ihog and Boi are absolutely required for the Hh signaling pathway. We surmise that the essential role for ihog and boi in Hh signaling was not discovered previously because they are redundant with one another, and therefore refractory to conventional forward genetic screens in Drosophila.
A previous study of the lethal mutation ihogKGreported defects that mimicked those caused by mutations of the Hh signaling pathway [9]. That ihogKGdisrupts gene(s) in addition to ihog is indicated by the fact that zygotic mutation of ihogKGalone is lethal, while the null allele ihogDC1is not. ihogKGwas reported to disrupt Ptc expression in the wing disc, and the patterning of wing veins [9], but we did not observe these effects with ihogDC1null mutations (Figure 4A'', B''). In addition, germline clones of ihogKGwere found to disrupt patterning of the embryonic cuticle [9]. Therefore, it was unexpected that our maternal/zygotic ihog DC1null mutants survived at least to pupal stages, and that there were no overt Hh-like phenotypes in the occasional flies that managed to escape the lethal phase and reach adulthood. Indeed, such flies looked remarkably normal. Perhaps the phenotypes observed in ihogKGare rare, or due to the disrupted function of the neighboring gene CG10158 or another gene in addition to ihog.
In Drosophila, genetic experiments have suggested that Ptc is the Hh receptor [38] and subsequent work has shown Ptc co-localization with Hh in S2 cells [39]. This has led to the model that Ptc binds Hh, though experiments have yet to demonstrate direct physical contact. In vertebrates, expression of the Ptc ortholog Ptc1 in cells promoted Sonic hedgehog (Shh) binding, suggesting that Ptc1 is a receptor for Shh [40, 41]. However, as noted by the authors in those studies, Shh could use additional receptors and Ptc could affect their ability to bind to Shh. Addressing the important question of whether Ptc/Ptc1 bind directly to Hh/Shh is likely to require purified proteins in cell-free systems, a challenge for transmembrane proteins with complex topologies like Ptc. Interestingly, Ihog and Boi have been shown to bind directly to Hh [9, 10]. Here we found that clones mutant for both ihog and boi phenocopied the effects of smo mutations for Hh signaling. Thus, our data demonstrate that Ihog and Boi are essential components of the Hh pathway. Coupled with our finding that Ihog and Boi act upstream of Smo and sequester Hh activity, this raises the possibility that Ihog and Boi are Hh-binding components of the Hh receptor complex. While preparing this manuscript, another research group came to a similar conclusion [42]. Using biochemical experiments, they further suggest that Ihog/Boi, together with Ptc and Hh, form a complex and that the presence of Ihog/Boi in this complex is essential to allow Ptc to bind to Hh. However, since purified proteins were not used in these experiments, it remains to be determined whether the interaction between Hh and Ptc is direct. Alternatively, Ptc could affect the ability of an additional protein (perhaps Ihog or Boi) to recruit Hh. Thus, the question as to whether Ptc binds directly to Hh, in the absence or the presence of Ihog/Boi, remains to be elucidated. Additional experiments will be required to understand the exact contributions of Ihog, Boi and Ptc to the reception and transduction of the Hh signal. Nonetheless, our experiments and those of Zheng and colleagues identify Ihog and Boi as essential components of a Hh receptor complex.
In addition to Ihog and Boi, other membrane-tethered Hh-binding proteins, such as the glypican family members, have been identified [8, 43–45]. Although it is possible that other developing tissues may use different Hh-binding molecules as sensors for Hh, our data indicate that Ihog and Boi are critical to elicit Hh signaling and that, at least in developing imaginal tissues, no other molecule can compensate for loss of Ihog and Boi.
Ihog and Boi: transmembrane IgSF proteins with potential to mediate cell affinity
In addition to the role of Ihog and Boi in wing cell fate specification (Zheng et al. [42] and this study), we show for the first time that Ihog and Boi are required for maintenance of the antero-posterior boundary of the wing disc. Formation of this boundary is thought to involve cell-surface recognition molecules responsible for affinity among anterior cells. Since ci mutant clones lose affinity for the anterior compartment [37], it is likely that such adhesion molecules are transcriptionally regulated by the Hh pathway. Despite broad screening efforts to identify these molecules [46], they remain unidentified. While the loss of cell affinity in boi;ihog double mutant clones is likely a consequence of loss of Hh signaling, it is an intriguing possibility that Ihog and/or Boi could also be Ci-regulated recognition molecules promoting the differential compartment-specific cell affinity. Consistent with this hypothesis, the vertebrate orthologs of Ihog and Boi, called Cdon (Cell adhesion molecule-related/down-regulated by oncogenes) and Boc (Brother of Cdon), can interact with each other and form higher-order cell-surface complexes with the well-characterized adhesion molecules N- and M-cadherin [7, 47]. These complexes mediate cell-cell interactions between muscle precursor cells and are enriched at sites of contact between myoblasts. Similar to Cdon and Boc, Ihog and Boi could mediate cell-cell interactions, and might play a direct role in compartment-specific cell affinity.
Ihog and Boi and their vertebrate orthologs in nervous system development
Although Cdon and Boc have also been shown to play a role in Hh signaling in vertebrates [9, 48–50], their absolute requirement for Hh signaling has not been demonstrated, and mouse embryos mutant for both cdon and boc do not display a smo-like phenotype (our unpublished data). Additionally, Hh binds to Ihog/Boi in a manner that is different from Shh binding to Cdon and Boc [10, 51], suggesting that a direct extrapolation of our work in flies to Cdon and Boc could be subject to added complexities.
Nevertheless, there are enough interesting parallels between Ihog and Boi and their vertebrate orthologs in the developing nervous system to warrant further investigation. We have shown here that Ihog and Boi are essential for the differentiation of R-cells in the developing fly visual system. Interestingly, overexpression studies in chick have shown that Boc and Cdon promote Shh-induced differentiation in the neural tube and that Cdon is required for Shh-dependent cell fate specification in the neural tube [48, 50]. Furthermore, Boc has been shown to be essential for axon guidance through the activation of a non-canonical, transcription-independent Shh signaling pathway mediated by the activity of Src-family kinases [49, 52, 53]. It will be interesting to determine whether Ihog and Boi also function in axon guidance and whether they may contribute to non-canonical Hh signaling in Drosophila.
We conclude that the transmembrane proteins Ihog and Boi are essential components of the Hh signaling pathway. Ihog and Boi act upstream of Smo and sequester Hh activity, raising the possibility that Ihog and Boi are Hh-binding components of the Hh receptor complex. These findings advance our understanding of the molecular signaling mechanism underlying the function of Hh.
Materials and methods
Fly stocks
ihogDC1was generated by Flippase (FLP)-mediated recombination between Flippase recognition target (FRT) sites in pBac{RB}CG10158e02576 and pBac{RB}iHoge02142 [54, 55]. After out-crossing to w1118, the resulting chimeric pBac element was excised, and the predicted deletion of 3,479 bp was confirmed by DNA sequencing and RT-PCR (Figure 2D). A deficiency stock that uncovers the ihog locus (Df(2L)Exel7029) was also used, and is designated ihogDfherein.
boiC1was identified in a screen for mutations that suppress ectopic wing veins caused by apGAL4-driven expression of P{EP}EP1447, a UAS-based P-element situated upstream of the boi gene (Figure S1A). Briefly, starved EP1447 male flies were exposed to 25 mM ethylmethanesulfonate (EMS) overnight, then crossed to apGAL4virgin females. boiC1was identified among approximately 15,000 adult progeny screened for suppression of ectopic wing veins. boiC1proved to be a viable mutation, and so PCR and DNA sequencing were used to compare the boi coding region in boiC1homozygotes with the parental line EP1447. An induced G-to-A transition was found in boiC1, resulting in a nonsense mutation at Trp626.
To create UAS-ihog::myc, the entire coding sequence of ihog (minus stop codon) was PCR-amplified from an ihog cDNA (GH03927), and cloned into pCR2.1-TOPO (Invitrogen, Carlsbad, CA, USA). This fragment was excised and cloned into p5MT2Stp (BamHI/SalI) to add five copies of the myc epitope to the carboxyl terminus. UAS- ihog::myc was generated by KpnI/NotI excision and cloning into pUASt. Transgenic flies were generated in a w- background by standard microinjection procedures (BestGene Inc., San Diego, CA, USA).
Mosaic analysis
Mosaic adult eyes were obtained by crossing boiC1/C1; ihogDC1, FRT40A/+ females to FRT40A, l(2)CL-L1; ey-FLP males [14]. Wing and eye disc clones marked by the absence of GFP were generated by crossing boiC1/C1; ihogDC1, FRT40A/+ females to Ubi-GFP, FRT40A; hs-FLP males. In this way, ihogDC1/DC1clones would be generated in boiC1/+heterozygotes (female progeny) or boiC1/-hemizygotes (male progeny). Embryos were collected for 16 hours, raised for 24 hours (25°C), then the larvae were heat shocked at 38°C for 1 hour and further raised (25°C) through L3.
Wandering L3 larvae were dissected and fixed according to standard procedures, just prior to pupation. Monoclonal antibodies obtained from the Developmental Studies Hybridoma Bank included: mouse anti-Ptc (dilution 1:50), mouse anti-Smo (1:50) and mouse anti-Elav (1:2,000). Other antibodies used were rabbit anti-GFP (1:1,000; Molecular Probes, Eugene, OR, USA) and rat anti-Ci155 (1:2,000) [19]. Secondary antibodies were: Alexa Fluor 488 conjugated goat anti-rabbit (1:300); Alexa Fluor 647 conjugated goat anti-mouse (1:300); and Alexa Fluor 588 conjugated goat anti-rat (1:300), all from Molecular Probes.
In situ hybridization
A digoxigenin (Dig)-labeling kit (Roche, Indianapolis, IN, USA) was used to synthesize cRNA probes from a BglII-digested ihog cDNA (GH03927) and an EcoRI-digested boi cDNA (SD07678), using T7 and Sp6 RNA polymerases, respectively. Prepared L3 larvae were hybridized with probes overnight at 55°C using standard procedures, and visualized using anti-Dig-AP (1:1,000; Roche).
RT-PCR was performed on total RNA isolated from adult flies. The ihog primer set was designed to amplify a band of 411 bp from the UAS-ihog::myc transgene, but not ihogDC1/DC1mutants (5'-CCCTGAGCAAGTGTGGAGAT-3'; 5'-CTCTAGGCGAGTACCGATGC-3'). An Actin5C fragment of 586 bp was co-amplified as a control (5'-GAGCGCGGTTACTCTTTCAC-3'; 5'-ATCCCGATCCTGATCCTCTT-3').
Brother of Cdon
Brother of Ihog
base pair
Cell adhesion molecule-related/down-regulated by oncogenes
Cubitus interruptus
fibronectin type III
green fluorescent protein
immunoglobulin superfamily
Interference hedgehog
third instar
morphogenetic furrow
reverse transcriptase PCR
Sonic hedgehog
Varjosalo M, Li SP, Taipale J: Divergence of hedgehog signal transduction mechanism between Drosophila and mammals. Dev Cell. 2006, 10: 177-186. 10.1016/j.devcel.2005.12.014.
Lum L, Beachy PA: The Hedgehog response network: sensors, switches, and routers. Science. 2004, 304: 1755-1759. 10.1126/science.1098020.
Jiang J, Hui CC: Hedgehog signaling in development and cancer. Dev Cell. 2008, 15: 801-812. 10.1016/j.devcel.2008.11.010.
Dessaud E, McMahon AP, Briscoe J: Pattern formation in the vertebrate neural tube: a sonic hedgehog morphogen-regulated transcriptional network. Development. 2008, 135: 2489-2503. 10.1242/dev.009324.
Charron F, Tessier-Lavigne M: Novel brain wiring functions for classical morphogens: a role as graded positional cues in axon guidance. Development. 2005, 132: 2251-2262. 10.1242/dev.01830.
Roignant JY, Treisman JE: Pattern formation in the Drosophila eye disc. Int J Dev Biol. 2009, 53: 795-804. 10.1387/ijdb.072483jr.
Kang JS, Mulieri PJ, Hu Y, Taliana L, Krauss RS: BOC, an Ig superfamily member, associates with CDO to positively regulate myogenic differentiation. EMBO J. 2002, 21: 114-124. 10.1093/emboj/21.1.114.
Lum L, Yao S, Mozer B, Rovescalli A, Von Kessler D, Nirenberg M, Beachy PA: Identification of Hedgehog pathway components by RNAi in Drosophila cultured cells. Science. 2003, 299: 2039-2045. 10.1126/science.1081403.
Yao S, Lum L, Beachy P: The ihog cell-surface proteins bind Hedgehog and mediate pathway activation. Cell. 2006, 125: 343-357. 10.1016/j.cell.2006.02.040.
McLellan JS, Yao S, Zheng X, Geisbrecht BV, Ghirlando R, Beachy PA, Leahy DJ: Structure of a heparin-dependent complex of Hedgehog and Ihog. Proc Natl Acad Sci USA. 2006, 103: 17208-17213. 10.1073/pnas.0606738103.
Wolff T, Ready DF: Pattern formation in the Drosophila retina. The Development of Drosophila melanogaster. Edited by: Bate M, Martinez-Arias A. 1993, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2: 1277-1325.
Baker N, Bhattacharya A, Firth L: Regulation of Hh signal transduction as Drosophila eye differentiation progresses. Dev Biol. 2009, 335: 356-366. 10.1016/j.ydbio.2009.09.008.
Strutt D, Mlodzik M: Hedgehog is an indirect regulator of morphogenetic furrow progression in the Drosophila eye disc. Development. 1997, 124: 3233-3240.
Stowers RS, Schwarz TL: A genetic method for generating Drosophila eyes composed exclusively of mitotic clones of a single genotype. Genetics. 1999, 152: 1631-1639.
Dominguez M: Dual role for Hedgehog in the regulation of the proneural gene atonal during ommatidia development. Development. 1999, 126: 2345-2353.
Heberlein U, Wolff T, Rubin GM: The TGF beta homolog dpp and the segment polarity gene hedgehog are required for propagation of a morphogenetic wave in the Drosophila retina. Cell. 1993, 75: 913-926. 10.1016/0092-8674(93)90535-X.
Ma C, Zhou Y, Beachy PA, Moses K: The segment polarity gene hedgehog is required for progression of the morphogenetic furrow in the developing Drosophila eye. Cell. 1993, 75: 927-938. 10.1016/0092-8674(93)90536-Y.
Kalderon D: The mechanism of hedgehog signal transduction. Biochem Soc Trans. 2005, 33: 1509-1512. 10.1042/BST20051509.
Aza-Blanc P, Ramirez-Weber FA, Laget MP, Schwartz C, Kornberg TB: Proteolysis that is inhibited by hedgehog targets Cubitus interruptus protein to the nucleus and converts it to a repressor. Cell. 1997, 89: 1043-1053. 10.1016/S0092-8674(00)80292-5.
Motzny CK, Holmgren R: The Drosophila cubitus interruptus protein and its role in the wingless and hedgehog signal transduction pathways. Mech Dev. 1995, 52: 137-150. 10.1016/0925-4773(95)00397-J.
Strutt DI, Mlodzik M: The regulation of hedgehog and decapentaplegic during Drosophila eye imaginal disc development. Mech Dev. 1996, 58: 39-50. 10.1016/S0925-4773(96)00555-2.
Lawrence PA, Morata G: Compartments in the wing of Drosophila: a study of the engrailed gene. Dev Biol. 1976, 50: 321-337. 10.1016/0012-1606(76)90155-X.
Tabata T, Schwartz C, Gustavson E, Ali Z, Kornberg TB: Creating a Drosophila wing de novo, the role of engrailed, and the compartment border hypothesis. Development. 1995, 121: 3359-3369.
Zecca M, Basler K, Struhl G: Sequential organizing activities of engrailed, hedgehog and decapentaplegic in the Drosophila wing. Development. 1995, 121: 2265-2278.
Torroja C, Gorfinkiel N, Guerrero I: Mechanisms of Hedgehog gradient formation and interpretation. J Neurobiol. 2005, 64: 334-356. 10.1002/neu.20168.
Strigini M, Cohen SM: A Hedgehog activity gradient contributes to AP axial patterning of the Drosophila wing. Development. 1997, 124: 4697-4705.
Chen Y, Struhl G: Dual roles for patched in sequestering and transducing Hedgehog. Cell. 1996, 87: 553-563. 10.1016/S0092-8674(00)81374-4.
Mullor JL, Calleja M, Capdevila J, Guerrero I: Hedgehog activity, independent of decapentaplegic, participates in wing disc patterning. Development. 1997, 124: 1227-1237.
Aza-Blanc P, Kornberg TB: Ci: a complex transducer of the hedgehog signal. Trends Genet. 1999, 15: 458-462. 10.1016/S0168-9525(99)01869-7.
Denef N, Neubuser D, Perez L, Cohen SM: Hedgehog induces opposite changes in turnover and subcellular localization of patched and smoothened. Cell. 2000, 102: 521-531. 10.1016/S0092-8674(00)00056-8.
Zhu AJ, Zheng L, Suyama K, Scott MP: Altered localization of Drosophila Smoothened protein activates Hedgehog signal transduction. Genes Dev. 2003, 17: 1240-1252. 10.1101/gad.1080803.
Nakano Y, Nystedt S, Shivdasani AA, Strutt H, Thomas C, Ingham PW: Functional domains and sub-cellular distribution of the Hedgehog transducing protein Smoothened in Drosophila. Mech Dev. 2004, 121: 507-518. 10.1016/j.mod.2004.04.015.
Jia J, Tong C, Wang B, Luo L, Jiang J: Hedgehog signalling activity of Smoothened requires phosphorylation by protein kinase A and casein kinase I. Nature. 2004, 432: 1045-1050. 10.1038/nature03179.
Dahmann C, Basler K: Compartment boundaries: at the edge of development. Trends Genet. 1999, 15: 320-326. 10.1016/S0168-9525(99)01774-6.
Blair SS, Ralston A: Smoothened-mediated Hedgehog signalling is required for the maintenance of the anterior-posterior lineage restriction in the developing wing of Drosophila. Development. 1997, 124: 4053-4063.
Rodriguez I, Basler K: Control of compartmental affinity boundaries by hedgehog. Nature. 1997, 389: 614-618. 10.1038/39343.
Dahmann C, Basler K: Opposing transcriptional outputs of Hedgehog signaling and engrailed control compartmental cell sorting at the Drosophila A/P boundary. Cell. 2000, 100: 411-422. 10.1016/S0092-8674(00)80677-7.
Ingham PW, Taylor AM, Nakano Y: Role of the Drosophila patched gene in positional signalling. Nature. 1991, 353: 184-187. 10.1038/353184a0.
Lu X, Liu S, Kornberg TB: The C-terminal tail of the Hedgehog receptor Patched regulates both localization and turnover. Genes Dev. 2006, 20: 2539-2551. 10.1101/gad.1461306.
Stone DM, Hynes M, Armanini M, Swanson TA, Gu Q, Johnson RL, Scott MP, Pennica D, Goddard A, Phillips H, Noll M, Hooper JE, de Sauvage F, Rosenthal A: The tumour-suppressor gene patched encodes a candidate receptor for Sonic hedgehog. Nature. 1996, 384: 129-134. 10.1038/384129a0.
Marigo V, Davey RA, Zuo Y, Cunningham JM, Tabin CJ: Biochemical evidence that patched is the Hedgehog receptor. Nature. 1996, 384: 176-179. 10.1038/384176a0.
Zheng X, Mann RK, Sever N, Beachy PA: Genetic and biochemical definition of the Hedgehog receptor. Genes Dev. 2010, 24: 57-71. 10.1101/gad.1870310.
Ayers KL, Gallet A, Staccini-Lavenant L, Therond PP: The long-range activity of Hedgehog is regulated in the apical extracellular space by the glypican Dally and the hydrolase Notum. Dev Cell. 2010, 18: 605-620. 10.1016/j.devcel.2010.02.015.
Gallet A, Staccini-Lavenant L, Therond PP: Cellular trafficking of the glypican Dally-like is required for full-strength Hedgehog signaling and wingless transcytosis. Dev Cell. 2008, 14: 712-725. 10.1016/j.devcel.2008.03.001.
Williams EH, Pappano WN, Saunders AM, Kim MS, Leahy DJ, Beachy PA: Dally-like core protein and its mammalian homologues mediate stimulatory and inhibitory effects on Hedgehog signal response. Proc Natl Acad Sci USA. 2010, 107: 5869-5874. 10.1073/pnas.1001777107.
Vegh M, Basler K: A genetic screen for hedgehog targets involved in the maintenance of the Drosophila anteroposterior compartment boundary. Genetics. 2003, 163: 1427-1438.
Kang JS, Feinleib JL, Knox S, Ketteringham MA, Krauss RS: Promyogenic members of the Ig and cadherin families associate to positively regulate differentiation. Proc Natl Acad Sci USA. 2003, 100: 3989-3994. 10.1073/pnas.0736565100.
Tenzen T, Allen BL, Cole F, Kang JS, Krauss RS, McMahon AP: The cell surface membrane proteins Cdo and Boc are components and targets of the Hedgehog signaling pathway and feedback network in mice. Dev Cell. 2006, 10: 647-656. 10.1016/j.devcel.2006.04.004.
Okada A, Charron F, Morin S, Shin DS, Wong K, Fabre PJ, Tessier-Lavigne M, McConnell SK: Boc is a receptor for sonic hedgehog in the guidance of commissural axons. Nature. 2006, 444: 369-373. 10.1038/nature05246.
Zhang W, Kang JS, Cole F, Yi MJ, Krauss RS: Cdo functions at multiple points in the Sonic Hedgehog pathway, and Cdo-deficient mice accurately model human holoprosencephaly. Dev Cell. 2006, 10: 657-665. 10.1016/j.devcel.2006.04.005.
McLellan JS, Zheng X, Hauk G, Ghirlando R, Beachy PA, Leahy DJ: The mode of Hedgehog binding to Ihog homologues is not conserved across different phyla. Nature. 2008, 455: 979-983. 10.1038/nature07358.
Fabre PJ, Shimogori T, Charron F: Segregation of ipsilateral retinal ganglion cell axons at the optic chiasm requires the Shh receptor Boc. J Neurosci. 2010, 30: 266-275. 10.1523/JNEUROSCI.3778-09.2010.
Yam PT, Langlois SD, Morin S, Charron F: Sonic hedgehog guides axons through a noncanonical, Src-family-kinase-dependent signaling pathway. Neuron. 2009, 62: 349-362. 10.1016/j.neuron.2009.03.022.
Parks AL, Cook KR, Belvin M, Dompe NA, Fawcett R, Huppert K, Tan LR, Winter CG, Bogart KP, Deal JE, Deal-Herr ME, Grant D, Marcinko M, Miyazaki WY, Robertson S, Shaw KJ, Tabios M, Vysotskaia V, Zhao L, Andrade RS, Edgar KA, Howie E, Killpack K, Milash B, Norton A, Thao D, Whittaker K, Winner MA, Friedman L, Margolis J: Systematic generation of high-resolution deletion coverage of the Drosophila melanogaster genome. Nat Genet. 2004, 36: 288-292. 10.1038/ng1312.
Thibault ST, Singer MA, Miyazaki WY, Milash B, Dompe NA, Singh CM, Buchholz R, Demsky M, Fawcett R, Francis-Lang HL, Ryner L, Cheung LM, Chong A, Erickson C, Fisher WW, Greer K, Hartouni SR, Howie E, Jakkula L, Joo D, Killpack K, Laufer A, Mazzotta J, Smith RD, Stevens LM, Stuber C, Tan LR, Ventura R, Woo A, Zakrajsek I: A complementary transposon tool kit for Drosophila melanogaster using P and piggyBac. Nat Genet. 2004, 36: 283-287. 10.1038/ng1314.
We are grateful to Keith Murai for critical reading of the manuscript. We thank Steves Morin and Yimiao Ou for technical assistance; Yong Rao, David Hipfner, Scott Cameron and Laura Nilson for advice; and Tom Kornberg, Phil Beachy, Jin Jiang, Isabel Guerrero and Gary Struhl for reagents. Fly stocks were obtained from the Bloomington Stock Center and the Exelixis Collection at Harvard. This work was supported by the Canadian Institutes of Health Research (CIHR), the Natural Sciences and Engineering Research Council of Canada (NSERC), the Peter Lougheed Medical Research Foundation, the Fonds de Recherche en Santé du Québec (FRSQ), and the Canada Foundation for Innovation (CFI).
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The authors declare that they have no competing interests.
Authors' contributions
DC performed the experiments, participated in their design, analysis and interpretation, and drafted the manuscript. KC participated in the genetic screen, AL constructed the UAS-ihog transgene, and both helped in data analysis and interpretation. DJvM and FC conceived of the study, and participated in its design and coordination and drafted the manuscript. All authors read and approved the final manuscript.
Electronic supplementary material
Additional file 1: Ectopic wing veins caused by overexpression of Boi. (A) Wing from a EP1447/+; apGAL4/+ fly, showing an ectopic vein (arrow and magnified boxed area) located between veins L3 and L4. Additional wing defects were also observed, though these were variable (arrowheads). (B) Wing from a fly of the genotype EP1447, boiC1/+; apGAL4/+, showing complete suppression of the ectopic vein defect. (PDF 1 MB)
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Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Cite this article
Camp, D., Currie, K., Labbé, A. et al. Ihog and Boi are essential for Hedgehog signaling in Drosophila. Neural Dev 5, 28 (2010). https://doi.org/10.1186/1749-8104-5-28
DOI: https://doi.org/10.1186/1749-8104-5-28
- Anterior Compartment
- Wing Disc
- Wing Imaginal Disc
- Morphogenetic Furrow
- Compartment Boundary


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It's true that most of the civilized world uses standard cellular technology to stay in touch with one another — after all, with convenient networks like LTE, we can instantly communicate with someone else around the world. That's truly incredible, and something that shouldn't be taken for granted.
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"Working" is the theme of a new speaker series that will start this summer and run once a month through the course of the next year. It's inspired by the Studs Terkel book of that name in which "people talk about what they do all day and how they feel about what they do." There are many people within our congregation engaged in fascinating work. This series will give us an opportunity to learn more about what they do.
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Merida, Yucatan — On the last day of 2019, the state government established a security tax for all property owners in Yucatan.
The new levy helps the state maintain public safety technology, such as surveillance cameras. Some details were published in the Official Gazette, which lays out the laws of the land.
Taxes vary according to the government's UMA, a federal economic index. Right now, the 3.44 UMA security fee amounts to around 300 pesos, or US$16, every month. A 2020 UMA has not yet been established.
How the tax is collected has not been decided. The fee may be added to electric bills, according to state officials, although federal officials said that procedure was not feasible.
Federal, state and municipal authorities are exempt from the fee, as are property owners whose monthly consumption of basic services is 100 pesos or less.
Exemptions also include the owners or holders of properties that are in the irrigation districts and are built distribute water for agriculture and livestock.
Source: Diario de Yucatan


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Theater Review: Stalemated pair search for next move in 'Endgame'
Spring Green – When we last saw Brian Mani in an American Players Theatre opening this summer, he was giving a wrenching performance in "Death of a Salesman" as an exhausted Willy Loman, crippled by a lifetime's worth of mistakes that have alienated him from the people he loves most, including his beloved son, Biff.
In an APT season filled with stories involving the fraught relations between parents and their children, Mani is now at it again, giving a compelling performance as Hamm in the just-opened APT production of Samuel Beckett's "Endgame," which debuted eight years after "Salesman." It's being directed here by Aaron Posner.
Much as Willy pines for a vanishing natural world from the confines of an increasingly crowded and claustrophobic Brooklyn, Hamm expresses nostalgia for earth and sky, woods and fields, mammals and creatures of the deep.
He'll never see any of these phenomena again, and not just because he's blind. Nature is dead, confining him to the small and nearly airless room in which he wiles away his days in some post-apocalyptic future. Unable to stand and restricted to an armchair on castors, his survival depends on Clov (David Daniel), who is in turn unable to sit, thanks to his stiff and crippled leg.
It quickly becomes clear that this deformed pair shares an equally debilitating relationship.
Although Hamm had taken Clov in as a boy and raised him as a surrogate son, years spent ordering him about – arbitrarily and even abusively – have taken their toll. "You loved me once," Hamm ruefully states, early on. Clov acknowledges as much, while simultaneously admitting that he no longer does.
So why does Clov stay, putting up with Hamm's abuse while threatening to leave?
Beckett admitted during rehearsals for the original production that Hamm and Clov were surrogates, respectively, for Suzanne Déchevaux-Dumesnil and himself, entangled in a relationship for nearly 50 years, even though both of them periodically wanted to leave it.
They didn't, for some of the same reasons Clov stays with Hamm: guilt, inertia, fear of the unknown and, for all the resentment he feels toward this father figure, the threadbare remains of onetime affection, worn to a habit that he has neither the will nor the imagination to break.
Daniel registers all of this, easily slipping into the role of the dependent child Clov once was and in some ways still is. Daniel's Clov can be sullen; he is also passive-aggressive. But he nevertheless chooses the devil he knows over a world he doesn't.
For his part, Mani makes clear that curmudgeonly as Hamm can seem, he's a bit of a marshmallow underneath; there's a reason we're told he has a heart bleeding in his head.
Hamm wants to love Clov better, but he's much too insecure. Even as he tries to tell himself that things with Clov are getting better, he's busy making them worse; he pushes Clov away because he's already convinced himself that Clov is going to leave.
Might things end differently, in this relationship or any other? Beckett explores as much through periodic interruptions involving Hamm's own parents, Nagg (John Pribyl) and Nell (Sarah Day).
They're living out their final days in two rubbish bins – an apt symbol for how we warehouse and then ignore the old. As with Hamm in relation to Clov, they look back with nostalgia on earlier days together.
But even as their bodies fail, Pribyl and Day give us poignant glimpses of the affection Nagg and Nell still feel, suggesting what every chess player knows: sure as the end itself may be, the game played in getting there has numerous variations, reflecting the choices we make along the way.
"Endgame" continues through Oct. 16 at APT's Touchstone Theatre. For tickets, call (608) 588-2361 or visit americanplayers.org. Read more about this production at TapMilwaukee.com.


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2 comments on "Self Portrait with Banjo"
irasmith Says:
Friday, February 23, 2007 @5:19:05 PM
Hi Phil, Nice work, watercolor, or pastel?
pickinchik Says:
Thursday, December 9, 2010 @6:45:33 AM
Very nice!! Didn't know that was you. My hubby is an artist too. He's a graphic artist but he's from the old school where he actually does a whole bunch of pencil, ink, and only colors in the computer.
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For latest updates about our work SUBSCRIBE to our Newsletter.
We launched an e-Library at Tihar and Computer Training Centres at Tihar, Gurgaon and Faridabad prisons, as we continue to extend our partnerships with more prisons across the nation. In 2014, we also joined hands with more partner institutes.
TKWS, Institute of Banking and Finance
Katha ( NGO), Govindpuri
India Vision Foundation is a voluntary non-profit, non-government organization registered as a Trust in India vide No. 4595 dated August 1, 1994. The foundation was born out of Ramon Magsaysay award (Equivalent to Asia's Nobel Peace Prize) conferred to Dr. Kiran Bedi, the first lady IPS (Indian Police Services) of India, for forging 'positive relationships' between people and police through creative leadership. Foundation aspires to contribute towards a crime free society by initiating reformation programs for prison inmates and reintegration opportunities to released inmates for the reduction in recidivism also facilitate welfare programs for their children to save them from becoming victims of their parental incarceration.
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text: Hello, everyone, We are happy to release our new 1/6 Girl NuoNuo, and bring the October Event. Doll Leaves October Event Time: 27th Sept ~ 30th Oct,2017 Content: During Event time, order Doll Leaves basic doll will get 12% OFF discount. (Accessories, makeup and special edifion are not included) Thank you very much for your support and love. New 1/6 Girl NuoNuo: DOLL LEAVES TEAM 26th Sept 2017


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Spring Arrives in Marion, North Carolina
Here along the eastern slopes of the Blue Ridge, the Bradford Pears are the first to show their blossoms. This is especially true in Marion, where these ornamental and showy trees are planted in profusion. Below is the view toward the Black Mountains and Mt. Mitchell.
Even though Marion has a population of just over 8,000, the estates and historic homes from the turn of the last century are, alone, worth the visit. A stroll around the business corridor can reveal hidden gems.
The second floor balcony and the wraparound Veranda make this Mountain Vernacular dowager a site to behold. The wall shows the stone craftsmanship that can be found all over town. After all, today, Marion is the ornamental stone capital of the East, and ships truckloads of finely shaped building rock all over the country.
With a nod to the palatial Plantations of the Old South, this hilltop masterpiece on Montevista Drive is just a short stroll from Main Street uptown.
The 1880's architectural frivolity below is on a corner just one block from Main Street. Like many of the historic homes in Marion, it has just gone through a renovation. It's unique for the large Queen Anne inspired turret above the porch and the triple fanlight dormer in the center. Again, notice the intricate rock wall.
The round turret continues on the first floor as a Parlour, and the large porch follows the shape of the turret.
An intricate wooden fan is above the beveled glass front door. I would find a way to conceal the downspouts that were obviously a later addition.


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The upgraded Strykers, nicknamed "Dragoons" to match the nickname of the 2nd Cavalry Regiment, are equipped with 30 mm autocannons that allow them to go toe-to-toe with armored opponents. Previously, the 2nd Cavalry's Strykers were only equipped with M2.50-caliber machine guns, which wouldn't do much against armor.
The autocannon can be equipped with high-explosive, anti-armor and air-burst rounds, and have twice the range of the.50-cals.
"The threat that we would face in Europe is mechanized based," said 2nd Cavalry Regiment commander Col. Patrick Ellis to Stripes. "If we have a chance encounter with a BMP, this makes us much more lethal."
The BMP is short for Boyevaya Mashina Pekhoty, or "Infantry Fighting Vehicle" in Russian. The BMP-3, the backbone of Russian armor, is equipped a 100 mm low-velocity gun. Meanwhile, the BTR-82 armored personnel carrier is usually equipped with either a 30 mm autocannon similar to the Dragoon's or a 120 mm mortar gun.
The 30 mm will give the Stryker more punching power in a hypothetical engagement against Russian armor, but Ellis emphasized that the guns were purely a precautionary measure. For starters, the Stryker is not really a match for the BMP to begin with: it's a fast and versatile troop carrier first and foremost, not meant to battle armored vehicles.
"Were not going to be chasing tanks, that's not what we're here for," Ellis said. "We are valued for our mobility, and ability to move large amounts of infantry forward, quickly."
But the 2nd made no bones about the Stryker delivery. When the delivery was announced back in August, Deputy Commander Lt. Col. Troy Meissel said that the need for more firepower was "directly attributable to Russian aggression, and we are actively working with our foreign partners in how to help shape our formation."
Nevertheless, the Dragoons said that the new Strykers served an unspoken purpose beyond the hypothetical of US-Russia hostilities: morale. "You're laying that hate [on a target] with a bigger round. It's doing a lot more damage and you're getting better effects," said Staff Sgt. Randall Engler in August.
The Stryker is meant to strike a balance between the speed and utility of a Humvee with a bit more armor. It can carry as many as nine passengers, but is best known for the wide variety of tasks it can be geared toward: mobile artillery launches, engineer transportation, chemical weapon reconnaissance, etc.
The 2nd Cavalry is based out of Vilseck, Germany, one of EUCOM's two permanent combat brigades, along with the 173rd Airborne. The unit traces its lineage back to the War of 1812, and counts several notable figures among its alumni — including HR McMaster, the National Security Advisor to US President Donald Trump.


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$100 million more sought for Exxon Valdez cleanup
A federal judge in Alaska will hear arguments Friday on whether the Exxon Mobil Corp. owes another $100 million to remove oil remaining on the Prince William Sound shoreline from the 1989 Exxon Valdez tanker spill.
The motion was filed by retired University of Alaska professor Rick Steiner, who is trying to force action on a claim filed in 2006 by the state and federal governments. It argues the oil is degrading too slowly and continues to harm wildlife.
Exxon argues it doesn't owe any more money. It paid $900 million in restitution in a 1991 settlement.
But the settlement also had a clause allowing the state and federal governments to later claim up to $100 million more from Exxon if there were unforeseen damages.
[Source: Forbes, AP, Anchorage, Alaska, 03Mar11]
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|This document has been published on 20mar11 by the Equipo Nizkor and Derechos Human Rights. In accordance with Title 17 U.S.C. Section 107, this material is distributed without profit to those who have expressed a prior interest in receiving the included information for research and educational purposes.


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I heard a wonderful story some years ago about two Englishmen who lived in France – rural France, in the Saintes area – in the 1990s. They were enjoying coverage of an England–France rugby match during the Five Nations series in the local tavern where they were known and on the whole accepted. Unfortunately, our Englishmen made the mistake of crowing too soon – for much of the match England was in the lead, and they were, of course, cock-a-hoop about this. But in the end the French came with a late surge and won the match. This was a mite embarrassing for our Englishmen, one can imagine, and it was made worse by the action of one of the French fans who had been drinking in the tavern at the same time. He swaggered over to their table, leaned down and thrust his face close to that of one of the Englishmen, eyeballing him at close range.
"Hah!" he exclaimed, in basic English, with a strong French accent. "That for Agincourt."
Now the Englishman dined out on the story in grand style, seeing this as evidence of the pettiness of the French and their inability to understand the essential camaraderie of sport. But when you look at the words of the French national anthem, the Marseillaise, dating of course from the days of the French revolution, but still sung with fervour, before rugby matches and on the occasion of French victory in the Olympics for example, it all makes a bit more sense. The Marseillaise is, not to put too fine a point on it, rather a paranoid confection. Not only do we have the warlike and rousing refrain "Aux armes, citoyens!" calling all French people to fight for their country, but this call is based on the conviction that the enemy is at the gate, threatening the people and all they care for. The interesting thing is not so much the adoption of the Marseillaise in the first place, at a time when the threat was real and most of Europe was at war or about to be at war with revolutionary France, but that they still sing it, unexpurgated, full of xenophobism and fury. Why is that, I wonder?
Much is made of the Last Night of the Proms, and even of the mention of the Queen being 'victorious' in the British national anthem. These are sometimes said to exemplify the gung-ho imperialism still underpinning our national life. But when you remember that the vitriolic second verse of the national anthem has been quietly dropped (I haven't heard it sung in years), and that in the third the Queen is reminded that she is to 'defend our laws' as a condition that we continue to wish her to reign over us, you have quite a different picture. Not to mention the fact that the most heartfelt community singing at the Last Night of the Proms, and the place of honour at the end of proceedings, is reserved for Jerusalem, which is essentially, for all Blake's warlike language, a pledge for civil struggle, for social justice.
I mention these differences as observations. I have no idea at all what significance (if any) they have. But I do just wonder whether, if the French were to decide to dump the Marseillaise in favour of a more peaceful song, Europe might be more ready to believe in their commitment to a united Europe. I haven't studied the words of the national anthems of other European countries, and perhaps it would be an interesting piece of research. How much do the sentiments of a national anthem affect the attitudes of the people of a country towards their neighbours, and towards the wider world where there may well be enemies? Is it something that we should consider more carefully?


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I get asked often what I use on my skin. This has a two part answer. Tonight, I will give you part one.
The most important thing to note? Listen to your skin. One set of products is unlikely to fit your skin needs all year round.
What I use may not fit your skin, but rest assured I will help you find the right thing for you. Just ask.
I wash with two things. Soap and liquid soap. Organic, Unscented mostly or at least I don't ever let anything with artificial fragrance touch my face (or my body most of the time) Today I washed with Virginia's unscented glycerin soap (I have a large and varied stash of naturally scented soap in my house and I pick which one I want/ need)in the shower and smeared the Ellenoire Unscented Body Butter everywhere ...from head to toe. Tonight I will use the Ellenoire organic foamy soap and some Ellenoire wildcrafted Shea Butter. I use toner less often than I should, but my skin is wonky from chemo. I recommend that everyone use a toner at least once a day, best at bedtime. When I use a toner I use the ellenoire custom blended toner( we will help u pick one right for you)with witch hazel and rosewater on real cotton on just washed and still wet skin.
In summer I use the Suki line of moisturizers and brightening creams to moisturize.
I exfoliate at least twice a week. Mostly with the ellenoire sugar scrub with no added scent.
Did this make sense? It's late. I'll talk about skin nutrition sometime soon.
Keep asking questions...


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Bloggers often use these opportunities to attract the traffic and the fonts become a prominent option for them.
In every way possible as they become a solid foundation for bloggers to attract the readers in reading various blogs through their interesting content.
Actually the font also plays an equal role in attracting the audience as they hold a varied importance when it comes to making eye defining experience for the readers.
The font makes it more legible, crisp and efficient for reading any kind of material in any genre.
So to simplify your experience we are bringing to you the top 10 best readable fonts in terms of bloggers.
10 Easiest Fonts to Read:
The fonts are famous because it is considered as the most readable and adding to it is free as well as available in all the types like light, normal, bold and ultra bold.
It is a typeface serif fonts which are available through Google fonts.
The main part of blogging is not only the content but the fonts also set the mood to read for the audience.
It is necessary to opt for best blogging fonts and somewhere Merriweather is the premium choice when it comes to blog writing in any genre. It feels traditional while reading in those fonts.
It was basically originated by Julieta Ulanovsky.
The idea behind originating these fonts is the old posters and signs inspired them to create such a linguistic material of fonts.
The basic urban typography from the earlier twenties gives it the traditional old school look.
The way of this font can be made when you are writing short pieces with all the caps and the geometric simplicity gives it a fine edge.
It is actually best suited to give the feeling of trust to the readers of your blog and can build a mutual understanding with the words conveyed through this font.
Pluto Sans:-
It is basically a geometric sans serif which was originally designed by Hannes Von Dohren and was published in 2012.
Though it is less quirky than the original Pluto Sans but the playful character makes it more interesting.
While writing a creative piece or a funny content as the y and g characters come with the looping descenders.
It is a free font which helps you to connect through the audience in a quirky way and can creatively build a trust between audience and blogger. For creativity it is the premium content.
These fonts are of large-x height give it more legible look then any other fonts and has a unique design with a feel of a bit in use for novel writing.
These fonts can be used professionally for the literature related material and also in study related content. Fonts are fairly upright so it can be used for evident display in applications.
It has also got a certificate of excellence for the outstanding performance in type design for the year 2007. The font was exclusively created by Mitja Miklav who is prominently an Slovenian designer.
It is for free and in premium quality while in use.
The fonts are being designed by Robin Nicholas and Patrica Saunders which has been a Neo-grotesque sans serif which can be marketed sometimes by Arial MT.
This type of font comprises many styles Regular, italics, bold italics and in various other abbreviations. Arial was prominently replaced by calibri in 2007.
Arial fonts can be prominently used everywhere by default for many software related services.
Professionally for writing blogs it can be the best in use with the readability that has been easy comparatively.
Helvetica is a widely used sans serif typeface which was developed in 1957 by Max Miedinger and also got an input from Eduard Hoffman.
It is a neutral and eligible font which can be best discovered with its neutrality and legibility.
The wide approach of Helvetica is due to its take over in the major companies so it became more and more popular.
It basically makes the web more beautiful, fast and open through great typography. The documentary about the typography mentioned it is one of the premium in its own kind for the world.
It is a serif that is typeface which was designed in 1993 by Mathew Carter and addon to this it was hinted by Tom Rickner.
It is must but legible for small and low resolution screens and printed with precision and accuracy.
The most accurate font is easy to perceive to read, sharper and more legible than any other fonts of the world.
The blogger needs to be very accurate with the fonts and Georgia can be very well stood in the world of dimensions is web safe system bonds with the world.
It is the best condemned for the blogger when it sustains the legible icons and style makes it best for the use.
It is a geometric sans serif which was basically designed by Paul Renner. The geometric shapes and add-on in the new Frankfurt project was basically used for technical projects.
As it has a great balance geometrical space the free web based font used in films and videos exclusively can be of a great versatility in every sense.
It can actually create crisp and clean form of content with razor sharp apexes as it can be used for design and commercial purposes.
It is a premium font used for creative purposes and can satisfy in each and everything possible.
It is a sans serif typeface family which was designed by the Warsaw based designer. These fonts were actually released in 2015.
Lato is a font which is widely used by 9.6 million websites and is actually considered as the third most searched font once in the Google fonts.
Lato is basically available as an open source in each and every sense possible and a free font which is best for professional resumes.
For the blogger it can be a best deal to use such fonts as for the professional people it is best for the use.
It is a sans serif with the rounded terminals. These fonts are basically designed by Andrew Panglinawan and used geometric shapes and designs.
They can use the fonts freely in your products and project it to print digital, commercial or otherwise they are free in use for the professionals.
It is a great font for headlines on television and news as it can thicker the font weight in every sense possible.
It is actually available for an open source font which derives its own derivation and crisp which belongs to every important document in this world that is all the reason to use Quicksand.
As we have seen the top 10 fonts in every space available and cracks mostly all the needs of your own blog.
Whether you write in a technical genre or professional or a highly creative blog everything can summed up with these fonts.
The top 10 blogging fonts have the capability to attract the traffic on your blogging site by creating a high on reach market for the bloggers and help them to grow more in every sense.
As per your convenience, you can use anyone for the mentioned above fonts to enhance your blogs in every capable way.


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This is a Sponsored Post written by me on behalf of Nexx Inc. All opinions are 100% mine.
I've been in blogging for almost 3 years and I must say that I really learned a lot. I am also very thankful to all my friends who keep on supporting all throughout. These friends motivate me to continue my passion~ blogging. So far, I am handling 16 blogs and I try my best to update them regularly. Aside from my blogs I also have 4 websites with my own domain. I never thought that I will reach this point that I will be hosting my own sites. It all started when a friend offered me to buy a domain because I can benefit a lot from it. At first, I am in doubt because I really don't know anything about domains. All I know is that there's a payment involves in owning a domain and it is very hard to maintain. Everything made clear to me after my friend explained all the details that I should know about domains. I am happy and satisfied with what's going on with my sites and I am very thankful that I was able to manage everything. And guess what? I am thinking of buying another one! Yup! The more the merrier, right? LOL. Anyway, I found a great web hosting site that offers a cheap yet very reliable web hosting services. Nexx is probably the great place to start your brand new domain. You can get a full year of Unlimited Web Hosting and one free .COM domain name for only $10! Not only that, I also found out that in each 10$ Nexx Hosting plan, you get unlimited diskspace, unlimited bandwidth, unlimited email accounts and just like what I said earlier, one free domain! Not yet satisfied? Here's more, in each 10$ Nexx Hosting plan, you'll also get 50$ Google Adwords Credits and 50$ Facebook Ads Credits. This is very ideal especially if you want to promote your site online and a great start to showcase your online business, if any. Also included in the plan is the 24/7 Customer Support. It also offers a "One-Click Wordpress and Blog installations".
All Nexx hosting services are powered by a top tier North American data center which utilizes clustered server technology, allowing proper resource allocation at all times so customers' websites will never go down or slow down. These are too many features for an amazing low price of 10$. So if I am to choose where to buy my new domain and avail a hosting plan, I'll go for Nexx!


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Only 50 will be built.
Specialty Vehicle Engineering was once known as SLP.
Can be purchased from your local Chevy dealer.
Specialty Vehicle Engineering, once known as SLP (Street Legal Performance), has just put the finishing touches on their latest creation: the 2021 Yenko/SC Chevrolet Silverado. Not only is it the most powerful limited production truck from GM, but it also looks the part.
The 2021 Yenko/SC Chevrolet Silverado is loaded with an impossibly long list of features. The key to making it all come together however is the custom-built 800-horsepower supercharged LT-1 6.2-litre V8 engine which will be matched up with a heavy-duty 6-speed automatic transmission with various performance upgrades. The Yenko/SC Chevrolet Silverado will be available in any factory colour and in either crew cab or double cab Custom models with 4WD.
Here's a sample of what else will be included:
Brembo front brakes with 6-piston calipers.
BDS lift kit that raises the truck 4 inches.
Higher-rated front and rear Fox performance shocks
Exterior will features YENKO Off-Road 1/4 panel graphics, hood inserts, and more.
Unique 12-spoke 20″ x 10″ aluminum alloy one-piece wheels in matte finish with rim edge finished in silver, black, or body color.
Nitto Ridge Grappler LT295/60R20 off-road performance tires.
Custom high-impact composite tailgate panel overlay with embossed YENKO/SC logo in gloss finish body color.
Custom front grille bar with stamped YENKO/SC logo.
And much more!
Only 50 will be assembled so get shopping now.


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Villarreal have reportedly made a 'tentative approach' to Santi Cazorla, hopeful that they can lure him back to Spain providing he is able to regain full fitness.
The midfielder is out of contract in the summer and Arsenal have yet to offer him a new deal as they wait to see if he is able to actually play again.
With Arsene Wenger leaving and a new manager not yet appointed, any contract renewals will most likely have to be delayed regardless of other circumstances.
Cazorla, for his part, is focusing on getting fit but it's hard to see him turning down a new deal at Arsenal should one come his way.
He will no doubt feel some sort of loyalty towards the club that has stood by him over the past 18 months unlike others who have graced Arsenal's treatment room for long periods of time before buggering off as soon as they got fit.
At his press conference ahead of Arsenal's penultimate fixture, Arsene Wenger confirmed that the Spaniard will not be fit in time to play any part in Arsenal's last fixture of the season on Sunday. He also said that Laurent Koscielny will not return before 'December at best'.
The report of Villarreal's interest comes via the Times [£] and AS.


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The Michael Vick story hit the news before I left the Atlanta area Tuesday morning. When Vick's picture appeared on the TV screen in the lobby of the Hyatt Place Hotel in Johns Creek one man looked up and scowled.
"I can't believe they're going to let him out of prison next year," he said. "They should leave him locked up and throw away the key."
Others nodded in agreement.
"I take it you're not a Vick fan," I said.
"I was," he said. "We've had season tickets to Falcons games for years. My engineering degree is from Virginia Tech. I was proud. No more. Vick is an example of what is wrong with professional sports. He's an example of what has gone wrong with Virginia Tech."
"Why do you say that?"
"When I was at Tech in the 60s, the emphasis was on education, not football. That was before they hired Frank Beamer and let him build a football program around street thugs."
I've heard Tech's football teams called thugs before but usually from alumni from other schools — not from graduates from the Blacksburg university.
During the six-hour drive back to Floyd, I had a lot of time to think about the concern from an alumni of Tech. Yes, Virginia Tech's image has not only been been damaged by Michael Vick's dog-fighting habit but also by the antics of his brother and other football players whose troubles have been front-page news.
Beamer is a winning coach of a football program that brings a lot of money into the university and such schools too often look the other way when money is involved. Indiana put up with the antics of Bobby Knight for too many years because he won national championships.
Beamer thought Michael Vick would bring a national football championship to Tech. More than one cop in the area has told me that arrests of Vick were quashed while he attended Tech. Tech officials intervened to stop an investigation into Vick's dog fighting activities while he played in Blacksburg. Papers filed by federal prosecutors during the investigation of Vick's dog fighting activities show he was actively involved in the despicable activity while attending Tech and that Tech officials knew about it.
Beamer admitted he knew that brother Marcus Vick had been arrested on a traffic charge — a violation of his probation with the school — but withheld that information from University officials so the younger Vick could play in a bowl game where he embarrassed the school on national television by stomping on an opposing player. Marcus Vick was finally kicked out of the program and spent a short-lived career with the Miami Dolphins. He copped a plea to a DUI charge in Norfolk this week.
Beamer's behavior in concealing Vick's arrest resulted in more scrutiny of his program by school administrators and, this season, he suspended starting receiver Zach Luckett after the player was arrested in Montgomery County for driving drunk and on a suspended license. In September, Luckett got five days in jail for the DUI conviction — his second in five years. In the Michael Vick era, Luckett would probably still be playing for Beamer.