The absence of otic AP asymmetries when both Fgf and Hh signalling were inhibited from 10 to 20S suggests that, in zebrafish, Fgf and Hh are the major early otic AP patterning signals. However, manipulations of retinoic acid (RA) signalling in the chick have recently also been shown to result in AP mirror image duplications of the whole inner ear (Bok et al., 2011). High RA levels appear to specify posterior otic development (in particular Tbx1 expression), and low levels are required for anterior gene expression (including markers of neurogenesis) (Bok et al., 2011). A similar positive regulation of otic tbx1 expression by RA (and negative regulation of neurogenesis) has recently been reported in the zebrafish, although mirror image duplications were not observed (Radosevic et al., 2011). Manipulation of RA signalling is also known to affect earlier stages of zebrafish otic development, but only indirectly; notably, early RA inhibition delays the onset of hindbrain fgf3 expression, and so interferes with otic induction (Hans and Westerfield, 2007). Any interaction of RA with the Fgf and Hh signalling pathways between the 10 and 20S stages in the zebrafish remains to be determined.
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While studies in zebrafish, Xenopus, chick and mouse embryos have all shown a role for Hh signalling in the ventralization of the eye [12,17-19], the developmental window during which the Hh pathway controls eye DV patterning and the temporal changes in the response of eye cells to Hh signals have been only partially investi- gated. In particular, there is a lack of information on the function of Hh signalling during the transition between eye field and optic vesicle stages, which is likely to be a critical window in the establishment of DV polarity, based on the dynamic expression patterns shown by relevant transcription factors . In this study, we therefore used various molecular tools to activate or inhibit Hh signalling during specific stages of Xenopus eye development and we assessed the consequences of these manipulations on the DV polarity of the eye. All the employed experimental ap- proaches suggest that the Hh pathway controls global DV patterning of the eye region, contributing to the specifica- tion of OS, VR and DR domains, as early as gastrula/ neurula embryonic stages. Concomitantly with the emer- gence of the optic vesicle, the influence of Hh signalling on DR and VR fates decreases, as shown by increased re- sistance of the DR to Hh-dependent ventralization and by Hh-independent maintenance of VR fates. In contrast, Hh signalling continues to support OS gene expression during optic vesicle stages and this prolonged regulatory input is required for the maintenance of proper OS size.
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We therefore examined proliferation of the ptc cells by phospho-histone H3 (pH3) staining. In neutral control clones, 0.5±0.9% (3 days ACI) to 1.1±1.1% (2 days ACI) of all homozygous cells were pH3 positive (Fig. 2G; supplementary material Fig. S5). As expected (Cheng et al., 2011; Hardy et al., 1979), all somatic mitoses occurred adjacent to the hub. By contrast, at both time points, ~2.5% of all ptc mutant cells (2 days ACI, 2.5±1.2%; 3 days ACI, 2.5±0.5%) were pH3 positive (ptc versus control at 3 days ACI, P<0.05, t-test; supplementary material Fig. S5). About one-third of these excess mitoses (27/71) occurred away from the hub (Fig. 2H). These results implicate Hh in CySC proliferation, resembling the situation in the ovary, where Hh signalling drives FSC proliferation (Forbes et al., 1996; Hartman et al., 2010; Zhang and Kalderon, 2001).
In order to explore the signalling outputs in this system, we constructed a mathematical model capturing the essence of the Hh signalling pathway (see Materials and Methods). In this model, Ptc represses Hh signalling targets unless it binds Hh. As Ptc is one of the targets of the pathway, Hh binding to Ptc releases the repressive action of Ptc and results in its upregulation (Nakano et al., 1989). Sens is included as a target in the model, although this does not imply that Sens is a direct target. Expression of Elav in Sens-expressing precursors follows irreversibly, and to reflect the loss of Sens expression observed in Elav cells, we have also included a negative feedback from Elav to Sens (see Fig. 4A). The dynamics of Hh production and dispersion in the model were calibrated using measured Hh:GFP profiles determined experimentally (Fig. S4; and see Table S1 for values, units and source of parameters). The intrinsic variability of the system is modelled by introducing a 20% variability in all parameters of the model (see Materials and Methods). In brief, the model simulates a two- dimensional array of 10×10 cells (see Fig. S5 and Movies 1 and 2) that respond to an external concentration of Hh. Hh is being secreted at one of boundaries of the system and diffuses through the array. The response to Hh in each cell is simulated by numerically solving a set of ordinary differential equations (ODEs) that model a simplified version of the Hh signalling pathway (Fig. 4A, see Materials and Methods). With no further assumptions, the model simulations confirmed the prior expectation: the accumulation of Elav cells was non-linear and differentiation was often not completed during the developmental period allowed (40 h) (Fig. 4B). The fact that the model was unable to reproduce the experimental observations indicated that our understanding of the signalling dynamics was missing some important process. Owing to the key relevance of Ptc as both Hh receptor and target
Subsequently, however, we have established that whereas a loss of Hh function does not affect the otic DV and ML axes in zebrafish (Hammond et al., 2003), increasing Hh levels by shh mRNA injection causes an expansion of ventromedial (VM) otic territories at the expense of dorsolateral (DL) domains. To investigate further, we analysed the otic phenotypes of a panel of lines carrying mutations in genes encoding inhibitors of the Hh pathway: ptc1, ptc2, su(fu) (sufu – ZFIN), dzip1 and hip, all of which are expressed in and around the developing otic vesicle. These lines provide a series with increased Hh signalling throughout the embryo (Koudijs et al., 2008; Koudijs et al., 2005). Ptc (Patched), the Hh receptor, represses Hh pathway activity in the absence of Hh ligand (Chen and Struhl, 1996) (reviewed by Ingham and McMahon, 2001). ptc1 is expressed in a posteroventromedial domain of the zebrafish otic vesicle and ptc2 in a wider ventral domain (Hammond et al., 2003). Hip (Hedgehog interacting protein) is a membrane-bound protein that binds to the Hh ligand and prevents it binding to the Ptc receptor (Chuang and McMahon, 1999; Ochi et al., 2006). hip is expressed in a complex pattern in the zebrafish, initially concentrated towards the anterior of the otic vesicle (Hammond and Whitfield, 2009). Dzip1 (Daz interacting protein 1) and Su(fu) (Suppressor of fused)
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During vertebrate brain development, induction and progressive posteriorization of neurectoderm is followed by a phase of regionalization (reviewed by Lumsden and Krumlauf, 1996; Wilson and Houart, 2004; Kiecker and Lumsden, 2005). This may involve specialized groups of cells in ‘signalling centres’ (reviewed by Rhinn and Brand, 2001), the best-characterized of which are the anterior neural border (ANB) (Houart et al., 2002) (reviewed by Wilson and Houart, 2004), the roof plate (reviewed in Chizhikov and Millen, 2004), the floor plate (reviewed in Strähle et al., 2004) and the midbrain-hindbrain boundary (MHB). The cells that release signal molecules from such centres are often located at boundaries between distinct territories, e.g. the MHB forms at the interface between the mesencephalon and anterior rhombencephalon, whereas the ANB lies at the boundary between the telencephalon and anterior epidermal ectoderm. The zona limitans intrathalamica (ZLI), a narrow transverse region between the prethalamus (also known as the ventral thalamus) and the thalamus (also known as the dorsal thalamus) (Kuhlenbeck, 1937; Shimamura et al., 1995) also bears the hallmarks of a signalling centre. Fate mapping experiments in chick have shown that the ZLI is cell lineage restricted at its boundaries and is thus a true developmental compartment (Zeltser et al., 2001; Garcia-Lopez et al., 2004). Furthermore, the ZLI is the only structure in the alar plate that expresses signal molecules of the Hedgehog family (Hh) (Figdor and Stern, 1993; Puelles and Rubenstein, 2003). Well-described functions of Hh signalling from basal and floor plates are ventralization of the neural tube (reviewed by Briscoe and Ericson, 1999; Jessell, 2000), promotion of growth and proliferation (Britto et al., 2002; Ruiz i Altaba et al., 2002), and formation of the hypothalamus (Chiang et al., 1996; Mathieu et al., 2002). The
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Although cilia in mice lacking Evc look normal on stan- dard microscopy  the possibility remained that the Hh signalling defect could be secondary to a structural abnormality of cilia. We therefore examined cilia struc- ture in chondrocytes using transmission electron micro- scopy (TEM). In the basal body region, cilia from mutant chondrocytes showed the normal configuration of 9 triplet microtubules with transition fibers radiating from the triplets at the distal end of the basal body (Fig- ure 6, blue arrowheads). The champagne glass structures that connect the microtubule doublets to the ciliary membrane seen in the transition zone  are present in the mutant chondrocytes (Figure 6, green arrow- heads). Sections through the mid-portion of the cilia show 9 microtubule doublets with normal orientation and structure. In the distal region of the cilia there is some collapse of the microtubule ring in chondrocyte cilia from both wild-type and Evc -/- mouse (Figure 6, red arrowheads), a feature that has been reported before in normal cilia [21,22]. All observed cilia showed com- plete triplet microtubule structure in the basal body and doublet microtubules in the ciliary region. No structural differences were observed between the cilia from wild- type and Evc -/- chondrocytes.
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A revised model of floorplate formation in zebrafish Taken together, our analyses of the mol –/– mutant and other studies allow us to revise existing models of floorplate formation in zebrafish (Fig. 10). Nodal signalling induces MFP (and floorplate-specific genes such as shh and foxa2) through Madh/Smad and FoxH1/Fast1/Sur transcription factors. This occurs in the absence of Foxa2 function, implicating other transcription factors, perhaps including Her9 (Latimer et al., 2005), downstream of Nodal in the earliest induction of floorplate identity. Downstream of Nodal activity, Foxa2 function is required for maintained expression of Fox and Hh family genes and for differentiation of the floorplate. Hh signals produced in the MFP subsequently contribute to the induction of foxa2 (and foxa) expression in more lateral cells. Foxa2 activity is required for lateral expansion of the floorplate, probably owing both to non-autonomous roles (e.g. regulation of Hh production in the MFP) and to activity within the LFP itself. Hh signalling also spreads further dorsally to induce and pattern adjacent ventral CNS cell types, including cranial motoneurones, serotonergic raphé neurones and oligodendrocytes. In the absence of Foxa2 function, Hh activity Fig. 10. Summary of proposed role for Foxa2, Nodal and Hh
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Thus, in both vertebrates and invertebrates, Hh signaling controls the expression of target genes by modulating the activity of the downstream Gli/Ci transcription factors. Studies in Drosophila have identified a large number of proteins that are involved in the regulation of this Gli/Ci activity (reviewed by McMahon, 2000; Ingham and McMahon, 2001; Nybakken and Perrimon, 2002). Within target cells, Ci forms a protein complex with Fused kinase (Fu) (Monnier et al., 1998; Stegman et al., 2000) and Costal2 (Cos2) (Robbins et al., 1997; Sisson et al., 1997) that is tethered to microtubules. Activated Smo signals to this cytoplasmic protein complex, resulting in the release of Ci and active transport into the nucleus where it can activate Hh target genes (Ohlmeyer and Kalderon, 1998; Ding et al., 1999; Kogerman et al., 1999; Methot and Basler, 2000; Murone et al., 2000; Wang et al., 2000). The transport of Ci to the nucleus is mediated by another protein, Suppressor of Fused [Su(Fu)]. In addition to such a positive regulation in response to Hh ligands, a negative pathway that includes cAMP-dependent protein kinase (PKA) also regulates Hh signaling (Li et al., 1995; Pan and Rubin, 1995; Lepage et al., 1995; Hammerschmidt et al., 1996). In the absence of Hh ligands, PKA directly phosphorylates Ci to promote its proteolytic cleavage, generating the repressor isoform (Chen et al., 1998; Chen et al., 1999; Price and Kalderon, 1999). Studies in vertebrates have shown that these intracellular components of the Hh signaling pathway have been largely conserved
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sector of G(221) models is assumed, and the breaking of the symmetry is produced in two stages. We study W → hH ± production in p-p collisions at √ s = 8 TeV. This analysis is dedicated to W → hH ± decay channel, where h is neutral and H ± is charged, and we calculate the partial width for W → hH ± within G(221) model. We further consider the ﬁnal state consisting of two same-sign leptons, MET and four jets, from which two are b-jets, arising from the decays of H ± and h and compare for this channel the predictions of G(221) models with those of SUSY published by ATLAS Collaboration
Construction of high-fidelity Cas9 variant-encoding plasmids and the HH-ribozyme-fused sgRNA-encoding plasmid eCas9-1.1- and Cas9-HF1-encoding plasmids (p3s- eCas9-1.1, Addgene #104172; p3s-Cas9-HF1, Addgene #104173) were created via site-directed mutagenesis of a WT Cas9 construct (p3s-Cas9-HN, Addgene #104171). HH-ribozyme sgRNA constructs were cloned via ligation of annealed oligonucleotides that included a HH-ribozyme sequence and a protospacer sequence into a plasmid (pRG2, Addgene #104174) in which sgRNA expression is under the control of the U6 promoter.
There is increasing evidence that the Wnt and HH signaling pathways cross-talk or intersect with the Notch and bone morphogenic protein pathways [43, 44]. The Wnt signaling pathway can also control Gli3 in the HH pathway , and HH can in turn antagonize Wnt signaling in the colon [46, 47]. Cross-talk between the Wnt and HH pathways has been also demonstrated in a gastric cancer cell line . Furthermore, many studies have proposed several mecha- nisms underlying the resistance to molecular target therapies, including TKI. Over-activation of other ErbB family receptors, including HER2, has been shown as the common mechanism triggered by the EGFR-directed therapeutic antibody Cetuximab . In addition, a recent study has also provided a rationale for co-tar- geting insulin-like growth factor 1 receptor and ALK in ALK fusion-positive lung cancer . Furthermore, another study has reported cross- talk between KIT and fibroblast growth factor receptor 3, which promotes gastrointestinal
The switch between active and inactive states of the HH pathway involves rapid translocation of SMO. The SMO protein is the key positive regulator of the HH pathway, and GLI family proteins play a critical role in the regulation of HH signaling pathway activity. Despite a strong link between SMO expression, HH pathway activity, and cancer development, the basis for SMO gene regulation has not been well characterized. Therefore, an investigation of the mechanisms controlling the expression of SMO and additional HH pathway genes may provide valuable insight into HH signaling alterations associated with cancer development. SMO also is the major target for pharmaceutical agents that modulate HH pathway activity [17-19], such as vismodegib  and sonidegib . We previously studied SMO peptides, and found that specific lipopeptides can serve as effective inhibitors [22,23].
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nucleus to regulate gene expression (Figure 1B). There are also some molecules that are required for Hh signaling pathway activation, such as, pitchfork (Pifo), the G protein-coupled receptor associated sorting protein 2 (Gprasp2) and Growth Arrest Specific 8 (Gas8) are essential components of an Hh induced ciliary targeting complex able to regulate Smo translocation to the primary cilium 20, 21 . Members
To determine whether Smo activation resulted in the activation of Gli2 ΔCLR in vivo, we examined the morphology, Hh target gene expression and spinal cord patterning in Gli2 ΔCLRKI/− ;Ptch1 –/– double mutants. At E9.5, Ptch1 –/– mutant embryos failed to turn and exhibited severe neural tube defects (Fig. 5B; Goodrich et al., 1997). The turning and neural tube defects were partially rescued in Gli2 ΔCLRKI/− ;Ptch1 –/– and Gli2 –/– ;Ptch1 –/– double mutants, and some double mutants were noticeably larger than Ptch1 –/– mutants (Fig. 5B). Hh target gene Gli1 was expressed in a ventral-to-dorsal gradient in the E9.5 wild-type spinal cord (Fig. 5C). However, as reported previously, Gli1 expression in the floor plate, the ventral- most part of the spinal cord, was downregulated by prolonged exposure to extremely high levels of Shh through a feedback mechanism (Ribes et al., 2010). Gli1 expression was downregulated in the entire Ptch1 mutant spinal cord, consistent with very high levels of Hh pathway activation (Fig. 5C). In Gli2 ΔCLRKI/− ;Ptch1 –/– double mutants, Gli1 expression was restored in the dorsal spinal cord, suggesting that Hh pathway activation was less than that in Ptch1 mutants (Fig. 5C). Interestingly, the expression pattern of Gli1 in the Gli2 ΔCLRKI/− ;Ptch1 –/– double mutant spinal cord was similar to that in Gli2 ΔCLRKI/− ;Ptch1 –/– double mutants (Fig. 5C), suggesting that the reduction of Hh pathway activation in these mutants resulted from a disruption of Gli2 ΔCLR activation.
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Abstract. Kikianty and Dragomir in 2008 introduced the p-HH-norms on the Cartesian product of two copies of a normed space, which are equivalent to the well-known p-norms. In this paper, notions of orthogonality in terms of 2-HH-norms are introduced. The main properties of these orthogonalities are discussed. Several characterizations of inner product spaces are established, as well as the characterization of strictly convex spaces.
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miRNA – target pathway interactions were performed for miRNA 21 and their targeted gene IL1B by using mirwalk database, their interaction was found in apoptosis and MAPK signalling pathway which are previously link with DR. furthermore for the identification of their link with diabetic retinopathy KEGG disease pathway analysis was performed.Which shows MAPK signalling and apoptosis both pathways are affected in case of diabetic retinopathy. 8. A comparative miRNA target prediction
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AnxB11 (b) driven by ap.Gal4 with a dorsal expression domain (marked by GFP), leaving the ventral domain in Wild Type conditions as an internal control. Note there is no change in fluorescence levels at the dorsal posterior compartment compared to the ventral side (a,b). (c) Boxplot showing the ratio between relative intensity of the mean gray value in the dorsal (RNAi treated) versus the ventral compartment (Wild type). The ratio is close to 1 in control, and in all RNAi cases except TSG101 and Vps22 where the ratio decreases due to accumulation of Hh in the RNAi treated cells.
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Currently, three categories of Hh pathway inhib- itors are under development: a) Smo inhibitors including cyclopamine, vitamin D3, vismodegib, SANT, BMS-833923, LDE225, and LEQ506, among which, the role of vismodegib in rectal cancer and ovarian cancer has been studied in phase II clinical trials ; SANT has been manufactured by several drug companies as a promising anti-cancer reagent ; b) The Shh ligand antagonists, i.e. robotnikinin, which bind to the N-terminal fragments of Shh, blocking translation of Hh target genes . c) Gli inhibi- tors, including GANT58, GANT61, and HPI-1, -2, -3 and -4, which block Gli-mediated gene trans- lation. Both GANT58 and GANT61 can inhibit the growth of implanted prostate cancer . Additionally, both itraconazole (a commonly used antifungal drug that can act on Smo) and arsenic trioxide (an anti-cancer drug that inhib- its Gli) can block Hh signaling. In some clinical trials, itraconazole has been used to treat basal cell carcinoma . Our study revealed that Hh signaling promotes liver regeneration after hep- atectomy and also promotes the growth of liver implanted tumors, suggesting Hh signaling may be a new target for the prevention and treat- ment of HCC recurrences and metastases. Some commonly used drugs (e.g. vitamin D3 and itraconazole) have been shown to exert anti-cancer effects by blocking Hh signaling [27, 28]. Therefore, the prevention and treat- ment of HCC recurrence by blocking Hh signal- ing is worthy of further study. Our findings may thus provide new evidence for the prevention and treatment of HCC recurrence following hepatectomy.
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The confirmation of these findings was found in many preclinical and clinical models. For example, an inactivat- ing mutation in the Ptch1 was found in about 85% of spo- radic BCCs . These observations have been supported by the fact that in Ptch1 heterozygous mice the formation of UV-induced BCC was more frequently observed . Activating Smo mutations that reduce its inhibition by Ptch1 have been found in 10% of sporadic BCCs. Rare mutations of the Sufu gene (<10% somatic) have also been found . However, although the Sufu is a tumor suppressor gene, its targeted inactivation in mouse skin does not result in BCC development, suggesting that the Sufu heterozygosity is not sufficient for tumorigenesis . Furthermore, it has been found that the hyperactivation of Hh signaling after Gli over- expression and activation of atypical protein kinase C (aPK- C)-ι/λ (GLI1-positive regulator) can promote BCC devel- opment regardless of the upstream components . Gli2 gene amplification is seen in 8% of BCCs, suggesting that the increase in the number of Gli2 copies can induce tumorigen- esis as well . In 30% cases of BCC, no mutations in Hh signaling pathway genes can be found . Given all the above results, it can be concluded that, in most instances, BCC is a disease related to the aberrant activation of Hh signaling path- way that leads to tumor cell proliferation and survival.
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