Regulation of Calvarial Osteogenesis by Concomitant De-repression of GLI3 and Activation of IHH Targets

Loss-of-function mutations in GLI3 and IHH cause craniosynostois and reduced osteogeneiss, respectively. In this study, we show that ihh ligand, the receptor Ptch1 and Gli transcription factors are differentialyy expressed in embryonic mouse calvaria osteogenic condenstions. We show that in both ihh(-/-) and Gli3(Xt-J/Xt-J) embroyonic mice, the normal gene expression architecture is lost and this results in disorganized calvarial bone developement. RUNX2 is a master regulatory transciption factor controlling osteogenesis. In the absence of Gli3, RUNX2 isoform II and IHH are upregulated, and RUNX2 isoform I downregulated. This is consistent with the expandeed and aberant osteogenesis observed in Gli3Xt-J/Xt-J mice, and consistent RunX2-t expression by relatively immature osteoprogenitors. ihh-/- mice exhibited small calvarial bones HH target genes, Ptch1 and Gli1, were absent. This indicates that IHH is the functional HH ligand, and that it is not compensated by another HH ligand. To decipher the roles and potential interaction of Gli3 and ihh. we generated ihh-/-; gli3Xt-J/Xt-J compound mutant mice. Even in the absence of ihh, Gli3 deletion was sufficient to induce aberrant precocious ossification across the developing suture, indicating that the carniosyostosis pehnotype of Gli3Xt-J/Xt-J mice is not dependent on IHH ligand. Also we found that ihh was not required for Runx2 expression as the expression of RUNX2 target genes was unaffected by deletion of Ihh. To test whether RUNX2 has a role upstream of IHH we performed RUNX2 siRNA knock down experiements in WT calvarial osteoblasts and explants and found that Ihh expression is suppressed. Our results show that IHH is the functional HH ligand in the embroynic mouse calvaria osteogenic condensations, where it regulates the progression of osteoblastic differentation. As GLI3 represses the expression of Runx2-II abd Ihh, and also elevats the Runx2-I expression, and as IHH may be regulated by RUNX2 these results raise the possibility of a regualtory feedback circuit to control calvarial osteogenesis and suture patency. Taken together RUNX2-controlled osteoblastic cell fate is regulated by IHH through concomitant inhibition of GLI3-repressor formation and activation of downstreams targets.Peer reviewe

MAGIC and H.E.S.S. detect VHE gamma rays from the blazar OT081 for the first time: a deep multiwavelength study

https://pos.sissa.it/395/815/pdfPublished versio

Regulation of Calvarial Osteogenesis by Concomitant De-repression of GLI3 and Activation of IHH Targets

Loss-of-function mutations in GLI3 and IHH cause craniosynostosis and reduced osteogenesis, respectively. In this study, we show that Ihh ligand, the receptor Ptch1 and Gli transcription factors are differentially expressed in embryonic mouse calvaria osteogenic condensations. We show that in both Ihh−/− and Gli3Xt−J/Xt−J embryonic mice, the normal gene expression architecture is lost and this results in disorganized calvarial bone development. RUNX2 is a master regulatory transcription factor controlling osteogenesis. In the absence of Gli3, RUNX2 isoform II and IHH are upregulated, and RUNX2 isoform I downregulated. This is consistent with the expanded and aberrant osteogenesis observed in Gli3Xt−J/Xt−J mice, and consistent with Runx2-I expression by relatively immature osteoprogenitors. Ihh−/− mice exhibited small calvarial bones and HH target genes, Ptch1 and Gli1, were absent. This indicates that IHH is the functional HH ligand, and that it is not compensated by another HH ligand. To decipher the roles and potential interaction of Gli3 and Ihh, we generated Ihh−/−;Gli3Xt−J/Xt−J compound mutant mice. Even in the absence of Ihh, Gli3 deletion was sufficient to induce aberrant precocious ossification across the developing suture, indicating that the craniosynostosis phenotype of Gli3Xt−J/Xt−J mice is not dependent on IHH ligand. Also, we found that Ihh was not required for Runx2 expression as the expression of RUNX2 target genes was unaffected by deletion of Ihh. To test whether RUNX2 has a role upstream of IHH, we performed RUNX2 siRNA knock down experiments in WT calvarial osteoblasts and explants and found that Ihh expression is suppressed. Our results show that IHH is the functional HH ligand in the embryonic mouse calvaria osteogenic condensations, where it regulates the progression of osteoblastic differentiation. As GLI3 represses the expression of Runx2-II and Ihh, and also elevates the Runx2-I expression, and as IHH may be regulated by RUNX2 these results raise the possibility of a regulatory feedback circuit to control calvarial osteogenesis and suture patency. Taken together, RUNX2-controlled osteoblastic cell fate is regulated by IHH through concomitant inhibition of GLI3-repressor formation and activation of downstream targets

Therapeutic effect of nanogel-based delivery of soluble FGFR2 with S252W mutation on craniosynostosis.

Apert syndrome is an autosomal dominantly inherited disorder caused by missense mutations in fibroblast growth factor receptor 2 (FGFR2). Surgical procedures are frequently required to reduce morphological and functional defects in patients with Apert syndrome; therefore, the development of noninvasive procedures to treat Apert syndrome is critical. Here we aimed to clarify the etiological mechanisms of craniosynostosis in mouse models of Apert syndrome and verify the effects of purified soluble FGFR2 harboring the S252W mutation (sFGFR2IIIcS252W) on calvarial sutures in Apert syndrome mice in vitro. We observed increased expression of Fgf10, Esrp1, and Fgfr2IIIb, which are indispensable for epidermal development, in coronal sutures in Apert syndrome mice. Purified sFGFR2IIIcS252W exhibited binding affinity for fibroblast growth factor (Fgf) 2 but also formed heterodimers with FGFR2IIIc, FGFR2IIIcS252W, and FGFR2IIIbS252W. Administration of sFGFR2IIIcS252W also inhibited Fgf2-dependent proliferation, phosphorylation of intracellular signaling molecules, and mineralization of FGFR2S252W-overexpressing MC3T3-E1 osteoblasts. sFGFR2IIIcS252W complexed with nanogels maintained the patency of coronal sutures, whereas synostosis was observed where the nanogel without sFGFR2S252W was applied. Thus, based on our current data, we suggest that increased Fgf10 and Fgfr2IIIb expression may induce the onset of craniosynostosis in patients with Apert syndrome and that the appropriate delivery of purified sFGFR2IIIcS252W could be effective for treating this disorder

Camera Calibration of the CTA-LST prototype

International audienceThe Cherenkov Telescope Array (CTA) is the next-generation gamma-ray observatory that is expected to reach one order of magnitude better sensitivity than that of current telescope arrays. The Large-Sized Telescopes (LSTs) have an essential role in extending the energy range down to 20 GeV. The prototype LST (LST-1) proposed for CTA was built in La Palma, the northern site of CTA, in 2018. LST-1 is currently in its commissioning phase and moving towards scientific observations. The LST-1 camera consists of 1855 photomultiplier tubes (PMTs) which are sensitive to Cherenkov light. PMT signals are recorded as waveforms sampled at 1 GHz rate with Domino Ring Sampler version 4 (DRS4) chips. Fast sampling is essential to achieve a low energy threshold by minimizing the integration of background light from the night sky. Absolute charge calibration can be performed by the so-called F-factor method, which allows calibration constants to be monitored even during observations. A calibration pipeline of the camera readout has been developed as part of the LST analysis chain. The pipeline performs DRS4 pedestal and timing corrections, as well as the extraction and calibration of charge and time of pulses for subsequent higher-level analysis. The performance of each calibration step is examined, and especially charge and time resolution of the camera readout are evaluated and compared to CTA requirements. We report on the current status of the calibration pipeline, including the performance of each step through to signal reconstruction, and the consistency with Monte Carlo simulations

Purification of soluble forms of FGFR2 and their biological activities <i>in vitro</i>.

<p>(A, B) The expression levels of purified sFGFR2IIIc and sFGFR2IIIc^S252W proteins (A), as well as that of Fgf2-His (B), were analyzed by western blot analysis. (B) Pull-down assay for the binding of Fgf2-His with sFGFR2IIIc-FLAG and sFGFR2IIIc^S252W-FLAG. Fgf2 interacted with sFGFR2IIIc-FLAG and sFGFR2IIIc^S252W-FLAG. (C) Dimerization between sFGFR2IIIc^S252W and FGFR2IIIc, FGFR2IIIc^S252W, or FGFR2IIIb^S252W was determined by immunoprecipitation assays using anti-FGFR2 antibodies that recognized the cytoplasmic region of Fgfr2, followed by western blot analysis using anti-FLAG antibodies. The formation of heterodimers between sFGFR2IIIc^S252W-FLAG and membrane-bound FGFR2IIIc-FLAG, FGFR2IIIc-FLAG, FGFR2IIIc^S252W-FLAG, or FGFR2IIIb^S252W-FLAG was confirmed.</p

Analysis of the inhibitory effects of sFGFR2IIIc^S252W on the premature fusion of coronal sutures.

<p>(A) The effects of nanogel-crosslinked hydrogels incorporating sFGFR2IIIc^S252W on the coronal sutures of AS mice were determined using a tissue culture system. Mature bones are shown in purple, resulting from <i>in situ</i> hybridization using the bone sialoprotein (<i>Bsp</i>) probe. Yellow lines on HE-stained images show the contours of the parietal and frontal bony edges, whereas a black arrowhead shows the existence of FLAG-tagged proteins in the calvarial tissue, as determined by immunohistochemistry using anti-FLAG antibodies. The images of blue fluorescence (4′, 6-diamidino-2-phenylindole [DAPI], staining the nucleic acids), green fluorescence (bromodeoxyuridine [BrdU], incorporated into cellular DNA during proliferation), and red fluorescence (rhodamine-stained nanogels) were merged. Administration of sFGFR2IIIc^S252W/nanogel complex maintained the patency of the coronal sutures in AS mice (n = 4/4; AS mice Nos. 9 and 10 are shown as representative examples); however, synostosis was observed on the side where only the nanogel was applied (n = 4/4). Control mice (n = 2/2; control mouse No. 8 is shown as a representative example) did not show any fusion between the frontal and parietal bones. Scale bar = 200 µm. f, frontal bone; p, parietal bone. (B) The ratios of BrdU-positive cells in AS mice and littermate controls are shown. Although no significant difference was observed, the ratio tended to be decreased in the suture tissue of AS mice, compared to the control littermates. Statistical analysis was performed using ANOVA and the Student-Newman-Keuls test.</p