INTRODUCTION

Identifying the cellular and molecular mechanisms whereby osteoblast cells are induced is centrally important in understanding the organizational principles underpinning a functional skeletal system.Deviation from the tight temporal and spatial regulation of osteoblast differentiation leads to a broad range of devastating diseases,such as craniosynostosis(premature suture fusion),heterotopic ossification(HO),and osteoporosis.In development,osteoblast differentiation is controlled by one of the two essential bone formation processes:intramembranous and endochondral ossification.During intramembranous ossification,mesenchymal progenitor cells differentiate directly into osteoblast cells,while during endochondral ossification,osteoblast differentiation is preceded by cartilage formation.The mechanisms underlying differential regulation of osteoblast differentiation in these two distinct ossification processes,although important,remain largely unknown.Molecular and cellular analyses of skeletal genetic diseases with abnormal osteoblast differentiation have provided important insights in the regulation of osteoblast induction,and in this study,we have focused on intramembranous ossification during craniofacial development.Progressive osseous heteroplasia(POH)(OMIM#166350)and Albright's hereditary osteodystrophy(AHO,OMIM 103580)are caused by lossof-function mutations in the GNAS gene,which encodes the stimulatory alpha subunit,Gαs,heterotrimeric G protein that transduces signals from G protein-coupled receptors(GPCRs).1-2 POH and AHO are characterized by progressive extra-skeletal bone formation through an intramembranous process.3-4In contrast,activating mutations of GNAS in McCune-Albright Syndrome(MAS)causes fibrous dysplasia(FD)(OMIM#174800)characterized by reduced ossification and bone marrow fibrosis.Studies of both POH and FD have identified the novel roles of GPCR/Gαssignaling in inhibiting Hedgehog(Hh)signaling while enhancing Wnt/β-catenin signaling in the regulation of osteoblast differentiation from mesenchymal progenitors.1-2,5-6Activated Gαssignaling has been found to reduce osteoblast maturation during endochondral bone formation,2,5while loss of Gαssignaling in committed osteoblasts resulted in severe osteoporosis characterized by impaired endochondral and intramembranous ossification due to accelerated differentiation of osteoblasts into osteocytes and decreased commitment of mesenchymal progenitors to the osteoblast lineage in association with attenuated Wnt signaling.7 In humans,Gαssignaling likely plays important roles in normal craniofacial development as both AHO and FD patients exhibit severe cranialbone defects.In AHO patients,craniofacial malformation such as craniosynostosis has been observed8and FD patients show craniofacial hyperostosis,which is characterized by polyostotic sclerosis and cystic changes in craniofacial bone.9 However,the cellular and molecular mechanisms underlying craniofacial bone defects in AHO or FD remained unknown.This is largely due to poor understanding of intramembranous ossification,which has hampered therapeutic development.

Calvarium development is tightly regulated at both molecular and cellular levels.10-12Mammalian cranium,or neurocranium,is the upper and back part of the skull.It protects the brain and supports sensory organs such as the ear and viscerocranium that support the face.The neurocranium can be divided into calvarium and chondrocranium,which grows to be the cranial vault that surrounds the brain and the skull base,respectively.Calvarium is composed of fl at bones:frontal bones,parietal bones,the interparietal,part of occipital bone,and squamous parts of temporal bone,12-13all undergo intramembranous ossification.By cell lineage analysis in mice,frontal bones show major contribution from the neural crest(NC)cells,while parietal and inter-parietal bones originate from head mesoderm.14-16Osteoblasts within calvarial bone primordia differentiate and secrete matrix,so that the calvarial bones grow and become mineralized12and finally meet at suture lines or fontanels.Cranial malformations are often progressive and irreversible,many of which need aggressive surgical management to prevent or mitigate severe impairment such as misshapened head or abnormal brain growth.10For instance,craniosynostosis(premature suture closure)is a significant medical problem and one of the most common cranial defects that affects 1 in 2500 individuals and requires surgical correction.Identifying molecular pathways that control cranial bone formation and growth is critically important in targeted therapeutic development.17

As the ectopic bone in POH and AHO patients forms through intramembranous ossification,we hypothesized that the Gαs-Hh signaling axis identified in POH and AHO is a common pathway that critically regulates intramembranous ossification both in normal skull development and in HO patients.In addition,as Gαs signaling also regulates Wnt/β-catenin signaling during osteoblast differentiation and maturation,2,5we hypothesized that Gαs signaling regulates intramembranous ossification by regulating both Hh and Wnt/β-catenin signaling.When Hh and Wnt/βcatenin signaling were manipulated in the mesenchymal progenitor cells or committed osteoblast cells,it has been found that while Hh signaling is required to induce osteoblast differentiation from osteoblast progenitor cells,Wnt/β-catenin signaling acts in the following steps to induce osteoblast commitment but inhibits further osteoblast maturation and ossification.18-19Hh signaling is required for osteoblast differentiation,20-21but its role in cranial bone development was not clear.Loss of Gαssignaling induces osteoblast differentiation by activating Hh signaling during ectopic intramembranous bone formation under pathological conditions.1However,loss of Indian Hedgehog(Ihh)ligand completely abolishes endochondral ossification but not intramembranous ossification during craniofacial bone formation.20 Furthermore,blocking ligand-dependent Hh signaling activity by removing a Hh receptor Smoothened(Smo)using the Wnt1-Cre mouse line resulted in severely reduced,but not completely abolished,formation of NC-derived craniofacial bone formation.22 Smo is required to transduce the signaling from all Hh ligands:Ihh,Sonic Hedgehog(Shh),and Desert Hedgehog(Dhh).23Therefore,it appears to be differential regulation and/or requirement of Hh signaling during intramembranous ossification compared to endochondral ossification.We hypothesized that additional Hh-ligand-independent mechanisms may regulate craniofacial bone formation and Gαssignaling is a strong candidate given the craniofacial malformation found in AHO and FD patients.

Here we have tested whether the mechanisms underlying pathological osteoblast induction by Gαssignaling loss in genetic diseases may also be applied to normal intramembranous bone development.We found that Hh signaling was differentially regulated during cranial and long bone formation by Gαsand Hh ligands.Both Hh and Wnt/β-catenin signaling mediated Gαs signaling in cranial bone development and small molecule inhibitors of Hh and Wnt/β-catenin signaling rescued craniofacial phenotypes in POH and FD mouse models,respectively.Therefore,insights gained by studying abnormal bone formation in genetic diseases allowed us to identify novel cellular and molecular mechanisms underlying normal craniofacial bone formation and therapeutic targets to treat craniofacial birth defects.

RESULTS

Reduced Gαssignaling correlates with increased Hh signaling in the developing cranial bone

To test the possibility that Hh signaling differentially regulates intramembranous and endochondral bone formation,we completely blocked ligand-dependent Hh signaling activity in both intramembranous and endochondral bone formation by removing Smo using the Prrx1-Cre mouse line.Prrx1-Cre targets the future posterior frontal bone,parietal,and inter-parietal bones that originate from the head mesoderm(Fig.S1a,b).14-16,24In the Prrx1Cre;Rosa 26tdTomatoembryo at embryonic day 18.5(E18.5;Fig.S1a),Prrx1Cre-targeted cells shown by tdTomato expression contributed to posterior frontal bones,parietal bones,interparietal bone,coronal suture,lambdoid suture,sagittal suture,anterior fontanel,and posterior fontanel at E18.5(Fig S1a).Craniofacial elements derived from NC are shown in green,whereas the ones from mesodermal origin are shown in blue in a modified schematics according to a previous study22(Fig.S1b).On this schematics,the Prrx1 lineage was indicated in red.These data indicate that the Prrx1 lineage are not only largely derived from mesodermal origin but also overlap with NC origin in the posterior frontal bone.In addition,the Prrx1 lineage cells include all Osx+cells and other surrounding progenitor cells in the targeted region(Fig.S1c).We found that,in the Prrx1-Cre;Smof/f embryos,while endochondral bone formation in the limb was similarly blocked as in the Ihh-/- embryos,20cranial bone formation controlled by intramembranous ossification was mildly affected,with good morphology and mildly reduced sizes(Fig.1a).These results indicate thatligand-dependentand ligandindependent Hh signaling pathways both regulate skull bone development and their relative contributions to the formation of skull bones from different origins may vary.We therefore examined Hh signaling activity in the developing cranial bone using a LacZ “knock in”allele of Ptch1.19As Ptch1 is a transcriptional target of Hh signaling,25LacZ expression in the Ptch1LacZ/+mice,which can be visualized by X-gal staining,serves as an in vivo readout of Hh signaling activity.19X-gal staining of Ptch1LacZ/+embryos at the E15.5 and E18.5 was performed.Analyses were focused on the frontal and parietal bones,in which ossification extended gradually from the basolateral region to the apex of the skull during development(Fig.1b).Strong X-gal staining was first found in the basal-lateral region at E15.5,extended to the apex of the skull at E18.5 in the Ptch1LacZ/+embryo(Fig.1c).Interestingly,when Smo was removed by Prrx1-Cre,Hh signaling activities were still present,albeit weakly reduced when X-gal staining was performed in the E18.5 Prrx1-Cre;Smof/f;Ptch1Lacz/+and the control Ptch1Lacz/+developing skulls(Fig.1d).These results indicate that Hh signaling is strongly activated during intramembranous bone formation and suggest that Ihh-and Smo-independent activation of Hh signaling may control osteoblast differentiation during normal craniofacial bone development and growth.

其中两电平VSC的接地需求包括2个方面：(1)交流侧滤波器的接地需求；(2)直流侧电容的接地需求。由于VSC电平数少，采用高频脉宽调试方式，开关频率高，换流器出口将含有较大的高次谐波，需在变压器阀侧加装较小容量的高频谐波滤波器。VSC在直流线路上有集中电容，可采取电容中点直接接地或通过组件接地的方式，如图1所示。

Activation of Gαssignaling inhibits cranial bone formation

von Kossa staining

To determine Gαssignaling activity at the osteogenic front in the Prrx1-Cre;Gnasf(R201H)/+mutants,we examined the levels of pCREB and total CREB at E15.5(Fig.2d).Indeed,in a location similar to the osteogenic front in the wild-type control,we found that,in mutants,while there was no change of the total CREB levels,pCREB levels were increased.In addition,Osx expression was not detected,while Gli1 expression was reduced in the mutant(Fig.2d,S6b).These results indicate that GnasR201H expression resulted in increased PKA activity,reduced Hh signaling,and delayed osteoblast differentiation.Bone formation was further analyzed at P0 by quantitative real-time PCR(qRT-PCR)analysis of gene expression in the formed parietal bone as illustrated (Fig.S2a-c).Early osteoblastdifferentiation was examined by alkaline phosphatase(Alp)and Osx expression,which were both increased in the Prrx1-Cre;Gnasf(R201H)/+mutants compared to the control(Fig.2e).However,markers for mature osteoblasts such as Collagen 1a1(Col1a1)and Osteopontin(Opn)were reduced in expression in the Prrx1-Cre;Gnasf(R201H)/+mutant(Fig.2e).Therefore,Gαsactivation by GnasR201Hexpression in osteochondral progenitor cells during cranial bone formation inhibited Hh signaling,delayed osteoblast induction in early development,and blocked osteoblast maturation later in the formed cranial bone.

Gαssignaling has been shown to inhibit Hh signaling downstream of Smo by inhibiting Gli activities during ectopic bone formation in POH patients and in brain tumors.1,26It is likely that the role of Gαssignaling in ectopic bone formation reflects its underappreciated function in regulating normal intramembranous ossification through inhibiting Hh signaling,as both ectopic bone in POH and normal cranial bones undergo intramembranous ossification.To test this hypothesis,we examined Gαsand Hh signaling at the osteogenic front of the developing parietal bone at E15.5 by immuno fluorescence staining(Fig.1e).Osteoblast differentiation was marked by an early osteogenic differentiation marker Osterix(Osx)expression,27which was gradually increased from the osteogenic front to the mature bone,indicating a gradient of osteogenic differentiation.Gαssignaling was shown by detecting CREB phosphorylation(pCREB),which is a readout of protein kinase A(PKA)activation by Gαssignaling.Phosphorylated CREB(pCREB)was gradually reduced while the total CREB levels did not change,suggesting that Gαssignaling was gradually decreased from the osteogenic front as the bone matured.Hh signaling was examined by the expression of Gli1,a readout of Hh signaling.28Importantly,Gli1 expression was increased while pCREB levels were reduced from the osteogenic front to the mature bone.These results show that,while osteoblast induction is associated with Hh signaling activation,there is an inverse correlation between Gαsand Hh signaling during cranial bone formation.

The change in cranial bone formation caused by Gαsactivation led us to test the hypothesis that Gnas removal in osteochondral progenitors may cause accelerated cranial bone formation in embryonic development and craniosynostosis after birth.We therefore removed Gnas in early osteochondral progenitors by generating the Prrx1-Cre;Gnasf/fmice.Opposite to what we had found in the Prrx1-Cre;Gnasf(R201H)/+mutants(Fig.2),the Prrx1-Cre;Gnasf/fmutants at E18.5 showed accelerated bone expansion from base to apex of the skull and increase in parietal and inter-parietal bone formation compared to the littermate control of Prrx1-Cre;Gnasf/+,Gnasf/f,or Gnasf/+embryos or mice,which are phenotypically indistinguishable from the wild-type ones(Fig.3a,b).Increased bone formation was also observed in the postnatal cranial bone leading to loss of sutures and therefore craniosynostosis(Fig.3c).Premature suture closure may have led to reduced head width,though head length was also slightly reduced in the Prrx1-Cre;Gnasf/fmutant mice compared to the controls(Fig.3c).The CI was reduced compared to that of the control,suggesting that loss of Gαssignaling may have caused a condition of dolichocephly,which can be a result of premature fusion of the sagittal suture.At the osteogenic front of the forming parietal bone at E15.5,Osx expression was accelerated and Gli1 expression was increased,but CREB phosphorylation was almost undetectable though total CREB levels were normal compared to the control(Fig.3d,S6c).The increase in osteoblast differentiation was further confirmed by qRT-PCR.At P0,expression of Alp,Osx,Col1a1,and Opn were increased(Fig.3e).Therefore,loss of Gαs signaling by removing Gnas in early osteochondral progenitor cells activated Hh signaling and accelerated osteoblastic differentiation and ossification during cranial vault development.Importantly,although loss of Gαssignaling accelerated osteoblast differentiation,the formed bone was of low quality with reduced mineral density and increased porosity(Fig.3c,S3a,b).We found that this is likely due to increased osteoclast differentiation indicated by tartrate-resistant acid phosphatase staining and this defect could be caused by more pronounced reduction in the expression of Osteoprotegerin(Opg)compared to the receptor activator of nuclear factor kappaB ligand(Rankl)(Fig.S3c,d).While Rankl is required for osteoclast differentiation,its activity is physiologically counterbalanced by the decoy receptor Opg.32-34 Taken together,Gαssignaling plays a key role in regulating osteoblast differentiation,maturation,and bone quality.

Gαsinhibits intramembranous ossification by inhibiting Hh signaling activity during cranial bone development

（6）优化工程施工资料的检查记录工作。航道项目具有隐蔽工程的施工占据主要地位的特点，工程档案记录显得尤为重要，需要全过程记录真实完整、闭环可追溯，隐蔽工程形成过程佐证资料齐全。目前作为施工企业还需在以下几个方面进行改进：

Hh signaling is not only activated during cranial bone formation but is also functionally important for cranial bone development.Hh signaling activation caused by a Ptch1 hypomorphic mutation led to craniofacial bone fusion,35while reduced Hh signaling due to loss of Gli2 function resulted in reduced intramembranous bone formation.36We next asked whether Gαscritically regulates intramembranous ossification during cranial vault bone formation by inhibiting Hh signaling.Upregulated Gli1 expression at the ossification front(Fig.1e)and its alteration by Gαssignaling(Figs 2d and 3d)indicated that Hh signaling mediated Gαs signaling activities in controlling intramembranous bone development.To further con fi rm this,we crossed the Ptch1LacZ/+allele19 with the Prrx1-Cre;Gnasf(R201H)/+or Prrx1-Cre;Gnasf/fmutants and visualized Hh signaling in Gαsgain-or loss-of-function mutant embryos,respectively,by X-Gal staining.In the nasal and anterior parts of frontal bones,where Prrx1Cre is not expressed,similar X-Gal staining pattern and intensities were found in the Prrx1-Cre;Gnasf(R201H)/+;Ptch1LacZ/+mutants at E16.5 and E18.5 compared to thecontrols(Fig.4a,S4a,c,e).However,in the posterior frontal bones,parietal,and inter-parietal bone areas where Prrx1Cre is expressed(Fig.S1a,b)as indicated by brackets in Fig.4a,b,GnasR201Hexpression led to larger space devoid of X-Gal staining in the apex of the skull.In the forming parietal bone,X-Gal staining was also reduced in the mutant compared to the control though islands of strong X-Gal staining were found in the mutant at later stages(Fig.4a,c).These island of cells might result from expansion of residual wild-type cells and/or wild-type reversal of mutant cells by epigenetic inactivation of the mutant GnasR201Hallele,which can happen in the imprinted Gnas locus.37-38These results indicate that Hh signaling is reduced by activated Gαssignaling(Fig.4a,c,Fig,S4a,c).

（教师点评：谁说“少年不识愁滋味”，只要我们用心思考，用心感受生活，一样可以有深刻的感悟。这篇文章展示出了作者的亲身体会，让我看到了六年级同学的思考深度。）

Conversely,in the Prrx1-Cre;Gnasf/f;Ptch1LacZ/+mutant embryos at E16.5 and E18.5,X-gal staining of the parietal and inter-parietal bones was enhanced and ectopically detected in the sagittal suture and posterior fontanel(Fig.4b,S4b).In addition,the forming parietal bone that was stained positive by X-Gal staining was expanded in size at E16.5 and E18.5 in the Prrx1-Cre;Gnasf/f;Ptch1LacZ/+embryo compared to the Ptch1LacZ/+control(Fig.4d and S4d).These results indicate that Hh signaling is upregulated by loss of Gαssignaling.Regulation of Hh signaling by Gαs signaling in the cranial bone was further confirmed by qRT-PCR analysis of the parietal bone tissue from the P0 mouse pups as shown in Fig.S2a-c.While GnasR201Hexpression decreased the expression of Hh signaling target genes Ptch1,Gli1,and Hip1,loss of Gnas upregulated the expression of these Hh target genes(Fig.4e,f).Importantly,expression of Hh ligand genes were not altered by loss of Gαssignaling(Fig.4g).Collectively,these data show that Gαssignaling inhibits Hh signaling during cranial vault bone formation without altering Hh ligand expression.

We next asked whether reduced Hh signaling mediated the effects of activated Gαssignaling in intramembranous ossification during cranial vault bone formation.We reasoned that the inhibitory effects of Gαssignaling in intramembranous ossification should be rescued by increasing Hh signaling if Hh signaling mediates the function of Gαssignaling.We therefore generated the Prrx1-Cre;Gnasf(R201H)/+;Ptch1LacZ/+mice as the LacZ insertion disrupts expression of Ptch119and Ptch1 is a negative regulatory component in the Hh signaling pathway.39We found that,at E16.5,ossification in the developing parietal bone of the Prrx1-Cre and Ptch1LacZ/+embryos was similar to the wild-type control.While ossification in the parietal bone of the Prrx1-Cre;Gnasf(R201H)/+embryos was reduced compared to the Prrx1-Cre and Ptch1LacZ/+controls,parietal bone formation was significantly rescued in the Prrx1-Cre;Gnasf(R201H)/+;Ptch1LacZ/+ embryos(Fig.5a).Conversely,we genetically reduced Hh signaling in the Prrx1-Cre;Gnasf/fmice by generating the Prrx1-Cre;Gnasf/f;Gli2f/+and the Prrx1-Cre;Gnasf/f;Gli2f/fmouse P0 pups(Fig.5b).Gli2 is the major Gli transcription factor that activates downstream target gene expression in the Hh signaling pathway.28Reduction of Gli2 led to mild reduction of cranial bone formation in the Prrx1-Cre;Gli2f/+and Prrx1-Cre;Gli2f/fmouse pups compared to the wild-type controls at P0(Fig.5b).Consistently,we found that increased ossification in the parietal and inter-parietal bones of the Prrx1-Cre;Gnasf/fpups was progressively rescued in the sagittal suture and posterior fontanel regions of the Prrx1-Cre;Gnasf/f;Gli2f/+and the Prrx1-Cre;Gnasf/f;Gli2f/fpups at P0 (Fig.5b).Furthermore,administration of a small molecule Gli inhibitor,arsenic trioxide(ATO),40to pregnantfemale mice inhibited ectopicbone formation in the posterior fontanel region in the Prrx1-Cre;Gnasf/fmouse pups at P0(Fig.5c).The rescuing effects are significant but not complete,which could be explained by at least two mechanisms.First,other Gli factors such as Gli3 also mediate transcription activities downstream of Hh signaling.Second,more than one signaling pathway is involved in Gnasregulated cranial bone formation,for example,we also found that Gnas regulates Wnt/β-catenin in this manuscript and other studies.2,5Taken together,our data indicate that Gαssignaling inhibits cranial bone formation by inhibiting Hh signaling(Fig.8d).

Gαsenhances Wnt/β-catenin signaling during intramembranous ossification of cranial bone development

Gαssignaling has been found to enhance Wnt/β-catenin signaling in the bone marrow stromal cells and during long bone development.2,5,7We therefore tested whether such regulation also occurs during cranial bone formation.We examined Wnt/β-catenin signaling by X-gal staining using tissues from the Axin2LacZ/+mice as Axin2 is a direct transcription target of Wnt/β-catenin signaling41-44(Fig.6a).In the Axin2LacZ/+control mice at P0,X-gal staining was detected in the parietal and inter-parietal bones and further upregulated in the forming sagittal suture and posterior fontanel(Fig.6a,b).We generated the Prrx1-Cre;Gnasf(R201H)/+;Axin2LacZ/+and Prrx1-Cre;Gnasf/f;Axin2LacZ/+mutant mice and found that X-gal staining was enhanced in the developing parietal and inter-parietal bone,particularly in the osteogenic front by GnasR201Hexpression,but it was reduced by loss of Gnas compared to the controls(Fig.6a,b).To further demonstrate that Wnt/β-catenin signaling is regulated by Gαs signaling,we examined the expression of Wnt/β-catenin signaling target genes Lef1 and Tcf1 by performing in situ hybridization and qRT-PCR(Fig.6c-e).Consistently,we found that Gαssignaling activation by GnasR201Hexpression expanded expression domains of Lef1 andTcf1 and enhanced their expression levels,while loss of Gαssignaling reduced Lef1 andTcf1 expression.These data indicate that Gαssignaling also enhances Wnt/β-catenin signaling during cranial bone formation.

应用SPSS 21.0对临床治疗过程中所取得的数据展开分析。使用了χ2对组间的差异性进行评判，采用Kaplan-Meier法探讨患者的服用阿帕替尼以后的生存情况[5]。

Given the critical role of Wnt/β-catenin signaling in osteoblast differentiation,we then tested whether Wnt/β-catenin signaling also mediates the effects of Gαssignaling in cranial bone formation.We hypothesized that reduction of Wnt/β-catenin signaling in the Prrx1-Cre;Gnasf(R201H)/+mice should ameliorate the defects of cranial bone formation.We therefore reduced Wnt/β-catenin signaling activity in mesenchymal progenitor cells by generating the Prrx1Cre;Gnasf(R201H)/+;Lrp6f/+mice45(Fig.7a).Lrp6 is a Wnt co-receptor dedicated to the Wnt/β-catenin signaling pathway.46-47Interestingly,although Gαsactivation and reduction of Lrp6 in Prrx1Cre-expressing cells both lead to reduced cranialboneformation compared to thecontrol(Fig.7a),cranial bone formation was increased in the Prrx1Cre;Gnasf(R201H)/+;Lrp6f/+mice at P0.These results are consistent with our observation in the long bones.5Furthermore,we found that administering LGK974,an inhibitor of Wnt secretion,48delayed ossification during cranial bone formation in the wild-type controls(Fig.7a,b).However,although cranial bone formation was delayed in both Prrx1-Cre;Gnasf(R201H)/+and Osx1-GFP::Cre;

X-gal staining

未来，区块链与IoT这2种技术将进一步融合，面向IoT安全的区块链技术也将逐渐兴起。共识效率更高、存储空间更小、绿色环保的安全区块链技术更能适应新型IoT技术的需求；而容错率高、鲁棒性强、去中心化的IoT安全管控势必会推进区块链应用技术的发展。

Gαssignaling activation in osteoblast lineage increases cartilage formation without changing cell fate specification

In both Prrx1-Cre;Gnasf(R201H)/+and Osx1-GFP::Cre;Gnasf(R201H)/+mice,we found that reduced ossification in the cranial bone was accompanied by increased cartilage formation as evidence by increased Safranin O staining compared to the controls(Fig.8a,S5a).It has been shown that osteoblast cells and chondrocytes are speci fi ed from their common osteochondral progenitors and loss of Osx or Wnt/β-catenin signaling leads to chondrocyte instead of osteoblast differentiation.27,49 We therefore asked whetherincreased cartilage formation wasdue to transdifferentiation of Osx+cells to chondrocytes when Gαs signaling was activated in the Osx1-GFP::Cre;Gnasf(R201H)/+mice.

模拟或角色扮演可以使学习者在模仿真实社交场景中扮演不同的角色，使真实交际能力得以增强,为以后在真实社会环境运用外语打下基础（周琳，2007）。在商务英语课堂，通过模拟真实的商务场景，让学习者进行角色扮演，亲自体验复杂的商务流程，运用前面所学的商务知识、语言和技能，这是体验学习的行动应用过程，学习者主动检验前面所学的知识是否有效。

All animal experiments were carried out according to protocols approved by the Harvard Medical School Institutional Animal Care and Use Committee.All mice have been described in the literature:Gnasf/f,29Prrx1Cre,24Gnasf(R201H)/+,5Osx1-GFP::Cre(Osx Cre),18Lrp6f/+,45Ptch1LacZ/+,19Smof/f,65and Gli2f/f.66The Axin2LacZ/+mice42were purchased from the Jackson Laboratories.Both male and female embryos were used in the analyses as sex could not be clearly identified in embryos or neonatal mice.Three or more littermate groups were examined and representative images are shown in fi gures.

DISCUSSION

In this study,we have identified critical roles of Gαssignaling in regulating normal intramembranous ossification during cranial bone development.Both gain-or loss-of-function mutations in GNAS have been found to cause severe cranial bone defects in MAS or POH human patients,respectively.8-9Our findings in this study support a model in which normal intramembranous bone development shares common underlying cellular and molecular mechanisms with ectopic intramembranous bone formation such as the one in POH.This model provides a new conceptualframework to furtheridentify basic regulatory mechanisms of normal bone formation in the cranium and test the contribution of bone development factors in ectopic bone formation and expansion under pathological conditions.The knowledge gained in these studies will facilitate development of therapeutic approaches for craniosynostosis,cleidocranial dysplasia,POH,and acquired HO.

There are major gaps in understanding the molecular mechanisms that distinguish intramembranous ossification versus endochondral ossification.We found here that regulation of Hh signaling exhibits major difference between long bone development in the limb and fl at bone formation in the skull.We have shown previously that Hh signaling is required before Wnt/βcatenin signaling during osteoblast differentiation.19It has been well established that ligand-dependent Hh signaling is absolutely required for endochondral bone formation.In the absence of Ihh or Hh receptor Smo,osteoblast cells cannot form during endochondral ossification.20-21Therefore,one critical function of preformed cartilage during endochondral bone formation is to provide Ihh required for osteoblast differentiation that first occurs in perichondrium adjacent to the pre-hypertrophic chondrocyte region.While Hh signaling is also activated during intramembranous ossification during cranial bone formation,surprisingly,Ihh and even Smo that mediates all ligand-dependent Hh signaling are not required for osteoblast differentiation.Consistent with previous findings in ectopic bone formation under pathological conditions that Gαssignaling inhibits Hh signaling downstream of Smo,1we found in this study that,during normal cranial bone formation,Gαssignaling plays a prominent role contributing to Hh signaling regulation in a ligand-independent manner during normalintramembranousbone formation.Theparathyroid hormone-related peptide(PTHrP)/PTH receptor PTH1R is predominantly coupled to Gαs54-55and plays important roles regulating osteoblast differentiation.It has been shown that,at P0,the Prrx1Cre;PTH1R fl/ flmice exhibited greatly enhanced mineralization in limbs and calvaria,56thus PTH1R may be one of the GPCRs that regulate Gαssignaling during cranial bone formation.PTH and locally produced PTHrP have pro-differentiating and pro-survival effects during bone formation.However,as continuous exposure to PTH reduces osteogenic differentiation,57-59 sustained PTH1R/Gαssignaling activation inhibits osteoblast maturation in the long bone.5-6Importantly,the function of PTH1R/Gαssignaling in endochondral bone formation is also mediated by its role in inhibiting chondrocyte hypertrophy of the growth plates,5,60-64 where Ihh is expressed in prehypertrophic chondrocytes.Therefore,an important difference between endochondral versus intramembranous bone formation is that PTH1R/Gαssignaling also indirectly inhibits Hh signaling by delaying Ihh expression in chondrocytes during endochondral bone formation.The dual roles of Ihh in controlling chondrocyte hypertrophy and osteoblast differentiation indicate that Ihh expression couples chondrocyte proliferation and hypertrophy with osteoblast differentiation from mesenchymal progenitors in the perichondrium during endochondral ossification.During cranial bone formation,however,the role of Ihh expression at the osteogenic front in osteoblastic induction is limited,and other signaling pathways such as Gαssignaling also control intramembranous ossification by regulating Hh signaling activities downstream of Smo.Therefore,the findings in this study provide a new conceptual framework to further understand the regulatory circuitry of craniofacial bone formation.

Hh signaling can be regulated by Hh ligand and other signaling component like Gαs.Bone can normally form when Hh and Wnt/β-catenin signaling levels are maintained in a certain range.Loss of Smo in the Prrx1+ mesenchymal cells blocked liganddependent Hh signaling,while the Gαs-regulated Hh or Wnt/βcatenin signaling is still intact.The mild bone phenotypes in the Prrx1Cre;Smof/fembryo allowed us to conclude that ligandindependent Hh signaling such as the one regulated by Gαs played significant roles in bone formation in the Prrx1-targeted skull tissues.Activated Gαssignaling in the Prrx1Cre;Gnasf(201H)/+mice not only reduced Hh signaling but also activated Wnt/βcatenin signaling in progenitor cells,and both led to reduced bone formation.It is foreseeable that abnormally activated Wnt/βcatenin signaling sensitized defects caused by Hh signaling reduction.Because of this,increasing Hh signaling by reducing Ptch1 will bring Hh signaling to a level closer to the normal one,thus reducing the phenotypic severity.Furthermore,Wnt/βcatenin signaling levels higher or lower than the normal range reduce bone formation.Gnas gain-of-function mutation causes reduced bone formation partially by enhancing Wnt/β-catenin signaling to levels above the normal range in the wild-type embryo.Therefore,reduction of Wnt/β-catenin signaling by deleting Lrp6 or administrating LGK974,which reduce bone formation in a wild-type background,brought down the Wnt/β-catenin signaling levels in the Prrx1Cre;Gnasf(201H)/+mutant closer to the normal range and thus the mutant phenotypes were rescued.

Gαssignaling regulates osteoblast differentiation by regulating both Wnt/β-catenin and Hh signaling.As we have shown in our previous studies,1-2,5the effect of gain or loss of Gαssignaling in osteoblast differentiation was mediated predominantly by Wnt/βcatenin orHh signaling,respectively.While Wnt/β-catenin signaling shown by the Axin2-LacZ expression was reduced in the loss of Gαssignaling mutant(Fig.6b),the bone phenotypes were mainly driven by elevated Hh signaling caused by Gαsloss(Fig.4b)even if Wnt/β-catenin signaling is reduced at the same time.However,the functional impacts of Gαsregulation of Wnt/βcatenin signaling is also different in intramembranous versus endochondral ossification.It appears that Gαsregulates endochondral ossification mainly through Wnt/β-catenin signaling while its role in osteoblast induction during intramembranous ossification is mainly determined by its regulation of Hh signaling.This explains the phenotypic discrepancy of bone formation caused by Gαssignaling activation in Osx+cells,which showed increased endochondral bone formation5while intramembranous ossification is severely inhibited with reduced cartilage dissolution(Fig.8a).

The less severe phenotypes of Prrx1Cre mutant than the OsxCremediated Gnas mutants(Fig.7b,c)are likely due to the different spatial and temporal onset of the Cre expression.Prrx1Cre targets earlier mesenchymal progenitor cells before Osx expression but mostly from the mesodermal origin,while OsxCre targets all osteoblast lineage cells regardless of their mesodermal or NC origins.As the gain-of-function Gnas promotes Wnt/β-catenin signaling,which is known to stimulate progenitor cell proliferation and reduce osteoblast maturation,Prrx1Cre-targeted mutation may lead to expansion of progenitor cells but reduced osteoblast maturation as has been shown in our studies of the Sox9CreER-mediated Gnas mutant.5In the OsxCre;Gnasf(R201H)/+mutant,there should be less progenitor cell proliferation due to the later onset of the Cre but more broadly affected skull bone tissues,leading to more severe phenotypes of reduced bone ossification.

The mouse models we have established allowed us to test small molecules that target Hh or Wnt signaling to ameliorate bone phenotypes caused by disrupted Gαssignaling.Craniofacial hyperostosis/FD in MAS or craniosynostosis/POH are still unmet medical challenges.The Hh and Wnt signaling pathways act downstream of Gαssignaling and both are important drug targets in many other diseases,including cancer.Therefore,some of the drugs already developed targeting Hh or Wnt signaling can be repurposed to treat bone defects including those in the craniofacial regions of FD or POH patients.Furthermore,our studies highlight the unparalleled opportunities offered by human genetic diseases in revealing fundamentally important genes and pathways in both human biology and pathology.Therefore,not only understanding the pathological mechanisms of genetic disease like POH and FD have allowed us to uncover a fundamental mechanism underlying a physiological bone formation process,the precise connection of pathological bone formation with normal bone development will provide invaluable insights in re fi ning disease diagnosis and development of treatment strategies.The mechanistic investigation of cranial bone development in FD and POH models has significantly expanded previous knowledge of Wnt and Hh signaltransduction in osteoblastdifferentiation and may provide new insights in other craniofacial bone diseases with similardefects,such ascraniometaphysealdysplasia and craniodiaphyseal dysplasia.

青贮方式为实验室塑料瓶罐装青贮，塑料瓶容积为1 L。将甜高粱与苜蓿按不同的比例进行混合青贮，甜高粱占比为0%、25%、50%、75%、100%，并将瓶内原料压实密封，每种比例混合青贮饲料制作10瓶，放在实验室避光保存备用。

MATERIALS AND METHODS

Mouse lines

Taken together,our data show that proper control of Gαs signaling is required to ensure normal cranial bone formation by regulating both Hh and Wnt signaling and reduction of Gαs signaling contributes to Hh signaling activation during normal intramembranous ossification,but not endochondral ossification.These results provide further insights in strategic development to treat FD,POH,and other related craniofacial bone malformation such as craniosynostosis.

The Osx+cells and their descendants were traced by TdTomato expression in the Osx1-GFP::Cre;Rosa 26tdTomatomice.50As the Osx1-GFP::Cre line expresses a Cre::GFP fusion protein,18osteoblasts are marked by green fluorescent protein(GFP)expression.Chondrocytes were detected by immuno fluorescence staining with Collagen type II(ColII)antibodies.We reasoned that,if there is a cell fate switch from osteoblasts to chondrocytes,some of the tdTomato+cells should also express ColII,otherwise there should be no overlap between tdTomato+and ColII+cells(Fig.8b).In the Osx1-GFP::Cre;Gnasf(R201H)/+;Rosa 26tdTomato mice,while there was large overlap between tdTomato and GFP expression,a few tdTomato+cells lost GFP expression.However,none of these cells were ColII+,indicating that the expansion of the ColII+ cell population is not a result of transdifferentiation of Osx+cells(Fig.8c,S6d).Interestingly,just as loss of Osx led to osteoblast conversion to chondrocytes,loss of Smo or β-catenin in early osteochondral progenitors during endochondralbone formation also led to similar osteoblasts to chondrocytes'cell fate changes.21,49,51-52However,although Gαssignaling activation in Osx+cells during intramembranous ossification resulted in expanded cartilage formation and reduced Hh signaling(Fig.4a,c,e),we found that expanded cartilage is not derived from Osx+cells in the Osx1-GFP::Cre;Gnasf(R201H)/+;Rosa 26tdTomatomice at P0(Fig.8c).The cartilage found in the Osx1-GFP::Cre;Gnasf(R201H)/+mutant skull looked similar to the one reported in the Membrane-type 1 matrix metalloproteinase(Mt1-mmp or Mmp14)mutant mice,53suggesting that the defect is in the cartilage remodeling rather than cell fate determination.Consistent with this notion,we found that,associated with altered bone formation in the Prrx1Cre;Gnasf(R201H)/+and Prrx1Cre;Gnasf/fmice,cartilage remodeling in the skull was also changed(Fig.S5a,b).To further con fi rm this,we examined the expression of Mt1-mmp and Mmp13 and found both were reduced in the Prrx1Cre;Gnasf(R201H)/+mutant parietal bone but increased in the Prrx1Cre;Gnasf/fmice compared to the control(Fig.S5c,d).

Skeletal preparation

Alcian blue staining for cartilage and Alizarin red staining for mineralized tissueswere performed asdescribed.67Brie fl y,embryos and early-postnatal mouse pups were collected and skinned carefully.Internal organs were then eviscerated and the embryos were placed in 100%ethanol overnight.Staining was performed according to a previously described protocol.18Some dorsal views of the skull were captured after removing mandibles and partial skull base(Fig S2d-e).

We next asked whether Gαssignaling indeed plays an important role in cranial bone formation.We hypothesized that,Gαs signaling activation by GnasR201Hexpression5in early osteochondral progenitor cells in mice should delay bone formation,while deletion of Gnas using a floxed loss-of-function Gnas allele(Gnasf)29should accelerate bone formation during embryonic development of the cranial vault.To test our hypothesis,the Gnasf(R201H)/+mice5were crossed with the Prrx1-Cre mouse line.24 Cranial bone formation was examined at E18.5 by skeletal preparation and von Kossa staining of the cranial bone section(Fig.2a,b).Compared to the littermate control of Prrx1-Cre or Gnasf(R201H)/+embryos or mice,which are phenotypically indistinguishable from the wild-type ones in development,the Prrx1-Cre;Gnasf(R201H)/+mutant embryos showed delayed ossification expansion from the base to the apex of the skull,therefore parietal or inter-parietal bone formation was reduced.Mineralization,assessed by von Kossa staining,was also reduced as indicated by the “sand patch”pattern in the Prrx1-Cre;Gnasf(R201H)/+mutants(Fig.2b).Reduced bone formation was also observed in the postnatal cranial bone at 2 months of age by micro-computed tomographic(μCT)scanning(Fig.2c).Similar to what has been reported in CT scans of human polyostotic fibrous dysplasia,30the Prrx1-Cre;Gnasf(R201H)/+ mutantskullshowed mixed-density fibrous dysplasia lesions with mixed lucencies and sclerosis(arrows in Fig.2c).Reduced bone formation was also manifested by reduced head length and width(Fig.2a,c).The mutant phenotype was further quantified by the cephalic index(CI)or cranial index,which is the ratio of the maximum width(biparietal diameter,side to side)of the head multiplied by 100 divided by its maximum length(occipitofrontal diameter,front to back).The CI of the Prrx1-Cre;Gnasf(R201H)/+mutant heads was increased comparing to the littermate control,suggesting that activated Gαs signaling may have caused brachycephaly.31

Cryostat sections were stained with 1%silver nitrate solution under a 60 W lamp for 1h.Slides were rinsed three times in distilled water.Sodium thiosulfate 5%was added to the slides for 5min.Slides were rinsed three times in distilled water and counterstained with 1%Safranin O or fast red for 5min.Slides were rinsed three times in 1%acetic acid or distilled water before mounted in mounting medium.

Histological and immunohistochemistry staining

Embryos and early-postnatal specimens were fixed in 4%(wt/vol)paraformaldehyde in phosphate-buffered saline(PBS).For immunohistochemistry,cryostat sections were blocked in 5%donkey serum and 0.3%Triton X-100 in PBS and performed with the following primary antibodies:rabbit anti-Osx(#sc-22536,Santa Cruz Biotechnology)1:500,rabbit anti-CREB1(#sc-25785,Santa Cruz Biotechnology)1:100,rabbit anti-pCREB(#06-519,Millipore)1:100,goat anti-Gli1(#sc-6153,Santa Cruz Biotechnology)1:50,and goat anti-ColII(#sc-7764,Santa Cruz Biotechnology)1:100.Secondary antibodies are:Alexa Fluor®488 donkey anti-goat(#A11055,Life Technologies),Alexa Fluor®568 donkey anti-rabbit(#A10042,Life Technologies),and Alexa Fluor®647 donkey antigoat(#A21447,Life Technologies).Signals of 647 were read by Cy5 filter and the signal is artificially shown in white color.Cryostat sections were mounted in mounting medium with 4,6-diamidino-2-phenylindole from Vector laboratories(H-1200).RNA in situ hybridization

RNA in situ hybridization was performed using digoxygeninlabeled anti-sense RNA probes as described before.68

Gnasf(R201H)/+mutants(Fig.7b,c),LGK974 administration to pregnant females rescued cranial bone formation in both Prrx1-Cre;Gnasf(R201H)/+and Osx1-GFP::Cre;Gnasf(R201H)/+mutants(Fig.7b,c).Taken together,we found that Gαssignaling activation in the mesenchymal progenitor cells or early osteoblast cells both led to activated Wnt/β-catenin signaling,which delayed ossification(Fig.8d).

Embryos were fixed in 0.5%formaldehyde and 0.1%glutaraldehyde and X-gal staining was performed according to previously described procedures.69

Quantitative real-time PCR

Total RNA from mouse calvarial bone tissue(Fig.S2a-c)was prepared using the TRIZOL reagent(Life Technologies)or RNeasy Mini Kit(Qiagen)according to the manufacturer's protocols.cDNA was synthesized from total RNA(1-3μg)using SuperScript II Reverse Transcriptase with random primer(Life Technologies).qRT-PCR were performed using SYBR Select Master Mix on StepOnePlus thermal cycler from Applied Biosystems.Expression levels were always given relative to glyceraldehyde 3-phosphate dehydrogenase(GAPDH).

GAPDH:Forward 5′-AGGTCGGTGTGAACGGATTTG-3′,Reverse 5′-TGTAGACCATGTAGTTGAGGTCA-3′;

Alk Phos:Forward 5′-CTTGACTGTGGTTACTGCTGAT-3′,Reverse 5′-GGAATGTAGTTCTGCTCATGGA-3′;

Osx:Forward5′-CCCACTGGCTCCTCGGTTCTCTCC-3′,Reverse 5′-GCTBGAAAGGTCAGCGTATGGCTTC-3′;

Col1a1:Forward 5′-CACCCTCAAGAGCCTGAGTC-3′,Reverse 5′-GTTCGGGCTGATGTACCAGT-3′;

Opn:Forward 5′-GGCATTGCCTCCTCCCTC-3′,Reverse 5′-GCA GGCTGTAAAGCTTCTCC-3′;

Lef1:Forward 5′-CTTCGCCGAGATCAGTCATCC-3′,Reverse 5′-AC GGGTCGCTGTTCATATTGG-3′;

Tcf1:Forward 5′-TCGAGAAGAGCAGGCCAAGT-3′,Reverse 5′-AG AGCACTGTCATCGGAAGGAA-3′;

Axin2:Forward 5′-CCATTGGAGTCTGCCTGTG-3′,Reverse 5′-GG ACACTTGCCAGTTTCTTTG-3′;

Ptch1:Forward 5′-ACCCGTCAGAAGATAGGAGAAG-3′,Reverse 5′-TTCCCAGAAGCAGTCCAAAGG-3′;

Gli1:Forward 5′-AATCCAATGACTCCACCACAAG-3′,Reverse 5′-G TCGAGGCTGGCATCAGAA-3′;

计算时不考虑结构自重产生的影响，荷载效应全部来自于体外预应力钢束。将预应力荷载等效为沿箱梁纵桥向作用的均布力作用于锚垫板上，所施加的荷载参数如表 1所示。

Hhip:Forward 5′-GGGAAAAACAGGTCATCAGC-3′,Reverse 5′-A TCCACCAACCAAAGGGC-3′;

Ihh:Forward 5′-ACGTGCATTGCTCTGTCAAGT-3′,Reverse 5′-C TGGAAAGCTCTCAGCCGGTT-3′;

(1) 随着支管关闭数量增加，流量改变量越大，造成的水锤升压越严重，因此，设计人员在设计时，应尽量的延缓末端阀门的关闭时间，并且要求尽量避免多条支管同时关闭。

Shh:Forward 5′-GATGACTCAGAGGTGCAAAGACAA-3′,Reverse 5′-TGGTTCATCACAGAGATGGCC-3′;

Dhh:Forward5′-CCCGACATAATCTTCAAGGATGA-3′,Reverse 5′-GCGATGGCTAGAGCGTTCAC-3′;

Mmp13:Forward 5′-TTCTGGTCTTCTGGCACACGCTTT-3′,Reverse 5′-CCAAGCTCATGGGCAGCAACAATA-3′;

Mt1-mmp: Forward 5′-CTGCCTACGAGAGGAAGGATGG-3′,Reverse 5′-ATTCAGGGCAGTGGACAGCG-3′.

本文对9例左冠状动脉异位起源于肺动脉的患者采取多普勒超声检查,结果与李文秀、耿斌、吴江等人[2]的研究结果相似,李文秀等人的研究结果为8例患者的平均左室舒张末内径为(51.36±2.62)mm,平均左室收缩末内径为(34.06±1.92)mm,平均左室射血分数为(69.61±1.02)%,但是李文秀等人的研究将8例患者的结果全部采取表格的形式表现出来,数据清晰,更具研究性,需要本研究进行学习。

Small molecule treatment

ATO 5mg(Sigma Aldrich 202673-5 G)was dissolved in 15mL conical tube with 1mL of 1N NaOH.Autoclaved PBS 7mL was then added to the tube.HCl 1.2N was used to adjust the pH to 7.2,and PBS was added to make up the volume to 10 mL.LGK974(Toronto Research Chemicals L397640)was dissolved in DMSO and then diluted with corn oil before injection.Pregnant females were injected with ATO or LGK974 by intraperitoneal injection at a concentration of 5mg·kg-1or 1mg·kg-1,respectively,every other day.Equivalent volumes of vehicle were injected as control.Mice were injected with care with ATO during their pregnancy starting at E13.5 or LGK974 at E14.5.

Statistical analysis

Statistical significance was assessed using two-tailed Student's t test for comparisons between two groups.P values＜0.05 were considered significant.Data are presented as mean±standard deviations(SD)unless otherwise indicated.

ACKNOWLEDGEMENTS

We thank members of the Yang laboratory for constructive discussions and HSDM μCT core for μCT scanning.This study was supported by the NIH grants R01DE025866 from NIDCR and R01AR070877 from NIAMS.R.X.is supported by the 111 Project,MOE(B14038),China.

AUTHOR CONTRIBUTIONS

R.X.and Y.Y.designed research,analyzed data,and wrote the manuscript;R.X.performed research;S.K.K.generated Gnasf(R201H)/+ conditionalknock-in allele;T.Z.and B.G.participated in some supplemental experiments;R.X.and Y.Z.performed genotyping;X.Z.and Y.Y.designed and supervised the manuscript and the project.

奥巴马当政以来，共和党反对声音不断，在医改问题上，民主党支持医改，共和党反对医改。2012年7月11日美国国会众议院投票通过议案，取消总统奥巴马在2010年签署的医疗保险改革法案，不过，与先前多次投票一样，众院的努力注定会被民主党占多数的参议院阻挡。拿征税问题举例，经过经济学家计算，单纯增税或削减开支都无法清理债务，如果两党妥协，共和党同意增税，民主党同意削减开支，债务问题就有望解决。但是，在共和党总统候选人的辩论中，主持人问是否同意每削减10美元的联邦财政开支就增加一美元的税，主要候选人没有一个敢支持。

对照组运用红霉素治疗方式，红霉素（唐山红星药业有限责任公司，国药准字H13022717）口服治疗，3次/d，连续使用10 d作为一个疗程，连续使用3个疗程

ADDITIONAL INFORMATION

The online version ofthisarticle (https://doi.org/10.1038/s41413-018-0034-7)contains supplementary material,which is available to authorized users.

《中国微侵袭神经外科杂志（CMINSJ）》中的“日常生活能力量表（ADL）”共有14项，包括两部分内容：一是躯体生活自理量表，共6项：上厕所、进食、穿衣、梳洗、行走、洗澡；二是工具性日常生活能力量表，共8项：打电话、购物、备餐、做家务、洗衣、使用交通工具、服药、自理经济。评定结果可按总分、分量表分和单项分进行分析。总分最低14分，为完全正常；大于14分，有不同程度的功能下降，最高56分。单项分1分为正常，2～4分为功能下降。凡两项或两项以上≥3分，或总分≥22分，为功能有明显障碍。

Competing interests:The authors declare no competing interests.

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