| Skull is composed of calvarial bones,craniofacial bones and cranial base.They are formed through distinct development modes.The calvarial bones are formed through intramembranous ossification,in which the mesenchymal precursor cells between the opposing osteogenic fronts of sutures directly differentiate into bone-forming osteoblasts driving the growth of calvarial bones.Bones of the cranial base are formed through endochondral ossification.Endochondral ossification of the synchondroses is responsible for the growth and expansion of cranial base.Apert syndrome?AS?is a common genetic syndrome in humans characterized with craniosynostosis,is caused by one of two missense mutations in adjacent amino acids,Ser252Trp?S252W?and Pro253Arg?P253R?,of fibroblast growth factor receptor 2?FGFR2?.AS is characterized with precocious closure of cranial sutures,midfacial hypoplasia and syndactyly of hands and feet.Additionally,patients with AS usually have raised intracranial pressure and mental retardation.Although the skull morphology of AS have been detailed descripted,the reasons for the characteristic skull shape of AS are not fully clarified presently.It's generally considered that premature fusion of sutures is the original reason for the abnormal skull shape of CS patients.The premature closure of coronal suture is most commonly exhibited in AS patients,which is also observed in the mouse models mimic AS.Individuals with AS are reported to have cartilaginous abnormalities in their cranial base,including premature fusion of the spheno-ethmoidal synchondrosis and the spheno-occipital synchondrosis,suggesting an important role of endochondral ossification in the skull malformation of AS.Similarly,dysmorphology of the cranial base is one of the significant skeletal abnormalities in mice with ubiquitous or chondrocyte-specific expression of FGFR2 with Pro253Arg mutation.Therefore,it is speculated that the disturbed cartilaginous development may also play an important role in the malformation of skulls of AS patients or AS mouse models.Besides skull maldevelopment,AS patients also have abnormalities in nervous system such as mental retardation and brain dysmorphology.There are controversies about the reasons for the abnormal brain morphology of AS patients.Brain malformation in AS is generally considered as the result,at least in part,of the premature fusion of cranial sutures.Some researchers,however,by finding that the brain dysmorphology was developed before the fusion of calvarial sutures of AS mice,proposed that brain is primarily affected,rather than secondarily responding to skull dysmorphogenesis in AS mouse model.To date,the pathogenesis of the skull abnormalities of AS is still not fully clarified.It is important to understand the accurate role of these individual composing parts of head in the malformation of AS skulls.Current treatment for AS is surgical correction of the deformed skulls.Because of the progressive skull maldevelopment,the surgery for AS is often executed for several times,resulting in increased suffering and complications.Biological therapies targeting the FGF/FGFR signaling may bring better outcome.In fact,several studies have explored the effects of biological inhibition of the molecular pathways activated in AS on skull phenotypes.Yokota et al.found a soluble form of FGFR2 with Ser252Trp mutation can partially alleviate the phenotype of Apert mouse model by alleviating the premature closure of coronal suture in cultured calvarias and transgenic mice.PD98059,a MEK1 inhibitor,was found to reduce coronal suture fusion in cultured calvarias of Fgfr2+/P253R mice.Inhibiting the MEK1/2 by U0126 alleviates the craniosynostosis phenotypes in Fgfr2+/S252W/S252W mice.The above described molecules do not specifically antagonize the mutant FGFR2itself,which may bring undesired effects.RNA interference?RNAi?,in contrast,could be a powerful tool to specifically inhibit the expression of mutant alleles at transcriptional level.By RNAi,we could specifically silence the mutant allele to treat human diseases caused by gain-of-function mutations while leave the expression of the wild-type allele undisturbed.To dissect the differential roles of these components of head in the pathogenesis of the abnormal skull morphology of AS,we generated mouse strains specifically expressing mutant FGFR2 in chondrocytes,osteoblasts,and progenitor cells of central nervous system?CNS?by crossing Fgfr2+/P253R-Neo mice with Col2a1-Cre,Osteocalcin-Cre?OC-Cre?,and Nestin-Cre mice,respectively.We then quantitatively analyzed the skull and brain morphology of these mutant mice by micro-CT and micro-MRI using Euclidean distance matrix analysis?EDMA?.Besides,this study screened and found a siRNA specifically targeting to Fgfr2-P253R allele and revealed that it inhibited osteoblastic differentiation and matrix mineralization by reducing the signaling of ERK1/2 and P38 in cultured primary calvarial cells and calvarial explants from Apert mice(Fgfr2+/P253R).Furthermore,AAV9carrying shRNA?AAV9-Fgfr2-shRNA?against mutant Fgfr2 was delivered to skulls of AS mice.Results demonstrate that AAV9-Fgfr2-shRNA attenuated the premature closure of coronal suture and the decreased calvarial bone volume of AS mice.The study provides a novel practical biological approach,which will,in combination with other therapies including surgeries,help treatment of patients with AS,while provide experimental clues for the biological therapies of other genetic skeletal diseases.Methods:Part ?:Deformed skull morphology is caused by the combined effects of the maldevelopment of calvarias,cranial base and brain in Fgfr2-P253R mice mimicking human Apert syndrome.1.By Cre/loxp approach to obtain mutant mice with expression of activated FGFR2?Pro253Arg?in chondrocytes,osteoblasts,and CNS progenitor cells,and analyzed the whole mount and bone morphology of these mutants at postnatal 4-and 8-week using three-dimensional micro-CT and X-ray.2.Researching morphology of cranial bone,sutures,cranial base etc.of mutant mice by three-dimensional micro-CT,morphometric assay and histopathology.To dissect the brain morphology of Nestin-253 mice by microMRI.3.Comparing skull morphology of mutant mice with that of WT mice by quantitatively analyzed the skull and brain morphology of these mutant mice by micro-CT and micro-MRI using Euclidean distance matrix analysis.Part ?:Molecular therapy for Apert syndrome.1.Synthesizing a set of siRNAs according to mutant sequence.Each siRNA fully matches the Fgfr2-P253R mRNA but contains a C:C mismatch with wild-type mRNA.The siRNAs were individually transfected into primary osteoblasts from Apert mice for assessing their silencing effects on the expressions of mutant Fgfr2 by Real-time PCR and Western blot.2.Primary calvarial osteoblasts from Apert mice and their littermates were used to detect the changes of cellular proliferation,differentiation and matrix mineralization by MTT,ALP and Alizarin red staining.FGF related signaling pathways were detected following siRNA treatment by Western blot.3.The therapeutic effects of siRNA were further evaluated in cultured calvarias.evaluating the degree of suture closure,and bone morphology of calvarial bone by micro-CT,X-ray and histopathology.4.In vivo injection of AAV2-GFP?AAV5-GFP?AAV-DJ-GFP?AAV9-GFP virus on calvarias,Screening for AAV serotypes with high infection to calvarias by evaluating the GFP fluorescence.5.To evaluate the in vivo therapeutic effects of shRNA on the calvarial phenotypes of Apert mice,AAV9-Fgfr2-shRNA viruses were locally injected on calvarias of Apert mice.Calvarias of shRNA treated Apert and WT mice were dissected to evaluate the degree of suture closure,and bone morphology of calvarial bone by real-time PCR,three-dimensional reconstruction of micro-CT image,X-ray and histopathology.Results:Part ?:Deformed skull morphology is caused by the combined effects of the maldevelopment of calvarias,cranial base and brain in Fgfr2-P253R mice mimicking human Apert syndrome.1.Mouse strains that specifically expressing mutant FGFR2 in chondrocytes,osteoblasts,and progenitor cells of central nervous system were generated by crossing Fgfr2+/P253R-Neo mice with Col2a1-Cre,Osteocalcin-Cre?OC-Cre?,and Nestin-Cre mice,respectively.the three mutant mice exhibited normal body length,weight and normal skeleton by X-ray,Micro-CT.2.Skulls of Col2a1-253 mice showed Apert syndrome-like dysmorphology,such as shortened skull dimensions along the rostrocaudal axis,shortened nasal bone,and evidently advanced ossification of cranial base synchondroses.The OC-253 mice showed malformation in face at 8-week stage.Nestin-253 mice exhibited increased dorsoventral height and rostrocaudal length on the caudal skull and brain at 8 weeks,through X-ray,Micro-CT,microMRI.3.By quantitative EDMA analysis of the skulls and brain,results revealed that mutant FGFR2 in Col2a1 expressing cells mainly affected cranial base,sutures and midface.Mice with specific expression of mutant FGFR2 in osteoblasts?OC?showed mildly shortene d face.Specific expression of mutant FGFR2 in Nestin-expressing cells mainly led to increased rostrocaudal length and dorsoventral height of the caudal brain.All three mutant mouse stains with expression of mutant FGFR2 in specific tissue exhibited skull malformation,which indicates that mutant FGFR2 played direct role in maldevelopment of these tissues.Furthermore,it's noticed that the phenotypes of each mutant strain just partially resembled the skull malformation of EIIa-253 mice,suggesting that the malformation of specific tissues partially contributed to dysmorphogenesis of skull of AS mice.Part ?:Molecular therapy for Apert syndrome.1.A set of siRNAs designated as S1 to S11 were synthesized.Each siRNA fully matches the Fgfr2-P253R mRNA but contains a C:C mismatch with wild-type mRNA.real-time PCR and WB detection revealed that S2 exhibited strongest inhibitory effect on mutant FGFR2 whereas no significant inhibition on normal FGFR2.2.The expression level of FGFR2 was significantly decreased in S2 treated Apert osteoblasts compared with scramble siRNA.S2 treatment significantly decreased the phosphorylation levels of ERK1/2 and P38 of Apert osteoblasts compared with N.C treated Apert osteoblasts,indicating that S2 alleviated the activation of ERK1/2 and P38 signaling caused by FGFR2-P253R mutation.compared with that of WT osteoblasts,the expression levels of Runx2,Collagen 1,Osteocalcin and Osteopontin were increased in Apert osteoblasts in the absence of S2 treatment,which were significantly reduced by S2treatment.When compared with S2 treated WT group,the expression of Runx2 and Collagen 1 were significantly decreased in S2 treated Apert osteoblasts.Following S2treatment,WT osteoblasts showed no significant changes in mineralization compared with N.C siRNA treated osteoblasts,whereas the accelerated mineralization in the Apert osteoblasts was significantly reduced.3.The premature closure of coronal sutures in Apert calvarias was accelerated compared with that of WT calvarias,while was delayed by S2 treatment.The thickness of cultured parietal bone in Apert mice was 31.94%thinner than that of WT calvarias.With the treatment of S2,the decreased thickness of parietal bone was rescued in Apert calvarias.S2 treated Apert calvarias had a 24.78%increase in the thickness of its parietal bone.Further analysis revealed that S2 efficiently attenuated the decrease of parietal bone volume in cultured parietal bone.4.AAV9-GFP injected calvarias had stronger GFP expression than that of other AAV-GFP injected calvarias,suggesting that AAV9 viral serotype is suitable for delivering genes to be expressed for a long term in calvarias.5.After shRNA injection,the expression of mutant Fgfr2 was dramatically reduced in Apert calvarias,indicating that AAV mediated shRNA worked efficiently in calvarias.Besides,the expressions of Collagen 1 and Runx 2 were significantly increased in Apert mice compared with that of WT mice,which were decreased after shRNA injection.Histological analysis revealed that shRNA treatment attenuated the overlapping area of osteogenic fronts in coronal suture of Apert skulls.Micro-CT analysis indicates that shRNA efficiently attenuated the decreased bone volume of parietal bone,while shRNA had no significant effect on the bone mineral density of Apert calvarias.Conclusions:1.Abnormal development in calvarias,cranial base and brain are all involved in the pathogenesis of the deformed skulls in AS mice,and there is direct effect of mutant FGFR2on brain development.This study provides new insight for the mechanistic understanding and clinical management of Apert syndrome.2.AAV9 viral serotype is suitable for delivering genes to be expressed in calvarias.3.Adeno-associated virus-mediated RNA interference against mutant allele attenuates abnormal calvarial phenotypes in an Apert syndrome mouse model. |
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