RAB23 coordinates early osteogenesis by repressing FGF10-pERK1/2 and GLI1
Ras-associated binding protein 23 ( RAB23 ) belongs to the RAB family of small GTPases , which function during several steps in cell vesicle trafficking ( Evans et al . , 2003 ) . RAB23 has 35–40% sequence homology with other RABs , it localizes to the plasma membrane and is proposed to regulate cargo internalization and relocation in the cell ( Evans et al . , 2003; Leaf and Von Zastrow , 2015; Lim and Tang , 2015; Olkkonen et al . , 1994 ) . RAB23 is unusual amongst RABs as it is involved in embryogenesis , for instance in heart and limb patterning , neural tube closure and skeletal development ( Eggenschwiler and Anderson , 2000; Eggenschwiler et al . , 2001; Fuller et al . , 2014; Günther et al . , 1994; Hor and Goh , 2018 ) . RAB23 negatively regulates Hh signaling during mouse neural tube development ( Eggenschwiler and Anderson , 2000 ) , and it controls nodal signaling independently of Hh signaling during vertebrate left-right patterning ( Fuller et al . , 2014 ) . RAB23 is involved in trafficking D1-type dopaminergic receptors ( Leaf and Von Zastrow , 2015 ) and kinesin-2 motor protein Kif-17 ( Lim and Tang , 2015 ) to the primary cilium . However , direct ciliary function of RAB23 is unclear as cilium length and function are reported normal in Rab23-/- mice ( Evans et al . , 2003; Fuller et al . , 2014 ) . Multiple mutations in RAB23 have been reported in patients with the autosomal recessive Carpenter syndrome; the majority of mutations result in nonsense-mediated decay while others include missense and in-frame deletions ( Jenkins et al . , 2011 ) . Carpenter syndrome ( CS , MIM #201000 ) or Acrocephalopolysyndactyly type II is a multi-organ developmental disorder with polysyndactyly , congenital heart defects , mental retardation , obesity and craniosynostosis as central features ( Carpenter , 1909; Jenkins et al . , 2007 ) . Craniosynostosis is the premature fusion of one or more craniofacial sutures that results in major disruption of face and skull growth . Mesenchymal cells in the center of the suture must be kept in an undifferentiated state to maintain suture patency , while progenitor cells at the osteogenic fronts proliferate and differentiate to facilitate bone growth . Suture biogenesis is dependent on the correct patterning of the skeletal elements , as well as the regulation of the mesenchymal stem cell niche , osteogenic condensation formation , osteoprogenitor proliferation and differentiation ( Rice and Rice , 2008; Twigg and Wilkie , 2015 ) . These developmental processes are regulated to permit coordinated craniofacial growth , without the fusion of the neighboring bones and consequent cessation of growth . Mutations in FGFRs are a major cause of syndromic craniosynostosis , with many of the mutations conferring a gain of function ( Hajihosseini , 2008; Wilkie et al . , 2017 ) . FGFRs isoforms show a distinct FGF ligand binding affinity and biological functions ( Ornitz and Itoh , 2015 ) . However , studies have shown that in an Apert syndrome ( FGFR2 mutation ) mouse model isoform FGFR2c loses ligand specificity , and is able to bind with cognate FGF10 ( Ibrahimi et al . , 2001; Ibrahimi et al . , 2004; Johnson and Wilkie , 2011; Yu et al . , 2000 ) . Interestingly , genetic knockdown of Fgf10 in this mouse model could rescue the premature fusion ( Hajihosseini et al . , 2009 ) . FGF signaling pathway members have not been linked to RAB23-mediated trafficking . However , study suggests that RAB23 resides in the plasma membrane and proposed to be involved in endocytosis ( Evans et al . , 2003 ) . In this context , RAB23 might have a direct role in growth factor receptor recycling and turnover , and therefore regulate the availability of the FGF receptors at the cell surface ( Langemeyer et al . , 2018; Zerial and McBride , 2001 ) . Similar to Carpenter syndrome , several craniosynostosis syndromes caused by mutations in FGFRs are characterized by patients exhibiting syndactyly and occasionally polysyndactyly ( Goos and Mathijssen , 2019; Mantilla-Capacho et al . , 2005 ) . Also , mutations in several hedgehog ( Hh ) pathway members cause polydactyly ( Malik , 2014; Ullah et al . , 2019 ) . Notably , Greig cephalopolysyndactyly syndrome ( MIM # 175700 ) is caused by haploinsufficiency of the Hh signaling negative regulator , GLI3 ( Vortkamp et al . , 1991 ) . In addition to the polysyndactyly , some patients with Greig cephalopolysyndactyly syndrome exhibit craniosynostosis , and the mouse model for Greig cephalopolysyndactyly syndrome ( Gli3Xt-J/Xt-J ) shows complete phenotypic penetrance for both craniosynostosis and polydactyly ( Hurst et al . , 2011; Rice et al . , 2010 ) . Also , a recurrent mosaic mutation in the hedgehog receptor Smoothened ( SMO ) causes craniosynostosis and polysyndactyly ( Twigg et al . , 2016 ) . These overlapping skeletal phenotypes caused by mutations in FGFRs , Hh regulators and RAB23 are suggestive of common etiological mechanisms . FGF and Hh signaling have well-defined roles during intramembranous osteogenesis . FGF signaling regulates many stages including mesenchymal condensation formation , osteoprogenitor proliferation and differentiation and activation of the osteogenic transcription factor RUNX2 ( Debiais et al . , 1998; Kim et al . , 1998; Ornitz and Itoh , 2015; Yoon et al . , 2014 ) . IHH positively regulates osteoprogenitor recruitment to the osteogenic front and GLI transcription factors regulate stem cell maintenance and osteoprogenitor proliferation ( Lenton et al . , 2011; Rice et al . , 2010; Veistinen et al . , 2012; Zhao et al . , 2015 ) . Interestingly , RAB23 regulates GLI1 in a Su ( Fu ) -dependent manner ( Chi et al . , 2012 ) and GLI1-positive cells have been identified in the suture as the main source of mesenchymal stem cells that plays crucial role in suture patency ( Zhao et al . , 2015 ) . The aim of this study was to determine the role of RAB23 during intramembranous bone development . Previously , it has not been possible to study skeletal development in RAB23 deficient mice due to their early lethality . Here , we generated RAB23 deficient ( Rab23-/- ) mice that survived until embryonic day ( E ) 18 . 5 , and this allowed us to investigate how RAB23 regulates bone formation . Similar to Carpenter syndrome patients , RAB23 deficient mice exhibited polysyndactyly and multiple craniosynostoses . We show that disruption of Rab23 leads to an upregulation of Pitx2 , Fgf10 and Fgfr1b expression , decreased p38 and enhanced pERK1/2-RUNX2 signaling along with elevated osteoprogenitor proliferation . In addition , Hh signaling was amplified with increased expression of GLI1 . During in vitro culture , inhibition of elevated pERK1/2 normalized osteoprogenitor proliferation , corrected the aberrant GLI1 and RUNX2 expressions , and rescued the lambdoid suture fusion . Our results suggest a novel role for RAB23 as an upstream regulator of both FGF10-pERK1/2 and Hh-GLI1 , and the additional regulation of GLI1 by pERK1/2 , to coordinate the initiation of osteogenesis .
Rab23 open brain 2 ( opb2 ) homozygous mutant mice did not produce RAB23 protein in primary cells , analyzed by western blot ( Figure 1—figure supplement 1 ) and will therefore be referred to as Rab23-/- mice . Rab23-/- C3 Heb/FeJ mice , generated through an ENU screen , survive until E12 . 5 rendering full analysis of organogenesis impossible ( Eggenschwiler et al . , 2001; Kasarskis et al . , 1998 ) . To obviate the gestation lethality before skeletal development , we backcrossed Rab23-/- C3 Heb/FeJ mice onto the C57Bl/6 strain . After six generations , the survival of the Rab23-/- homozygote embryos were prolonged to E18 . 5 , and this allowed us to study skeletal development of these mice . Rab23-/- mice at embryonic day E18 . 5 exhibited craniosynostosis in the coronal , parietal-temporal , fronto-nasal and in the lambdoid sutures when compared to their wild type ( Wt ) littermates ( Figure 1A–E ) . The prevalence of premature suture fusion in Rab23-/- mice represented as percentage and only parieto-temporal sutures showed bi-lateral suture fusion ( 100% ) in all the samples ( Figure 1F ) . Along with craniosynostosis , Rab23-/- mice showed skeletal patterning defects in the forelimbs and hindlimbs . Rab23-/- mice showed pre-axial polydactyly ( seven digits ) of the forelimb and preaxial polysyndactyly ( seven digits ) of the hindlimbs ( Figure 1G ) . Polydactyly and craniosynostosis were observed ( 100% ) in all Rab23-/- samples examined . At E18 . 5 , 5% of Rab23-/- lambdoid sutures exhibited unilateral suture fusion ( Figure 1F ) . Compared to Wt samples ( Figure 1I ) , unfused Rab23-/- lambdoid sutures showed abnormal bony protrusions from parietal bones toward interparietal bone ( Figure 1J ) or ectopic bones in the lambdoid suture ( Figure 1K ) . At this stage , Rab23-/- lambdoid sutures were narrower than Wt lambdoid sutures ( Figure 1L ) . As 95% of Rab23-/- lambdoid sutures were consistently patent at E18 . 5 they were chosen as a model to study the role of RAB23 in osteogenesis . Rab23-/- mice die neonatally , therefore , analysis of Rab23-/- lambdoid suture beyond the E18 . 5 stage was assessed by in vitro calvarial explant culture ( Rice et al . , 2003b; Figure 1M ) . Wt and patent Rab23-/- lambdoid sutures harvested for culture at E18 . 5 , Rab23-/- lambdoid sutures fused predictably after 3 days of culture ( Figure 1N , O ) . Rab23 mRNA expression was analyzed in whole calvaria and tissue sections by in situ hybridization and protein expression by immunohistochemical staining and western blotting . Rab23 was expressed in suture mesenchyme with the strongest signal in interfrontal , sagittal and coronal sutures and parietal bone side of the lambdoid suture ( Figure 1P–a ) . Analysis of tissue sections revealed Rab23 expression in the osteogenic fronts of the calvarial sutures and throughout the sutural mesenchyme ( Figure 1P–b ) . RAB23 was expressed in the lambdoid sutural osteogenic fronts and in the underlying cartilage . Co-expression with RUNX2 confirmed the cells in the osteogenic front to be osteoprogenitor or osteoblast ( Figure 1P–c ) . The Rab23 expression pattern was consistent with the phenotype of synostosed calvarial sutures observed in the Rab23-/- mice ( Figure 1B , C and E ) . Western blotting of proteins extracted from E15 . 5 Wt calvarial frontal , sagittal and lambdoid sutures along with their osteogenic fronts confirmed the presence of RAB23 protein in the suture ( Figure 1P–d ) . To investigate differentially expressed genes between Wt and Rab23-/- calvaria and to find candidate genes responsible for the craniosynostosis in the RAB23-deficient mice , we performed a microarray-based gene expression analysis of whole Wt and Rab23-/- calvaria samples excluding the skin and underlying brain at E15 . 5 ( Figure 2A ) . Our analysis revealed 223 significantly differentially expressed genes between the Wt and Rab23-/- calvaria ( t-test , p<0 . 05 ) ( Figure 2B , Supplementary file 1 , MIAME-compliant data has been deposited in GEO database as source data , GEO accession GSE140884 ) . Among these genes , 115 genes were upregulated ( Figure 2B , red ) and 108 genes were downregulated ( Figure 2B , green ) . Fgf10 was found to be overexpressed in the microarray in Rab23-/- calvaria ( Figure 2C ) . Fgf10 was selected as a candidate for further analysis as it has previously been shown to be expressed early in calvarial development and has been implicated in craniosynostosis pathogenesis ( Hajihosseini et al . , 2009; Veistinen et al . , 2009 ) . Fgf10 expression was elevated in whole calvarial mesenchyme , lambdoid sutural mesenchymal tissue and in calvaria derived ( CD ) mesenchymal cells at E15 . 5 as assessed by RT-qPCR ( Figure 2D ) . Our microarray analysis also showed that the transcription factor Pitx2 was elevated in Rab23-/- calvaria ( Figure 2C ) . Pitx2 is a well-documented upstream regulator of Fgf10 and may act in a reciprocal regulatory loop with FGF10 during early organogenesis ( Al Alam et al . , 2012 ) . We found that Pitx2 was overexpressed both in the Rab23-/- calvaria and in Rab23-/- lambdoid sutures ( Figure 2E ) . Pitx2 overexpression was further observed in Rab23-/- calvaria by whole-mount in situ hybridization in and surrounding the interparietal bone ( Figure 2F ) . We assessed the expression of FGF10-specific receptors Fgfr1b , Fgfr2b , also Fgfr2c in Wt and Rab23-/- lambdoid sutural mesenchymal tissue . Our analysis showed elevated Fgfr1b expression and a non-significant upregulation of Fgfr2b in Rab23-/- samples ( Figure 2G ) . FGFR1 protein levels were found to be elevated in Rab23-/- lambdoid sutures compared to Wt samples when analyzed by western blotting ( Figure 2H , I ) and by immunohistochemical staining ( Figure 2—figure supplement 1 ) . The expression of Fgfr2c was unchanged ( Figure 2G ) . To test whether the upregulation of Fgfr1b and Fgfr2b expression was due to the high levels of FGF10 ligand present in the Rab23-/- lambdoid suture , we isolated E15 . 5 wild-type calvaria derived ( CD ) mesenchymal cells and treated them with exogenous FGF10 ( 250 ng/ml ) for 3 hr . Fgfr1b and Fgfr2b expression levels were then assessed by RT-qPCR . We found a significant ( p=0 . 05 ) upregulation of Fgfr1b expression and a slight upregulation of Fgfr2b expression in the cultured CD mesenchymal cells after exogenous FGF10 stimulation , indicating FGFR1b as a target of FGF10 in the calvarial mesenchyme ( Figure 2J ) . FGF signaling has been shown to activate MAPK signaling pathway subtypes pERK and p38 in suture morphogenesis and osteoblast differentiation ( Kyono et al . , 2012; Pfaff et al . , 2016; Figure 3A ) . In Rab23-/- lambdoid suture , we found decreased p38 and elevated pERK1/2 and RUNX2 expressions compare to Wt samples ( Figure 3B–G ) . To test whether the upregulation of RUNX2 expression was due to the high levels of FGF10 present in the Rab23-/- lambdoid suture , we isolated E15 . 5 Rab23-/- calvaria derived ( CD ) mesenchymal cells and treated them with exogenous FGF10 ( 500 ng/ml ) for 2 and 4 hr . Runx2 expression levels were then assessed by RT-qPCR . We found a significant ( p<0 . 05 ) upregulation of Runx2 expression at both time points compare to untreated samples , indicating that RUNX2 as a target of FGF10 in the calvarial mesenchyme ( Figure 3H ) . Since ERK1/2 signaling regulates Hh and GLI expression , and RAB23 is a known negative regulator of Hh signaling ( Eggenschwiler et al . , 2001 ) , we further assayed Hh , Gli1 , Gli2 , and Gli3 transcripts extracted from Wt and Rab23-/- lambdoid sutural mesenchyme at E15 . 5 . We detected a dramatic increase in the expression of Hh ( Figure 3I ) and Gli1 ( Figure 3J ) , a sutural stem cell marker ( Zhao et al . , 2015 ) . Although Gli3 expression is not changed , a functional change in the ratio of GLI3FL to GLI3R may still possible ( Veistinen et al . , 2017 ) . As TGFβ-superfamily members have also been shown to regulate suture patency ( Kim et al . , 1998; Komatsu et al . , 2013; Pan et al . , 2017 ) , we assayed both pSMAD1/5/8 and pSMAD2/3 expressions but found no difference between Wt and mutant samples ( Figure 3—figure supplement 1 ) . As FGF10-pERK1/2 signaling and RUNX2 are known to regulate suture osteoprogenitor proliferation ( Kim et al . , 1998; Qin et al . , 2019 ) , we assessed co-expression of EdU and RUNX2 in the WT and Rab23-/- lambdoid suture . EdU and RUNX2 co-localized in the osteogenic fronts indicating that the proliferating cells are osteoprogenitors ( Figure 4A , B ) . We analyzed the proliferation in Wt and Rab23-/- lambdoid sutures ( Figure 4C–H ) . EdU pulsed Rab23-/- samples showed more proliferating cells in the osteogenic fronts compared to Wt ( Figure 4C–H ) . In addition , increased level of cell proliferation was observed in Rab23-/- derived mouse embryonic fibroblast ( MEF ) cells ( Figure 4I ) . Rab23-/- calvaria derived cells also had an elevated number of total cells at different passages ( P1 to P3 ) when compared to Wt calvaria derived cells ( Figure 4J ) . Increased proliferation in the sutures can result in a larger pool of osteoprogenitor cells and enhanced osteogenesis ( Lana-Elola et al . , 2007; Rice et al . , 2003b ) . Taken together , this data shows that RAB23 is needed to repress proliferation in undifferentiated mesenchymal cells . Our data suggest that Rab23-/- lambdoid suture undergoes fusion through aberrant FGF signaling , specifically by activating pERK1/2 signaling , RUNX2 and enhanced cell proliferation of osteoprogenitor cells . To test this , we aimed to normalize the Rab23-/- lambdoid suture by inhibiting the activation of MEK1/2 , a downstream component of the MAPK-ERK1/2 signaling pathway by using MEK1/2 inhibitor U0126 . We cultured E18 . 5 Wt and Rab23-/- whole calvaria in osteogenic growth medium up to 72 hr . To confirm that lambdoid sutures were patent at time point zero , the vital bone dye calcein green was used to detect the bone edges . Calcein green was administered to pregnant females 24 hr before samples were taken . Half of the Rab23-/- calvaria were treated with U0126 and half of the Rab23-/- calvaria samples were untreated ( Figure 5A , B ) . None of the untreated Rab23-/- ( n = 0/12 ) and 84 . 34% of U0126 treated Rab23-/- ( n = 11/12 ) lambdoid sutures were rescued ( Figure 5A , B ) . To confirm whether bones were fused or not , Wt , U0126 treated and untreated Rab23-/- samples were further analyzed in tissue sections and by µ-CT ( Figure 5A–C , Figure 5—figure supplement 1 and Figure 5—Videos 1–3 ) . None of the Wt lambdoid sutures were fused ( n = 0/12 ) . The same experiment ( n = 4 in each genotype ) was carried out with E17 . 5 samples and cultured for 4 days . Untreated Rab23-/- lambdoid sutures underwent suture fusion by day 4 , while U0126-treated Rab23-/- lambdoid sutures remained patent during the culture period ( Figure 5—figure supplement 2 ) . The effect of U0126 on sutural cells was also analyzed by cell death assay and no effect was observed in U0126-treated lambdoid sutural cells ( Figure 5—figure supplement 3 ) . U0126 treatment not only prevented suture fusion but it also normalized the level of osteoprogenitor cell proliferation ( Figure 5D–G ) . EdU incorporation revealed that U0126 treated Rab23-/- lambdoid sutures ( n = 8 ) exhibited reduced proliferation when compared to Rab23-/- controls ( n = 8 ) ( Figure 5D–G ) and normalized it to Wt levels ( n = 8 ) after 24 hr of culture ( Figure 5D , E ) . The proliferation in all genotypes decreased over time ( Figure 5D–G ) . This indicates that RAB23 regulates suture patency through a MAPK-MEK-ERK-driven mechanism . pERK1/2 regulates RUNX2 and GLI1 expression in calvaria derived mesenchymal cells . We analyzed the effect of the MEK1/2 inhibitor U0126 , cyclopamine and the combined action of cyclopamine and U0126 on pERK1/2 , RUNX2 and GLI1 expression levels in Rab23-/- CD cells at 6 , 24 and 48 hr of culture ( Figure 6 ) . After 6 hr of culture , the relative pERK1/2 levels were significantly reduced in U0126-treated Rab23-/- cells compare to untreated Rab23-/- cells . The downregulation of pERK1/2 continued in U0126-treated Rab23-/- cells at 24 and 48 hr of culture compare to the untreated Rab23-/- cells ( Figure 6A , B ) . Whereas , RUNX2 expression significantly reduced only after 48 hr of U0126-treated Rab23-/- cells ( Figure 6A , C ) . U0126-treated Rab23-/- cells showed gradual reduction of GLI1 expressions at 6 and 24 hr; however , it showed drastic reduction at 48 hr compared to untreated Rab23-/- cells ( Figure 6A , D ) . Further immunohistochemical expression of GLI1 was also found reduced in the U0126 treated Rab23-/- lambdoid sutures at 24 and 48 hr compared to untreated Rab23-/- lambdoid sutures ( Figure 6—figure supplement 1 ) . The effect of cyclopamine and combined action of cyclopamine and U0126 on these cells showed that cyclopamine has no effect on pERK1/2 expression and can reduce RUNX2 expression only after prolonged exposure ( 48 hr ) ( Figure 6E-H ) . However , the combined action of cyclopamine and U0126 caused a rapid downregulation of RUNX2 ( 24 hr ) ( Figure 6E-H ) . These findings collectively indicate that pERK1/2 regulates RUNX2 and GLI1 expressions . Whereas , RUNX2 is a predominant downstream target of pERK1/2 and a weak target of GLI1 .
The calvarial suture allows us to study many developmental processes that regulate osteogenesis and skull morphogenesis . Normal development is ensured by the correct temporal and spatial coordination of skeletal patterning , mesenchymal stem cell niche regulation , condensation formation , osteoprogenitor expansion , and osteoblast differentiation and function . In this study , we generated Rab23-deficient mice that survive into late gestation and they exhibit multiple suture craniosynostosis . We show that RAB23 regulates the sutural stem cell niche marker GLI1 , represses Hh , and inhibits osteoprogenitor expansion . RAB23 also represses FGF10-driven pERK1/2 signaling which has the multiple effect of directly regulating osteoprogenitor development , RUNX2-mediated differentiation and of repressing GLI1 . Therefore , RAB23 coordinates suture biogenesis by regulating FGF-ERK1/2-RUNX2 activity , as well as the activation of GLI1 . In an attempt to study complex developmental events , we have utilized both primary cells and organ culture approaches . However , one can also take a more cell biological approach using immortalized cells remote from the external factors and by using this standardized approach it may be easier to understand the effects of a specific intervention . This will be help deciphering the role of RAB23 , specifically during endocytosis . FGF10 signaling regulates early osteogenesis: it is expressed in the osteogenic condensation , and is implicated in the pathogenesis of several craniosynostotic models ( Twigg and Wilkie , 2015; Veistinen et al . , 2009 ) . Here , we show that Fgf10 , its receptor FGFR1b and the FGF activator Pitx2 , are all upregulated in Rab23-deficient calvaria . It is surprising that the FGFR1b splice form , which is normally expressed in epithelia , is upregulated in the mesenchymal suture . However , this is not without precedent . While FGFRc isoforms are predominantly expresses in the mesenchyme and b isoforms in the epithelium , their expression is not mutually exclusive and FGFR1b is known to be expressed by late embryonic murine lung mesenchymal cells and also in the zebrafish in the sutural mesenchyme ( Al Alam et al . , 2015; Rice et al . , 2003b; Rice et al . , 2004; Topczewska et al . , 2016 ) . Under pathological/force conditions , mis-expression of splice forms can occur . Occasionally , Apert craniosynostosis syndrome is caused by heterozygous Alu-insertions or large deletions in the FGFR2c domain . These rare mutations induce abnormal expression of the FGFR2b splice form alongside FGFR2c splice form in mesenchymal cells . This permits cells to respond to both b- and c-activating FGF ligands including FGF10 ( Bochukova et al . , 2009; Oldridge et al . , 1999 ) . Analyzing the MAPK pathway in Rab23-/- lambdoid sutures we show that p38 signaling is decreased and pERK1/2 increased . FGFR activation of pERK1/2 is important for several stages of osteoblastogenesis , including osteoprogenitor proliferation , and in the induction and stabilization of RUNX2 ( Choi et al . , 2008; Yoon et al . , 2014 ) . Augmentation of pERK1/2 signaling either through overactivation of FGFRs , downregulation of its inhibitors , or downregulation of ERF that regulates the export of pERK from the nucleus , all result in craniosynostosis ( Lee et al . , 2018a; Lee et al . , 2018b; Timberlake et al . , 2017; Twigg et al . , 2013 ) . Although RUNX2 is a substrate for both p38 and pERK1/2 , pERK1/2 can activate RUNX2 on average six times more efficiently than p38 ( Ge et al . , 2012 ) . In addition , ERK1/2 binding to RUNX2 through MAPK D site has greater affinity than p38 binding and more sensitive to osteoblast differentiation during calvaria explant or primary osteoblast culture ( Franceschi and Ge , 2017; Ge et al . , 2012 ) . The importance of pERK1/2 regulation of RUNX2 during osteogenesis is shown in this study by the reversal of the craniosynostotic phenotype in Rab23-/- mice . p38 has been implicated in the skull phenotypes of craniosynostotic mice where it may regulate the development and growth of cartilage elements ( Wang et al . , 2010; Wang et al . , 2012 ) . Opposing roles of MAPK pathway subtypes p38 and pERK1/2 in cartilage development are well documented ( Ma et al . , 2019; Oh et al . , 2000; Stanton et al . , 2003 ) . Commonality of etiological mechanisms is suggested by the overlapping skeletal phenotypes exhibited in Carpenter syndrome and Grieg cephalopolysyndactyly syndrome patients as well as their mouse disease models , Gli3Xt−J/Xt−J ( Rice et al . , 2010 ) and Rab23-/- ( this study ) . Specifically , postaxial polydactyly in the forelimb , preaxial polydactyly in the hindlimb and lambdoid and interfrontal suture craniosynostosis ( Eggenschwiler et al . , 2001; Jenkins et al . , 2007; Kalff-Suske et al . , 1999; Vortkamp et al . , 1991 ) . Collectively suggest that craniosynostosis and polysyndactyly are overlapping phenotypes of aberrant FGF and Hh signaling . Aberrant osteogenesis and suture obliteration in the Gli3Xt−J/Xt−J mouse is caused by concomitant derepression of GLI3 and augmentation of IHH which activates the osteogenic master regulatory transcription factor RUNX2 ( Veistinen et al . , 2017 ) . Analysis of early Rab23-/- embryos suggests that RAB23 also has a role in promoting the production of GLI3 repressor ( Eggenschwiler et al . , 2006 ) . In both Gli3Xt−J/Xt−J and Rab23-/- calvaria elevated Hh signaling results in an upregulation of the transcription factor Gli1 , and presumably the mesenchymal stem niche which it labels . Previously , we have shown that FGF10 upregulates Hh and its receptor Patch1 during facial development ( Rice et al . , 2004 ) . Here , we show FGF10 upregulates Runx2 . Our study revealed that inhibition of pERK1/2 prevented Rab23-/- lambdoid suture fusion by decreasing osteoprogenitor proliferation and GLI1 expression ( Figures 5 and 6 ) . We further showed that combined and individual inhibition of GLI1 and pERK1/2 in Rab23-/- calvarial cells downregulated RUNX2 and GLI1 ( Figure 6 ) and that this effect was exaggerated in Rab23-/- cells . Phenotypic analyses of compound Rab23;Smoothened early embryonic mice shows that RAB23 can regulate GLI independently of Smoothened ( Eggenschwiler et al . , 2006 ) , and the regulation of GLI1 by pERK1/2 suggests a mechanism how this might occur ( Figure 7 ) . Non-canonical regulation GLI1 through tyrosine kinase activity has been documented during carcinogenesis , with activation of GLI1 promoting stemness through an increase in transcriptional activity , nuclear localization or protein stability ( Pietrobono et al . , 2019 ) . However , the non-canonical repression of GLI1 has not been well described in a developmental context . We show that RAB23 represses both FGF and Hh/GLI signaling ( Figure 7 ) . RAB23 coordinates suture morphogenesis and controls suture patency through three mechanisms: FGF10-pERK1/2 signaling , the repression of Hh targets through a canonical pathway , and through the repression of GLI1 by pERK1/2 . GLI1 contributes in the regulation of the mesenchymal stem cell niche . FGF10-pERK1/2 and also Hh-GLI1 signaling regulate osteoprogenitor cell proliferation .
Animal experiments were approved by the University of Helsinki , Helsinki University Hospital and the Southern Finland Council Animal Welfare and Ethics committees . Permit ESAVI/11956/04 . 10 . 07/2017 ( external ) and KEK17-008 ( internal ) . Rab23opb2/opb2 ( Rab23-/- ) mice were originally in C3Heb/Fej background were crossed into C57Bl/6J mouse strain ( Charles River ) for six or more generations with speed congenic method . The animals for breeding were selectively based on highest amount of C57 SNP markers ( Illumina mouse chip array ) to speed up strain change . After four generations more than 89% of SNPs were on C57 . The genotype of all mouse and embryos were verified with PCR-based genotyping as previously described ( Eggenschwiler and Anderson , 2000; Kasarskis et al . , 1998 ) . Analysis of the RNA expression was performed using Illuminan Mouse WG-6v2 microchips . RNA was isolated from E15 calvaria from Rab23-/- embryos and Wt littermates using Machrey-Nagel Nucleospin RNA II isolation kit . Labeling and chip run were performed by the Technology Centre , Institute for Molecular Medicine Finland ( FIMM ) , University of Helsinki . The data analysis and normalization was performed using Chipster 3 . 16 . 0 . Fold change of genes is calculated by arithmetic mean in linear scale and shown in the volcano plot . Fold change >1 ( up-regulated gene ) , fold change < 1 ( down-regulated gene ) . The microarray dataset has been deposited in MIAME-compliant GEO public database as source data . Accession number: GEO accession GSE140884 . For quantitative PCR , RNA was isolated individually from whole calvaria and from lambdoid suture of Rab23-/- and Wt embryos at E15 . 5 . 1 µg of RNA from calvarial ( n = 3+3 ) and 500 ng of RNA from lambdoid suture ( n = 8+8 ) were reverse-transcribed into cDNA using random hexamer primer and RevertAid reverse transcriptase ( Thermo scientific ) . 2 µl of diluted cDNA and Brilliant III ultra-fast SYBR-green qPCR master mix with QuantiTect Primers ( Qiagen ) . We performed the experiments as previously described ( Sanz-Navarro et al . , 2019 ) . In single-gene in situ RNAscope transcripts appear in red . A customized 20ZZ probe named Mm-Rab23 targeting 45–1330 of NM_008999 . 4 was purchased from Advanced Cell Diagnostics to perform Rab23 expression analysis on mouse calvarial suture . Images were obtained using a bright field microscope ( BX61 , Olympus ) . In situ hybridizations on whole embryonic samples was performed using digoxigenin-UTP labeled Rab23 probe and Pitx2 probe according to the protocol and modification described ( Kim et al . , 1998 ) . Immunohistochemistry on lambdoid sutural samples was performed using primary anti-RAB23 , GLI1 , RUNX2 and FGFR1 antibody . After incubation with HRP conjugated or alexa fluor conjugated secondary antibody Enzmet detection kit was used for GLI1 and FGFR1 detection and alexa fluor conjugated 488 , 546 secondary antibody was used to detect RAB23 and RUNX2 signals . The samples were permeabilized by triton X-100 and antigen retrieval was performed by citrate buffer . All the other procedure was followed according to the protocol described ( Veistinen et al . , 2017 ) . WT and Rab23-/- embryos were taken out at E15 . 5 embryonic day . We and others have previously described the procedure of calvarial primary cells isolation ( Rodan and Noda , 1991; Veistinen et al . , 2017 ) . Cells were cultured in DMEM containing high glucose and supplemented with 10% FBS , glutamine , penicillin and streptomycin , maintained at 37°C with 5% CO2 . After maintaining the isolated cells in growth medium next passage is always used for experiments . E15 . 5 Wt calvaria derived mesenchymal cells were treated with exogenous FGF10 ( 250 ng/ml ) for 3 hr . Fgfr1b and Fgfr2b mRNA expression were then assessed by RT-qPCR . E15 . 5 Rab23-/- calvaria derived mesenchymal cells were treated with exogenous FGF10 ( 500 ng/ml ) for 2 and 4 hr . Runx2 mRNA expression were then assessed by RT-qPCR . RIPA buffer containing 1% SDS together with protease inhibitor and phosphatase inhibitor were used to lyse the lambdoid sutural tissue on ice . After brief sonication ( 10 s , twice ) , tissue lysates were centrifuged at full speed for 10 min to collect the protein supernatant . By using BCA protein assay kit , protein concentrations were measured for immunoblotting analysis . Equal amount of proteins from wild type and RAB23 deficient cells were subjected to prepare to separate on SDS-PAGE under reduced gel electrophoresis ( 4–20% Mini-PROTEAN TGX Gels , Bio-Rad ) . After transferring to nitrocellulose membrane , membranes were blocked by odyssey blocking buffer at room temperature for 3 hr . Membranes were then incubated with corresponding primary anti-RAB23 , anti-p38 , anti-phospho-p38 , anti-ERK , anti-pERK , anti-RUNX2 , anti-pSMAD1/5/8 , anti-GLI1 , anti-FGFR1 and anti-Tubulin antibodies and kept overnight at 4°C . Fluorophore-conjugated corresponding IRdye goat anti-rabbit 680LT or CW800 or anti-mouse 800CW secondary antibodies were used at room temperature for 1 hr . α-Tubulin was used to normalize protein expressions . In vitro inhibition of pERK1/2 in Rab23-/- calvaria derived primary cells ( E15 . 5 ) was performed by adding 5 µM of U0126 and 10 µM of cyclopamine for 6 , 24 and 48 hr . Membranes were scanned using an Odyssey infrared imaging system ( Odyssey; LI-COR Biosciences , model 9120 ) . Band intensity was determined using ImageJ ( NIH ) software . After removing skin and underneath brain calvaria from E18 . 5 Wt and Rab23-/- were cultured in an in vitro explant culture system . The calvaria was kept on top of the filter laid on metallic grid . The calvaria sample was supplemented with DMEM containing 10% FBS , dexamethasone , ascorbic acid and β-glyceraldehyde . Rab23-/- calvaria received 5 µM of U0126 treatment or DMSO as control . After 48 hr fresh medium and inhibitors were added . After 48 or 72 hr , samples were fixed with 4% PFA for subsequent analysis . For immediate calvarial bone and suture recognition vital bone dye calcein green was injected to mother mouse 1 day before the E17 . 5 or E18 . 5 embryo collection . Subsequent imaging in the culture was performed after alizarin red incorporation in the medium . Images were taken by Olympus SZX12 fluorescent microscope . The Alcian blue-Alizarin red staining of E18 . 5 specimens was performed as previously described ( Rice et al . , 2003a ) . For analysis of calvarial bone and sutures images were captured using Analysis software ( Soft Imaging System ) and Olympus BX41 microscope and analyzed in Adobe Photoshop CS4 . Mouse embryonic fibroblasts ( MEFs ) were isolated from E13 . 5 embryos ( noon of the vaginal plug detection day was considered E0 . 5 ) . Briefly , embryos were collected to sterile PBS . The head , liver and spleen were removed and used for histology , RNA isolation or for genotyping . The remaining tissue was cut into smaller pieces and dissociated with gentle pipetting in 0 . 25% Trypsin-EDTA solution . After 10 min incubation , any larger tissue pieces were discarded and remaining cell suspension was plated with rich MEF medium into two T25 flasks per embryo . The medium was changed daily until first passaging at confluency . The growth medium was DMEM , High Glucose , GlutaMAX ( Gibco ) supplemented with 10% FBS , 1x non-essential amino acids ( Gibco ) , 20 mM Hepes pH 7 . 3 and 1x penicillin-streptomycin . The cells were subcultured when 90% confluent to 750 , 000 live cells/T75 flask and only passages 1–6 ( active division ) were used for EdU pulsed proliferation analyses . The cell density and viability was assessed with Trypan Blue and Countess automated cell counter ( Invitrogen ) . Cell proliferation was assessed by prior EdU incorporation followed by EdU click reaction . The MEF cell lines were pulsed for 1 hr with 10 µM EdU ( Molecular probes , Invitrogen ) in DMSO dissolved to pre-warmed medium before distribution to cells for 1 hr EdU pulse and fixed with 4% paraformaldehyde . The EdU was detected with Click-iT EdU imaging kit , Alexa fluor 594 ( Molecular probes , Invitrogen ) according to manufacturer's instruction . The number of EdU positive cells was counted from three separate experiments from a minimum five different locations per cell line and compared to total cell number determined with Hoechst 33342 staining of nuclei . The mice were pulsed with 0 . 05 mg/g ( i . p . ) EdU in PBS 2 hr collection of tissue . After sacrificing E17 . 5 embryonic lambdoid sutures were collected and fixed o/n with 4% paraformaldehyde , and processed for paraffin sections . Paraffin-embedded lambdoid sutures were sectioned 7 µm size . The sections were deparaffinized and stained with same Click-iT kit . EdU click reaction procedure in brief , sectional explants were deparaffinized by xylene and rehydrated by a gradient of ethanol series . After washing with 2 mg/ml glycine , sutural sections were permeabilized with 0 . 5% Triton X-100 . After several washing in PBS , 10 µM EdU cocktail was used for click reaction for 30 min at dark room . After several PBS washing 5 µg/ml Hoechst was used for counter staining . Vectashield mounting medium was used to mount the slides . Slides were then imaged with fluorescence microscope with emission wavelength 615 nm . Cell counting: Cluster of proliferating cells ( red cell ) considered as osteogenic front . The middle of the scale was kept on the tip of each osteogenic front and then rest of the part was flanked towards the suture . The length of the scale is 145 µm and width 61 µm . Cells from skin , dura mater and cartilage were not included in counting . Apoptotic assay on tissue sections from in vitro cultured Wt , control Rab23-/- and U0126 treated Rab23-/- lambdoid suture was performed according to manufacturer protocol ( In situ apoptosis detection kit AB206386 ) . E18 . 5 WT and Rab23-/- whole head and samples from rescue studies were collected and fixed with 4% paraformaldehyde overnight . Samples were dehydrated by gradient ethanol series to 70% ( v/v ) , followed by processed for x-ray microtomography ( µCT ) imaging with Bruker SkyScan1272 ( desktop micro-CT system , Bruker microCT N . V . , Kontich , Belgium ) . Tomography 3D reconstructions were obtained using the program NRecon ( desktop micro-CT system , Bruker microCT N . V . , Kontich , Belgium ) . Paired student’s t-test has been applied to perform the statistics of all the data obtained from measurements , western blotting and gene expressions . Data represented as mean ± SD . p-Value less than 0 . 05 considered as statistical significant .
Mutations in the gene encoding Ras-associated binding protein 23 ( RAB23 ) cause Carpenter Syndrome , which is characterized by multiple developmental abnormalities including polysyndactyly and defects in skull morphogenesis . To understand how RAB23 regulates skull development , we generated Rab23-deficient mice that survive to an age where skeletal development can be studied . Along with polysyndactyly , these mice exhibit premature fusion of multiple sutures resultant from aberrant osteoprogenitor proliferation and elevated osteogenesis in the suture . FGF10-driven FGFR1 signaling is elevated in Rab23-/-sutures with a consequent imbalance in MAPK , Hedgehog signaling and RUNX2 expression . Inhibition of elevated pERK1/2 signaling results in the normalization of osteoprogenitor proliferation with a concomitant reduction of osteogenic gene expression , and prevention of craniosynostosis . Our results suggest a novel role for RAB23 as an upstream negative regulator of both FGFR and canonical Hh-GLI1 signaling , and additionally in the non-canonical regulation of GLI1 through pERK1/2 .
In many animals , the skull is made of several separate bones that are loosely joined during childhood and only fuse into one piece when the animal stops growing . A genetic disease called Carpenter syndrome causes the bones of the skull to fuse early in life , stopping it from growing correctly . Carpenter syndrome is often caused by changes to the gene responsible for making a protein called RAB23 . RAB23 helps move other molecules and cell components between different parts of the cell , and is therefore involved in a number of cellular processes . Previous studies suggest that RAB23 has a role in many parts of the body during development . Yet , it is unclear which cells in the skull depend on RAB23 activity and how this protein is controlled . To answer this question , Hasan et al . grew pieces of developing skull bones that had been taken from mice lacking the RAB23 protein in the laboratory . Examining these samples revealed that RAB23 is active in cells called osteoblasts that add new bone to the edge of each piece of the skull as it grows . Hasan et al . also found that RAB23 regulates two cellular signaling pathways – called the hedgehog pathway and the fibroblast growth factor pathway – that interact with one another and co-ordinate skull development . These findings show how RAB23 controls the growth and fusion of skull bones in developing animals . This could improve our understanding of the role RAB23 plays in other processes during development . It also sheds light on the mechanisms of Carpenter syndrome which may inform new approaches for treating patients .