Role of Gli3 during intramembranous calvarial bone development

The flat bones of the skull, the calvarial bones, develop by intramembranous ossification during which mesenchymal cells first condense and subsequently differentiate into osteoblasts. Sutures separate the calvarial bones and facilitate the synchronized growth of the underlying brain and the calvaria. Hedgehog (Hh) signalling has an indisputable role in craniofacial development as well as during endochondral ossification. Yet, little is known about its function during intramembranous ossification of the calvarial bones. GLI-Kruppel family member 3 (Gli3) is a zinc-finger transcription factor that mediates Hh signalling. In the absence of Hh ligand Gli3 is proteolytically cleaved into a repressor that inhibits transcription of Hh target genes. Mutations in GLI3 cause Greig cephalopolysyndactyly syndrome in humans, in which an infrequent, but significant feature is premature fusion of the metopic suture (interfrontal suture in mice). We have used Gli3 loss-of- function mouse (Gli3Xt-J/Xt-J) as a model to investigate the effects of aberrant Hh signalling during calvarial development. In my thesis I describe how loss of Gli3 causes craniosynostosis of the lambdoid as well as interfrontal sutures in mice. Elevated proliferation and ectopic differentiation of osteoprogenitors underlies this phenomenon. We were able to rescue craniosynostosis in these mice by two mechanisms. Firstly, by elevating fibroblast growth factor (Fgf) signalling in the suture prior to its fusion by imbedding Fgf2 soaked beads in tissue culture. This induced Twist1 expression, which inhibits function of ectopically expressed Runx2. Secondly, craniosynostosis was prevented by genetically reducing Runx2 activity by generating Gli3Xt-J/Xt-J;Runx2+/- mice, which normalized elevated levels of Bmp signalling in the affected sutures. We also put forward a model of how Hh signalling helps to maintain the integrity of bone margins during calvarial development. The repressor isoform of Gli3 inhibits Runx2 activity in the early osteoprogenitor cells. Runx2, on the other hand, activates Ihh expression in the mature osteoblasts, which then induces osteogenesis by inhibiting the function of Gli3 repressor. Our findings indicate that Gli3 and Hh signalling have an important role in mediating the location of osteoblast differentiation and the speed of bone formation in the developing calvaria. Uncovering the cellular and molecular mechanisms that underlie normal calvarial development, as well as pathological processes, is a vital step in developing treatment strategies for patients with craniosynostosis.Kallon litteät peitinluut kehittyvät ns. suoran luutumisen mekanismilla eli intramembranoottisesti: mesenkymaaliset solut muodostavat ensin tiivistymän, jonka jälkeen ne erilaistuvat asteittain luusoluiksi eli osteoblasteiksi. Kasvavia kallon peitinluita erottavat kallon saumat, joissa luiden aktiivinen kasvu jatkuu mahdollistaen alla olevien aivojen samanaikaisen kasvun. Kraniosynostoosi on patologinen tila, jossa yksi tai useampi kallon sauma luutuu ennenaikaisesti. Tällöin kasvu kyseisessä kallon saumassa loppuu, mikä johtaa kallon, silmäkuoppien ja kasvojen epäsymmetriseen kasvuun, sekä neurologisiin oireisiin. Toistaiseksi ainoa hoitokeino on luutuneen sauman kirurginen avaus. Hedgehog (Hh) viestintäreitillä on merkittävä rooli sekä kraniofakiaalisen kehityksen että endokondraalisen luutumisen aikana. Kuitenkin, sen mahdollisesta tehtävästä intramembranoottisten kallon peitinluiden kehityksessä tiedetään varsin vähän. Gli3 on transkriptiotekijä, joka säätelee Hh signalointia. Hh-viestimolekyylin puuttuessa Gli3 pilkotaan proteolyyttisesti lyhyempään repressori-muotoon, mikä estää Hh viestintäreitin kohdegeenien ilmentymistä. Ihmisellä mutaatiot GLI3 geenissä aiheuttavat Greig kefalopolysyndaktylia-oireyhtymän, jonka harvinainen, mutta merkittävä piirre on metooppisen sauman (interfrontaalisauma hiirellä) kraniosynostoosi. Tässä väitöskirjatyössä käytimme mallina Gli3-poistogeenistä hiirtä (Gli3Xt-J/Xt-J) tutkiessamme epänormaalin Hh signaloinnin vaikutusta kallon peitinluiden kehitykseen. Hiirellä Gli3 geenin puutos johti sekä lambda- että interfrontaalisaumojen kraniosynostoosiin. Kraniosynostoosin taustalta paljastui kiihtynyt solujen jakautuminen sekä ektooppinen osteoblastien erilaistuminen. Pystyimme estämään saumojen ennenaikaisen luutumisen Gli3-poistogeenisessä hiiressä kahdella mekanismilla. 1) Lisäsimme fibroblastikasvutekijäperheeseen kuuluvaa Fgf2-proteiinia kudosviljelmässä paikallisesti lambdasaumaan ennen sen luutumista. Fgf2 indusoi saumassa Twist1-geenin ilmentymisen, mikä puolestaan esti ektooppisesti ilmentyvän Runx2:den toiminnan. Runx2 on välttämätön osteoblastien erilaistumiselle. 2) Vähensimme myös Runx2-geenin aktiivisuutta poistamalla Gli3-poistogeenisestä hiirestä yhden kopion Runx2-geeniä (Gli3Xt-J/Xt-J;Runx2+/-), mikä normalisoi lisääntyneen luun morfogeeninen kasvutekijä (Bmp)-viestintäreitin aktiivisuuden affektoituneissa saumoissa. Väitöskirjassani kuvaamme mallin, jossa Hh viestintäreitti säätelee kallon peitinluiden muodostusta kallon saumassa. Gli3:n repressori-muoto ilmentyy hyvin varhaisissa osteoblastien esiasteissa, luun sauman puoleisella reunalla, jossa se rajoittaa näiden solujen erilaistumista estämällä Runx2:den toimintaa. Toisaalta, myöhemmässä osteoblastien erilaistumisvaiheessa Runx2 aktivoi Hh-viestimolekyylin, Indian hedgehogin, ilmentymisen, mikä puolestaan edistää osteoblastien erilaistumista estämällä Gli3-repressorin muodostusta. Tämän väitöskirjan tulokset osoittavat, että Gli3:lla ja Hh viestintäreitillä on merkittävä rooli kallon kehityksen aikana, sekä osteoblastien erilaistumispaikan että erilaistumisnopeuden säätelyssä. Normaalin kallon kehityksen ja patologisten tilanteiden taustalla olevien solu- ja molekyylitason mekanismien selvittäminen on välttämätön askel uusien hoitomuotojen kehityksessä kraniosynostoosipotilaille

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

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

Loss-of-function of Gli3 in mice causes abnormal frontal bone morphology and premature synostosis of the interfrontal suture

Peer reviewe

Noggin null allele mice exhibit a microform of holoprosencephaly

Holoprosencephaly (HPE) is a heterogeneous craniofacial and neural developmental anomaly characterized in its most severe form by the failure of the forebrain to divide. In humans, HPE is associated with disruption of Sonic hedgehog and Nodal signaling pathways, but the role of other signaling pathways has not yet been determined. In this study, we analyzed mice which, due to the lack of the Bmp antagonist Noggin, exhibit elevated Bmp signaling. Noggin−/− mice exhibited a solitary median maxillary incisor that developed from a single dental placode, early midfacial narrowing as well as abnormalities in the developing hyoid bone, pituitary gland and vomeronasal organ. In Noggin−/− mice, the expression domains of Shh, as well as the Shh target genes Ptch1 and Gli1, were reduced in the frontonasal region at key stages of early facial development. Using E10.5 facial cultures, we show that excessive BMP4 results in reduced Fgf8 and Ptch1 expression. These data suggest that increased Bmp signaling in Noggin−/− mice results in downregulation of the hedgehog pathway at a critical stage when the midline craniofacial structures are developing, which leads to a phenotype consistent with a microform of HP

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

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

Inactivation of IL11 Signaling Causes Craniosynostosis, Delayed Tooth Eruption, and Supernumerary Teeth

Craniosynostosis and supernumerary teeth most often occur as isolated developmental anomalies, but they are also separately manifested in several malformation syndromes. Here, we describe a human syndrome featuring craniosynostosis, maxillary hypoplasia, delayed tooth eruption, and supernumerary teeth. We performed homozygosity mapping in three unrelated consanguineous Pakistani families and localized the syndrome to a region in chromosome 9. Mutational analysis of candidate genes in the region revealed that all affected children harbored homozygous missense mutations (c.662C>G [p.Pro221Arg], c.734C>G [p.Ser245Cys], or c.886C>T [p.Arg296Trp]) in IL11RA (encoding interleukin 11 receptor, alpha) on chromosome 9p13.3. In addition, a homozygous nonsense mutation, c.475C>T (p.Gln159X), and a homozygous duplication, c.916_924dup (p.Thr306_Ser308dup), were observed in two north European families. In cell-transfection experiments, the p.Arg296Trp mutation rendered the receptor unable to mediate the IL11 signal, indicating that the mutation causes loss of IL11RA function. We also observed disturbed cranial growth and suture activity in the Il11ra null mutant mice, in which reduced size and remodeling of limb bones has been previously described. We conclude that IL11 signaling is essential for the normal development of craniofacial bones and teeth and that its function is to restrict suture fusion and tooth number. The results open up the possibility of modulation of IL11 signaling for the treatment of craniosynostosis