Possible roles of Runx1 and Sox9 in incipient intramembranous ossification.
We evaluated the detailed expression patterns of Runx1 and Sox9 in various types of bone formation, and determined whether Runx1 expression was affected by Runx2 deficiency and Runx2 expression by Runx1 deficiency. Our results indicate that both Runx1 and Sox9 are intensely expressed in the future osteogenic cell compartment and in cartilage. The pattern of Runx1 and Sox9 expression suggests that both genes could potentially be involved in incipient intramembranous bone formation during craniofacial development.


BACKGROUND
Runx1, a gene essential for hematopoiesis, contains RUNX binding sites in its promoter region, suggesting possible cross-regulation with Runx2 and potential regulatory roles in bone development. On the other hand, Sox9 is essential for chondrogenesis, and haploinsufficiency of Sox9 leads to premature ossification of the skeletal system. In this study, we studied the possible roles of Runx1 and Sox9 in bone development.


METHODS
Runx1, Runx2/Osf2, and Sox9 expression was evaluated by in situ hybridization in the growing craniofacial bones of embryonic day (E)12-16 mice and in the endochondral bone-forming regions of embryonic and postnatal long bones. In addition, we evaluated Runx2/Osf2 expression in the growing face of Runx1 knockout mice at E12.5 and Runx1 expression in Runx2 knockout mice at E14.5.


RESULTS
Runx1 and Sox9 were expressed in cartilage, and the regions of expression expanded to the neighboring Runx2-expressing osteogenic regions. Expression of both Runx1 and Sox9 was markedly downregulated on ossification. Runx1 and Sox9 expression was absent in the regions of endochondral bone formation and in actively modeling or remodeling bone tissues in the long bones as well as in ossified craniofacial bones. Runx2 expression was not affected by gene disruption of Runx1, whereas the expression domains of Runx1 were extended in Runx2(-/-) mice compared with wildtype mice.


CONCLUSIONS
Runx1 and Sox9 are specifically expressed in the osteogenic cell compartments in the craniofacial bones and the bone collar of long bones, and this expression is downregulated on terminal differentiation of osteoblasts. Our results suggest that Runx1 may play a role in incipient intramembranous bone formation.
Yamashiro
Takashi
T
Department of Orthodontics, Graduate School of Medicine and Dentistry, Okayama University, Okayama, Japan. yamat@md.okayama-u.ac.jp
Wang
Xiu-Ping
XP
Li
Zhe
Z
Oya
Shinji
S
Aberg
Thomas
T
Fukunaga
Tomohiro
T
Kamioka
Hiroshi
H
Speck
Nancy A
NA
Takano-Yamamoto
Teruko
T
Thesleff
Irma
I
D000818:Animals; D002356:Cartilage / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000378:metabolism; D050659:Core Binding Factor Alpha 1 Subunit; D050676:Core Binding Factor Alpha 2 Subunit; D050657:Core Binding Factor alpha Subunits; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015536:Down-Regulation; D005147:Facial Bones / Q000196:embryology / Q000378:metabolism; D006609:High Mobility Group Proteins / Q000378:metabolism*; D017403:In Situ Hybridization; D051379:Mice; D018345:Mice, Knockout; D009363:Neoplasm Proteins / Q000172:deficiency; D010006:Osteoblasts / Q000502:physiology; D010012:Osteogenesis / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D055758:SOX9 Transcription Factor; D014157:Transcription Factors / Q000172:deficiency / Q000378:metabolism*; D014457:Ulna / Q000196:embryology / Q000378:metabolism
D015815:Cell Adhesion Molecules; D050659:Core Binding Factor Alpha 1 Subunit; D050676:Core Binding Factor Alpha 2 Subunit; D050657:Core Binding Factor alpha Subunits; D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D009363:Neoplasm Proteins; C082838:Postn protein, mouse; D011518:Proto-Oncogene Proteins; C493726:Runx1 protein, mouse; D055758:SOX9 Transcription Factor; C527933:Sox9 protein, mouse; D014157:Transcription Factors