title: Hedgehog-Gli activators direct osteo-chondrogenic function of bone morphogenetic protein toward osteogenesis in the perichondrium.
abstract: Specification of progenitors into the osteoblast lineage is an essential event for skeletogenesis. During endochondral ossification, cells in the perichondrium give rise to osteoblast precursors. Hedgehog (Hh) and bone morphogenetic protein (BMP) are suggested to regulate the commitment of these cells. However, properties of perichondrial cells and regulatory mechanisms of the specification process are still poorly understood. Here, we investigated the machineries by combining a novel organ culture system and single-cell expression analysis with mouse genetics and biochemical analyses. In a metatarsal organ culture reproducing bone collar formation, activation of BMP signaling enhanced the bone collar formation cooperatively with Hh input, whereas the signaling induced ectopic chondrocyte formation in the perichondrium without Hh input. Similar phenotypes were also observed in compound mutant mice, where signaling activities of Hh and BMP were genetically manipulated. Single-cell quantitative RT-PCR analyses showed heterogeneity of perichondrial cells in terms of natural characteristics and responsiveness to Hh input. In vitro analyses revealed that Hh signaling suppressed BMP-induced chondrogenic differentiation; Gli1 inhibited the expression of Sox5, Sox6, and Sox9 (SRY box-containing gene 9) as well as transactivation by Sox9. Indeed, ectopic expression of chondrocyte maker genes were observed in the perichondrium of metatarsals in Gli1(-/-) fetuses, and the phenotype was more severe in Gli1(-/-);Gli2(-/-) newborns. These data suggest that Hh-Gli activators alter the function of BMP to specify perichondrial cells into osteoblasts; the timing of Hh input and its target populations are critical for BMP function.
authors: Center for Disease Biology and Integrative Medicine, The University of Tokyo, Tokyo 113-0033, Japan; Department of Sensory and Motor System Medicine, The University of Tokyo, Tokyo 113-0033, Japan. Electronic address: hojo@bmw.t.u-tokyo.ac.jp.
authors: Hironori
authors: H
authors: Hojo
authors: Center for Disease Biology and Integrative Medicine, The University of Tokyo, Tokyo 113-0033, Japan. Electronic address: soba@bmw.t.u-tokyo.ac.jp.
authors: Shinsuke
authors: S
authors: Ohba
authors: Hitachi Central Research Laboratory, Tokyo 185-8601, Japan.
authors: Kiyomi
authors: K
authors: Taniguchi
authors: Hitachi Central Research Laboratory, Tokyo 185-8601, Japan.
authors: Masataka
authors: M
authors: Shirai
authors: Center for Disease Biology and Integrative Medicine, The University of Tokyo, Tokyo 113-0033, Japan; Department of Sensory and Motor System Medicine, The University of Tokyo, Tokyo 113-0033, Japan.
authors: Fumiko
authors: F
authors: Yano
authors: Department of Sensory and Motor System Medicine, The University of Tokyo, Tokyo 113-0033, Japan.
authors: Taku
authors: T
authors: Saito
authors: Department of Sensory and Motor System Medicine, The University of Tokyo, Tokyo 113-0033, Japan.
authors: Toshiyuki
authors: T
authors: Ikeda
authors: Center for Disease Biology and Integrative Medicine, The University of Tokyo, Tokyo 113-0033, Japan; Department of Sensory and Motor System Medicine, The University of Tokyo, Tokyo 113-0033, Japan.
authors: Keiji
authors: K
authors: Nakajima
authors: Center for Disease Biology and Integrative Medicine, The University of Tokyo, Tokyo 113-0033, Japan; Department of Sensory and Motor System Medicine, The University of Tokyo, Tokyo 113-0033, Japan.
authors: Yuske
authors: Y
authors: Komiyama
authors: Center for Animal Resources and Development, Kumamoto University, Kumamoto 860-0811, Japan.
authors: Naomi
authors: N
authors: Nakagata
authors: Center for Animal Resources and Development, Kumamoto University, Kumamoto 860-0811, Japan.
authors: Kentaro
authors: K
authors: Suzuki
authors: Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan, Ann Arbor, Michigan 48109.
authors: Yuji
authors: Y
authors: Mishina
authors: Laboratory for Developmental Neurobiology, RIKEN Brain Science Institute, Saitama 351-0198, Japan; Common Resources Group, Okinawa Institute of Science and Technology, Okinawa 904-0412, Japan.
authors: Masahisa
authors: M
authors: Yamada
authors: Department of Bioengineering, The University of Tokyo, Tokyo 113-0033, Japan.
authors: Tomohiro
authors: T
authors: Konno
authors: Department of Sensory and Motor System Medicine, The University of Tokyo, Tokyo 113-0033, Japan.
authors: Tsuyoshi
authors: T
authors: Takato
authors: Department of Sensory and Motor System Medicine, The University of Tokyo, Tokyo 113-0033, Japan.
authors: Hiroshi
authors: H
authors: Kawaguchi
authors: Hitachi Central Research Laboratory, Tokyo 185-8601, Japan.
authors: Hideki
authors: H
authors: Kambara
authors: Center for Disease Biology and Integrative Medicine, The University of Tokyo, Tokyo 113-0033, Japan; Department of Bioengineering, The University of Tokyo, Tokyo 113-0033, Japan.
authors: Ung-Il
authors: UI
authors: Chung
mesh_terms: D000818:Animals; D019485:Bone Morphogenetic Proteins / Q000378:metabolism*; D002454:Cell Differentiation; D019070:Cell Lineage; D019902:Chondrocytes / Q000166:cytology*; D016000:Cluster Analysis; D005786:Gene Expression Regulation; D053823:Hedgehog Proteins / Q000378:metabolism*; D051741:Kruppel-Like Transcription Factors / Q000378:metabolism*; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D010006:Osteoblasts / Q000166:cytology; D010011:Osteocytes / Q000166:cytology*; D010012:Osteogenesis; D011994:Recombinant Proteins / Q000378:metabolism; D055758:SOX9 Transcription Factor / Q000378:metabolism; D055755:SOXD Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation; D000071676:Zinc Finger Protein GLI1
chemical_list: D019485:Bone Morphogenetic Proteins; C498520:Gli1 protein, mouse; D053823:Hedgehog Proteins; D051741:Kruppel-Like Transcription Factors; D011994:Recombinant Proteins; D055758:SOX9 Transcription Factor; D055755:SOXD Transcription Factors; C465570:Shh protein, mouse; C089186:Sox5 protein, mouse; C095939:Sox6 protein, mouse; C527933:Sox9 protein, mouse; D000071676:Zinc Finger Protein GLI1