title: HIF1alpha regulation of Sox9 is necessary to maintain differentiation of hypoxic prechondrogenic cells during early skeletogenesis.
abstract: During early stages of limb development, the vasculature is subjected to extensive remodeling that leaves the prechondrogenic condensation avascular and, as we demonstrate hereafter, hypoxic. Numerous studies on a variety of cell types have reported that hypoxia has an inhibitory effect on cell differentiation. In order to investigate the mechanism that supports chondrocyte differentiation under hypoxic conditions, we inactivated the transcription factor hypoxia-inducible factor 1alpha (HIF1alpha) in mouse limb bud mesenchyme. Developmental analysis of Hif1alpha-depleted limbs revealed abnormal cartilage and joint formation in the autopod, suggesting that HIF1alpha is part of a mechanism that regulates the differentiation of hypoxic prechondrogenic cells. Dramatically reduced cartilage formation in Hif1alpha-depleted micromass culture cells under hypoxia provided further support for the regulatory role of HIF1alpha in chondrogenesis. Reduced expression of Sox9, a key regulator of chondrocyte differentiation, followed by reduction of Sox6, collagen type II and aggrecan in Hif1alpha-depleted limbs raised the possibility that HIF1alpha regulation of Sox9 is necessary under hypoxic conditions for differentiation of prechondrogenic cells to chondrocytes. To study this possibility, we targeted Hif1alpha expression in micromass cultures. Under hypoxic conditions, Sox9 expression was increased twofold relative to its expression in normoxic condition; this increment was lost in the Hif1alpha-depleted cells. Chromatin immunoprecipitation demonstrated direct binding of HIF1alpha to the Sox9 promoter, thus supporting direct regulation of HIF1alpha on Sox9 expression. This work establishes for the first time HIF1alpha as a key component in the genetic program that regulates chondrogenesis by regulating Sox9 expression in hypoxic prechondrogenic cells.
authors.affiliation: Department of Molecular Genetics, Weizmann Institute of Science, PO Box 26, Rehovot 76100, Israel.
authors.forename: Roy
authors.initials: R
authors.lastname: Amarilio
authors.forename: Sergey V
authors.initials: SV
authors.lastname: Viukov
authors.forename: Amnon
authors.initials: A
authors.lastname: Sharir
authors.forename: Idit
authors.initials: I
authors.lastname: Eshkar-Oren
authors.forename: Randall S
authors.initials: RS
authors.lastname: Johnson
authors.forename: Elazar
authors.initials: E
authors.lastname: Zelzer
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D015687:Cell Hypoxia; D049109:Cell Proliferation; D002478:Cells, Cultured; D020219:Chondrogenesis; D005121:Extremities / Q000196:embryology; D018507:Gene Expression Regulation, Developmental; D006609:High Mobility Group Proteins / Q000235:genetics / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D051379:Mice; D055758:SOX9 Transcription Factor; D012863:Skeleton; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
chemical_list: C497443:Hif1a protein, mouse; D006609:High Mobility Group Proteins; D018398:Homeodomain Proteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; C489936:Prrx1 protein, mouse; D055758:SOX9 Transcription Factor; C527933:Sox9 protein, mouse; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A