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First published online 3 October 2007
doi: 10.1242/dev.008441

Development 134, 3917-3928 (2007)
Published by The Company of Biologists 2007
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HIF1{alpha} regulation of Sox9 is necessary to maintain differentiation of hypoxic prechondrogenic cells during early skeletogenesis

Roy Amarilio1, Sergey V. Viukov1, Amnon Sharir1, Idit Eshkar-Oren1, Randall S. Johnson2 and Elazar Zelzer1,*

1 Department of Molecular Genetics, Weizmann Institute of Science, PO Box 26, Rehovot 76100, Israel.
2 Molecular Biology Section, Division of Biology, University of California, San Diego, CA, USA.


Figure 1
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  		Fig. 1. HIF1{alpha} is expressed by hypoxic differentiating chondrocytes. (A) Sections of E12.5 autopod showing avascular cartilage. The vasculature was detected by immunofluorescence using anti-CD31 antibody (green); cartilage was detected by anti-collagen type II (red). (B,C) Sections through the elbow show hypoxic cells in the condensed mesenchyme at E11.5 (B) and E12.5 differentiating chondrocytes (C). (D,E) Section through the autopod at E12.5 (D) and E13.5 (E). Hypoxic cells were detected by immunofluorescence using Hypoxyprobe-1. (F) HIF1 detection in E13.5 digits by immunofluorescence using anti-HIF1{alpha} antibody. (G-J) Pgk1 expression was detected by in situ hybridization on sections, showing the humerus region (G) and the digits (I) of E12.5 control. The expression was lost in humerus, ulna (H) and digits (J) of Prx1-Cre-Hif1{alpha} autopods. Black arrows indicate Pgk1 expression in the interzone; red arrows indicate Pgk1 expression in the inner part of the digit. h, humerus; r, radius; u, ulna.

 

Figure 2
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  		Fig. 2. Skeletal abnormalities in Prx1-Hif1{alpha} mutants. (A-C) Alizarin Red and Alcian Blue staining of wild-type (top) and Prx1-Hif1{alpha} (bottom) mouse forelimbs. E18.5 littermates demonstrate severe retardation in limb skeleton formation (A) and joint fusion (B). Autopod skeletons (C) show cartilage formation at the periphery of the forming digits and joint loss in the Prx1-Cre-Hif1{alpha} limb. (D) Histological sections of E13.5 control (top) and Prx1-Cre-Hif1{alpha} (bottom) elbows. In the Prx1-Cre-Hif1{alpha} joint, the presence of apoptotic cells with shrunken cytoplasm and condensed nuclei is observed. (E,F) Histological sections through the digits revealed partial joint cavitation (E) and loss of phalanx (F). (G-I) MicroCT analysis of 21-day-old Prx1-Cre-Hif1{alpha} and WT autopod: dorsal view demonstrates severe retardation in the Prx1-Cre-Hif1{alpha} skeleton including bone deformation, lack of phalanges (bracket), and joint fusion. H and I show dorsal and lateral views, respectively, of digit 3. Yellow arrows indicate fused joints and fused sesamoid bones.

 

Figure 3
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  		Fig. 3. HIF1{alpha} is necessary for chondrocyte differentiation. (A,B) Histology sections of E13.5 control (top) and Prx1-Cre-Hif1{alpha} (bottom) autopods showing mesenchyme-like morphology of the cells in Prx1-Cre-Hif1{alpha} digits. (B) Higher magnification of the boxed area in A demonstrates lack of interzone formation. (C,D) In situ hybridization for Sox9. (C) Uniform expression of Sox9 in E11.5 mesenchymal condensation; (D) substantial reduction in Sox9 expression in the center of E12.5 Prx1-Cre-Hif1{alpha} digits. (E-H) Serial sections of E13.5 control and Prx1-Hif1{alpha} autopods were hybridized with RNA probes for Sox9 (E), Sox6 (F), collagen type II (Col2a1; G) and aggrecan (H). Yellow arrows indicate the interzone.

 

Figure 4
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  		Fig. 4. Reduced chondrocyte proliferation in Prx1-Hif1{alpha} digits. BrdU incorporation into forming digits of control (A) and Prx1-Hif1{alpha} (B) mice. (C) Quantification of BrdU incorporation reveals a 3.5-fold reduction in the percentage of dividing cells in the mutant (control, 20.99±1.65%; Prx-1-Hif1{alpha}, 5.94±0.62%. P<0.001, n=4).

 

Figure 5
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  		Fig. 5. Aberrant interzone formation in Prx1-Hif1{alpha} autopod. (A-D) In situ hybridizations for Gdf5. Whole-mount at E12.5 (A) and E13.5 (B) and sections at E13.5 (C) and E14.5 (D) show noticeable differences in the expression patterns between Prx1-Hif1{alpha} and control; white and black arrowheads indicate interzone area. (E) Noggin expression at E14.5. Red arrows indicate interzone area. (F-I) Higher magnification of the joint located between the first and the second phalanges of E15.5 control and Prx1-Hif1{alpha} autopods. (F) H&E staining. (G-I) Sections were hybridized with RNA probes for Gdf5 (G), Wnt9A (H) and Bmp2 (I).

 

Figure 6
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  		Fig. 6. Vegf expression and vasculature formation in Hif1{alpha}-deleted limb. Immunofluorescence of E12.5 sections of control (A) and Prx1-Cre-Hif1{alpha} (B) mouse limbs using antibodies for collagen type II (red) and CD31 (green). (C) Quantitative RT-PCR demonstrates reduction in Vegf mRNA level in Prx1-Cre-Hif1{alpha} E12.5 limbs (n=2).

 

Figure 7
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  		Fig. 7. HIF1{alpha} is necessary for mesenchymal cell differentiation under hypoxia. (A,B) Micromass culture of mesenchymal cells derived from E11.5 Hif1{alpha} floxed/floxed mouse embryos were infected with AdCre or AdGfp (control) and were cultured under normoxic (20% O2) or hypoxic (1% O2) conditions as indicated (gray, AdGfp; black, AdCre). Quantitative RT-PCR shows deletion of Hif1{alpha} (A) and loss of Pgk1 induction under hypoxia (B) in AdCre-infected cells relative to AdGfp-infected cells. (C) Alcian Blue staining (above) and immunohistochemical staining for collagen type II (Col2a1; below) reveal reduction in cartilage nodule formation in AdCre-infected cells cultured under hypoxia. (D,E) Substantial reduction in collagen II (D) and aggrecan (E) mRNA is detected by quantitative RT-PCR (n=3).

 

Figure 8
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  		Fig. 8. HIF1{alpha} directly regulates Sox9 expression. (A) Increase in Sox9 and Pgk1 mRNA level upon infection of mesenchymal cells with lentivirus encoding stabilized human HIF1{alpha} (n=2). (B,C) Substantial reduction in Sox9 mRNA and protein level in AdCre-infected cells was detected by quantitative RT-PCR analysis (B) and immunoblot assay (C). (D,E) Quantitative RT-PCR analyses for Sox6 (D) and Sox5 (E) show a marked decrease in AdCre-infected cells under hypoxia (n=3). (F) Schematic view of 3.0 kb Sox9 promoter region showing the position of four potential HIF1{alpha} binding sites relative to the transcription start site. (G,H) Chromatin immunoprecipitation assay of mouse Sox9 gene. Real-time PCR quantification of Hif1{alpha} binding to regions A-D (amplicons) is indicated by the percentage of total input chromatin DNA (n=2). (I) Immunoprecipitation assay with plasmids that contain control Sox9 promoter or plasmids that contain the Sox9 promoter with a four-nucleotide substitution in the HRE 398 (n=2).

 

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© The Company of Biologists Ltd 2007