Journal: bioRxiv

Article Title: Loss of Foxc1 and Foxc2 function in chondroprogenitor cells disrupts endochondral ossification

doi: 10.1101/2021.01.27.428508

Figure Lengend Snippet: SOX9 regulates expression of Foxc1 . (A) SOX9 expression was induced in mouse embryonic stem cells containing a doxycycline (Dox)-inducible cassette. Expression was induced for 48h by removal of Dox. Expression of Foxc1, Foxc2 Col2a and Sox9 mRNA was determined by qRT-PCR. Data are presented from three independent experiments. Error bars represent standard deviation. (B) SOX9 binding to regulatory elements in Col2a and Foxc1 was determined by Chromatin Immunoprecipitation (ChIP) in ATDC5 chondrocyte cells. Four Sox9 binding sites in the regulatory region of Foxc1 (Distal A-D) were identified from previous ChIP-seq experiments (Ohba et al 2015). (C) Foxc1 Distal regulatory elements were cloned into pGL4-luciferase reporters and transfected along with Sox9 in ATDC5 cells. Only Foxc1-Distal C was activated by Sox9 . * p-value <0.05.

Article Snippet: Mouse embryonic stem (mES) cells containing an inducible Sox9 or Foxc1 expression cassette ( , ) were obtained from either the Coriell Institute (Sox9) or Dr. Minoru Ko (Foxc1).

Techniques: Expressing, Quantitative RT-PCR, Standard Deviation, Binding Assay, Chromatin Immunoprecipitation, ChIP-sequencing, Clone Assay, Luciferase, Transfection

Journal: bioRxiv

Article Title: Loss of Foxc1 and Foxc2 function in chondroprogenitor cells disrupts endochondral ossification

doi: 10.1101/2021.01.27.428508

Figure Lengend Snippet: Elevated Foxc1 promotes chondrocyte differentiation of mouse embryonic stem cells (mES). Schematic of chondrocyte differentiation protocol for mES cells (Kawaguchi et al 2005). (B) Expression of FOXC1 was confirmed in mES cells containing a Dox-inducible flag-tagged Foxc1 expression cassette. (C) Chondrocyte differentiation in mES cells was monitored by measuring expression of Sox9, Sox6, Col2a, Runx2 and ColX expression. Data presented are the means from three biological replicates. Error bars represent standard deviation. * p-value <0.05

Article Snippet: Mouse embryonic stem (mES) cells containing an inducible Sox9 or Foxc1 expression cassette ( , ) were obtained from either the Coriell Institute (Sox9) or Dr. Minoru Ko (Foxc1).

Techniques: Expressing, Standard Deviation

Journal: bioRxiv

Article Title: Loss of Foxc1 and Foxc2 function in chondroprogenitor cells disrupts endochondral ossification

doi: 10.1101/2021.01.27.428508

Figure Lengend Snippet: Loss of Foxc1 expression in ATDC5 cells alters chondrocyte differentiation. (A) Deletion of Foxc1 expression was achieved through Crispr mutagenesis. Reduced protein levels observed in Foxc1 mutant cells (crFOXC1). (B) Wild type and crFOXC1 cells were differentiated for 21 days and chondrogenesis measured by Alcian Blue staining. The number of chondrogenic nodules were counted in wild type and mutant cells. (C) Levels of chondrocyte-expressed genes are affected in Foxc1 mutant ATDC5 cells after 21 days of differentiation.

Article Snippet: Mouse embryonic stem (mES) cells containing an inducible Sox9 or Foxc1 expression cassette ( , ) were obtained from either the Coriell Institute (Sox9) or Dr. Minoru Ko (Foxc1).

Techniques: Expressing, CRISPR, Mutagenesis, Staining

Journal: bioRxiv

Article Title: Loss of Foxc1 and Foxc2 function in chondroprogenitor cells disrupts endochondral ossification

doi: 10.1101/2021.01.27.428508

Figure Lengend Snippet: Foxc1 and Foxc2 are expressed in the perichondrium and resting zone of the growth plate. (A) Localization of Foxc1 and Foxc2 expression was determined in the 14.5 dpc hind limb by in situ hybridization. (B)Foxc1 and (C) Foxc2 mRNA expression in the proximal tibia at 16.5 dpc was abundant in the perichondrium. (D). Foxc1 and Foxc2 mRNA displayed both overlapping and distinct expression patterns in the developing limb. (E). Foxc1 transcripts (Red) were detected in the perichondrium and resting zone, while Foxc2(Green) was mainly expressed in the perichondrium. Low levels of Foxc1 and Foxc2 expression were detected in the proliferative zone (F) and hypertrophic zone (G).

Article Snippet: Mouse embryonic stem (mES) cells containing an inducible Sox9 or Foxc1 expression cassette ( , ) were obtained from either the Coriell Institute (Sox9) or Dr. Minoru Ko (Foxc1).

Techniques: Expressing, In Situ Hybridization

Journal: bioRxiv

Article Title: Loss of Foxc1 and Foxc2 function in chondroprogenitor cells disrupts endochondral ossification

doi: 10.1101/2021.01.27.428508

Figure Lengend Snippet: Col2-cre ablation of Foxc1 and Foxc2 expression in the developing limb. (A) (B) ROSA26 tm4(ACTB-tdTomato,-EGFP) (mTmG) mice were crossed to Col2-Cre mice to monitor Cre activity in the developing limb at 14.5 dpc. EGFP was only detected in limb of mice containing the Col2-cre transgene. (C)(D) To create chondrocyte-specific Foxc1 and Foxc2 mutant mice, Col2-cre mice were crossed with homozygous “floxed” Foxc1 and Foxc2 mice. No expression of Foxc1 and Foxc2 mRNA was detected by in situ hybridization in the developing humerus or proximal tibia in Col2-cre;Foxc1 Δ/Δ ;Foxc2 Δ/Δ mice at 16.5 dpc.

Article Snippet: Mouse embryonic stem (mES) cells containing an inducible Sox9 or Foxc1 expression cassette ( , ) were obtained from either the Coriell Institute (Sox9) or Dr. Minoru Ko (Foxc1).

Techniques: Expressing, Activity Assay, Mutagenesis, In Situ Hybridization

Journal: bioRxiv

Article Title: Loss of Foxc1 and Foxc2 function in chondroprogenitor cells disrupts endochondral ossification

doi: 10.1101/2021.01.27.428508

Figure Lengend Snippet: Col2-cre deletion of Foxc1 and Foxc2 causes skeletal hypoplasia. (A). Alizarin red and Alcian blue skeleton preps from 18.5 dpc embryos. Control embryos lack the Col2-cre transgene. (B) Disrupted endochondral ossification can be observed in the axial and appendicular skeleton of Col2-cre;Foxc1 Δ/Δ ;Foxc2 Δ/Δ embryos. (C)Length of ossification is reduced in the tibia at 18.5 dpc of Col2-cre;Foxc1 Δ/Δ ;Foxc2 Δ/Δ mice is reduced.

Article Snippet: Mouse embryonic stem (mES) cells containing an inducible Sox9 or Foxc1 expression cassette ( , ) were obtained from either the Coriell Institute (Sox9) or Dr. Minoru Ko (Foxc1).

Techniques: Control

Journal: bioRxiv

Article Title: Loss of Foxc1 and Foxc2 function in chondroprogenitor cells disrupts endochondral ossification

doi: 10.1101/2021.01.27.428508

Figure Lengend Snippet: Disrupted growth plate organization in Col2-cre;Foxc1 Δ/Δ ;Foxc2 Δ/Δ mutants. (A) Hematoxylin-Eosin staining of the 16.5 dpc proximal tibia growth plate. Enlarged sections are depicted with coloured boxes. (B) Length of the resting, proliferative and hypertrophic zone were measured in control (CON) or Col2-cre;Foxc1 Δ/Δ ;Foxc2 Δ/Δ (C1 ΔΔ ;C2 ΔΔ ) growth plate. (C) Chondrocyte proliferation in control of Col2-cre;Foxc1 Δ/Δ ;Foxc2 Δ/Δ was measured by Ki67 immunofluorescence.

Article Snippet: Mouse embryonic stem (mES) cells containing an inducible Sox9 or Foxc1 expression cassette ( , ) were obtained from either the Coriell Institute (Sox9) or Dr. Minoru Ko (Foxc1).

Techniques: Staining, Control, Immunofluorescence

Journal: bioRxiv

Article Title: Loss of Foxc1 and Foxc2 function in chondroprogenitor cells disrupts endochondral ossification

doi: 10.1101/2021.01.27.428508

Figure Lengend Snippet: Loss of Foxc1 and Foxc2 function in chondrocytes results in a general reduction of mRNA levels of genes expressed throughout endochondral ossification. (A) RNA from the rib cage from control or Col2-cre;Foxc1 Δ/Δ ;Foxc2 Δ/Δ embryos at 16.5 dpc was isolated and gene expression monitored by qRT-PCR. (B) Heat map of the top 25 genes down regulated in 16.5 dpc Col2-cre;Foxc1 Δ/Δ ;Foxc2 Δ/Δ rib RNA as determined by RNA-seq analysis. (C) Functional annotation genes down regulated Col2-cre;Foxc1 Δ/Δ ;Foxc2 Δ/Δ mutants.

Article Snippet: Mouse embryonic stem (mES) cells containing an inducible Sox9 or Foxc1 expression cassette ( , ) were obtained from either the Coriell Institute (Sox9) or Dr. Minoru Ko (Foxc1).

Techniques: Control, Isolation, Gene Expression, Quantitative RT-PCR, RNA Sequencing, Functional Assay

Journal: bioRxiv

Article Title: Loss of Foxc1 and Foxc2 function in chondroprogenitor cells disrupts endochondral ossification

doi: 10.1101/2021.01.27.428508

Figure Lengend Snippet: Altered chondrocyte differentiation in Col2-cre;Foxc1D/D;Foxc2D/D embryos. Gene expression patterns markers of chondrocyte differentiation were monitored in the proximal tibia growth plate at 16.5 dpc in control or Col2-cre;Foxc1 Δ/Δ ;Foxc2 Δ/Δ embryos. (A) SOX9 immunofluorescence (IF) SOX6 IF (C) Fgfr3 in situ hybridization (D) Ihh ISH (E) RUNX2 IF (F) COLX IF; (G) MMP13 IF. Length of RUNX2 (H) and COLX (I) positive cells in control vs Col2-cre;Foxc1 Δ/Δ ;Foxc2 Δ/Δ growth plates. Scale bar 200 mm.

Article Snippet: Mouse embryonic stem (mES) cells containing an inducible Sox9 or Foxc1 expression cassette ( , ) were obtained from either the Coriell Institute (Sox9) or Dr. Minoru Ko (Foxc1).

Techniques: Gene Expression, Control, Immunofluorescence, In Situ Hybridization

Journal: bioRxiv

Article Title: Loss of Foxc1 and Foxc2 function in chondroprogenitor cells disrupts endochondral ossification

doi: 10.1101/2021.01.27.428508

Figure Lengend Snippet: Impaired mineralization in Col2-cre;Foxc1 Δ/Δ ;Foxc2 Δ/Δ mutants. (A,A’-B,B’) Mineralization in the primary ossification center of control and Col2-cre;Foxc1 Δ/Δ ;Foxc2 Δ/Δ embryos determined by Von Kossa staining. (C-D) Levels of OSTEOPONTIN (Red) and OSTERIX protein (E,F,) COLLAGEN I and MMP13 localization (G-H) VEGFA localization.

Article Snippet: Mouse embryonic stem (mES) cells containing an inducible Sox9 or Foxc1 expression cassette ( , ) were obtained from either the Coriell Institute (Sox9) or Dr. Minoru Ko (Foxc1).

Techniques: Control, Staining