Volume 22, Issue 5, Pages (May 2012)

Slides:



Advertisements
Similar presentations
CXC chemokine ligand 12a enhances chondrocyte proliferation and maturation during endochondral bone formation  G.-W. Kim, M.-S. Han, H.-R. Park, E.-J.
Advertisements

Crucial Roles of MZF1 and Sp1 in the Transcriptional Regulation of the Peptidylarginine Deiminase Type I Gene (PADI1) in Human Keratinocytes  Sijun Dong,
Volume 138, Issue 1, Pages e3 (January 2010)
CXC chemokine ligand 12a enhances chondrocyte proliferation and maturation during endochondral bone formation  G.-W. Kim, M.-S. Han, H.-R. Park, E.-J.
M. Wang, H. Jin, D. Tang, S. Huang, M.J. Zuscik, D. Chen 
Volume 8, Issue 5, Pages (May 2005)
The homeodomain protein Cdx2 regulates lactase gene promoter activity during enterocyte differentiation  Rixun Fang, Nilda A. Santiago, Lynne C. Olds,
Zara E Khan, Timothy C Wang, Guanglin Cui, Alfred L Chi, Rod Dimaline 
M. Wang, H. Jin, D. Tang, S. Huang, M.J. Zuscik, D. Chen 
Volume 47, Issue 2, Pages (July 2012)
Volume 138, Issue 2, Pages (February 2010)
Volume 62, Issue 4, Pages (October 2002)
Volume 138, Issue 1, Pages e3 (January 2010)
Volume 16, Issue 6, Pages (December 2004)
M. Ushita, T. Saito, T. Ikeda, F. Yano, A. Higashikawa, N. Ogata, U
Volume 19, Issue 2, Pages (August 2010)
Volume 26, Issue 4, Pages (August 2013)
Volume 53, Issue 1, Pages (January 2014)
Volume 8, Issue 2, Pages (August 2001)
A. Woods, Ph. D. , D. Pala, M. Sc. , L. Kennedy, M. Sc. , S. McLean, B
M.M.-G. Sun, F. Beier  Osteoarthritis and Cartilage 
Volume 23, Issue 3, Pages (February 2013)
Wenqian Hu, Bingbing Yuan, Harvey F. Lodish  Developmental Cell 
Dysregulation of Chondrogenesis in Human Cleidocranial Dysplasia
Yin-Yang 1 Negatively Regulates the Differentiation-Specific Transcription of Mouse Loricrin Gene in Undifferentiated Keratinocytes  Xuezhu Xu, Yasuhiro.
Volume 13, Issue 6, Pages (December 2007)
Transcription Factor CTIP2 Maintains Hair Follicle Stem Cell Pool and Contributes to Altered Expression of LHX2 and NFATC1  Shreya Bhattacharya, Heather.
Regulation of α-Synuclein Expression by Poly (ADP Ribose) Polymerase-1 (PARP-1) Binding to the NACP-Rep1 Polymorphic Site Upstream of the SNCA Gene  Ornit.
Volume 11, Issue 1, Pages (July 2006)
Wnt/β-Catenin Signaling in Mesenchymal Progenitors Controls Osteoblast and Chondrocyte Differentiation during Vertebrate Skeletogenesis  Timothy F. Day,
Volume 41, Issue 4, Pages e5 (May 2017)
Jungmook Lyu, Vicky Yamamoto, Wange Lu  Developmental Cell 
Angiopoietin-like protein 2 promotes chondrogenic differentiation during bone growth as a cartilage matrix factor  H. Tanoue, J. Morinaga, T. Yoshizawa,
Transcriptional Regulation of ATP2C1 Gene by Sp1 and YY1 and Reduced Function of its Promoter in Hailey–Hailey Disease Keratinocytes  Hiroshi Kawada,
HBL1 Is a Human Long Noncoding RNA that Modulates Cardiomyocyte Development from Pluripotent Stem Cells by Counteracting MIR1  Juli Liu, Yang Li, Bo Lin,
Volume 22, Issue 2, Pages (February 2012)
Volume 143, Issue 1, Pages (October 2010)
Promotion Effects of miR-375 on the Osteogenic Differentiation of Human Adipose- Derived Mesenchymal Stem Cells  Si Chen, Yunfei Zheng, Shan Zhang, Lingfei.
Keratinocyte growth factor promotes goblet cell differentiation through regulation of goblet cell silencer inhibitor  Dai Iwakiri, Daniel K. Podolsky 
HBL1 Is a Human Long Noncoding RNA that Modulates Cardiomyocyte Development from Pluripotent Stem Cells by Counteracting MIR1  Juli Liu, Yang Li, Bo Lin,
Volume 70, Issue 5, Pages (June 2011)
MyoD Targets TAF3/TRF3 to Activate Myogenin Transcription
A Molecular Switch for Photoperiod Responsiveness in Mammals
Codependent Activators Direct Myoblast-Specific MyoD Transcription
Volume 10, Issue 3, Pages (September 2006)
Regulation of the Expression of Peptidylarginine Deiminase Type II Gene (PADI2) in Human Keratinocytes Involves Sp1 and Sp3 Transcription Factors  Sijun.
Volume 11, Issue 1, Pages (July 2018)
Volume 127, Issue 4, Pages (October 2004)
Volume 20, Issue 4, Pages (April 2011)
Xuepei Lei, Jianwei Jiao  Stem Cell Reports 
Volume 22, Issue 5, Pages (May 2012)
Volume 35, Issue 3, Pages (November 2015)
Drosophila Maelstrom Ensures Proper Germline Stem Cell Lineage Differentiation by Repressing microRNA-7  Jun Wei Pek, Ai Khim Lim, Toshie Kai  Developmental.
The Chick Transcriptional Repressor Nkx3
Volume 26, Issue 3, Pages (May 2007)
Hung-Chun Chang, Leonard Guarente  Cell 
Volume 9, Issue 9, Pages (May 1999)
Transcriptional Repression of miR-34 Family Contributes to p63-Mediated Cell Cycle Progression in Epidermal Cells  Dario Antonini, Monia T. Russo, Laura.
EBF2 Regulates Osteoblast-Dependent Differentiation of Osteoclasts
Volume 10, Issue 5, Pages (May 2006)
Volume 31, Issue 6, Pages (December 2009)
Volume 12, Issue 4, Pages (April 2007)
Mary O'Riordan, Rudolf Grosschedl  Immunity 
Volume 16, Issue 5, Pages (May 2009)
Volume 8, Issue 6, Pages (June 2017)
Volume 11, Issue 3, Pages (September 2012)
A Splicing-Independent Function of SF2/ASF in MicroRNA Processing
Volume 21, Issue 6, Pages (June 2012)
Volume 19, Issue 3, Pages (September 2010)
Zhen Zhang, Jamie M. Verheyden, John A. Hassell, Xin Sun 
Presentation transcript:

Volume 22, Issue 5, Pages 927-939 (May 2012) FoxA Family Members Are Crucial Regulators of the Hypertrophic Chondrocyte Differentiation Program  Andreia Ionescu, Elena Kozhemyakina, Claudia Nicolae, Klaus H. Kaestner, Bjorn R. Olsen, Andrew B. Lassar  Developmental Cell  Volume 22, Issue 5, Pages 927-939 (May 2012) DOI: 10.1016/j.devcel.2012.03.011 Copyright © 2012 Elsevier Inc. Terms and Conditions

Developmental Cell 2012 22, 927-939DOI: (10.1016/j.devcel.2012.03.011) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 Characterization of Chondrocyte-Specific Transcription Factor Binding Sites within the Chicken Collagen X Enhancer (b2) that Are Critical for Promoter Activity (A) Either upper sternal chondrocytes or fibroblasts isolated from chicken embryos were cotransfected with a collagen X luciferase reporter (b2-230-Luc) plus Smad1/Runx2/MEF2C expression vectors as indicated. The reporter is comprised of the b2 collagen X enhancer appended to a 230 bp proximal promoter region driving the expression of the Renilla Luciferase gene. In both this and subsequent experiments, Renilla Luciferase Units were normalized to the expression of a cotransfected SV40-Firefly Luciferase vehicle. Significance was calculated using Student's t test, ∗ denotes statistical significance at p ≤ 0.01. Error bars indicate standard error of the mean (SEM), where n = 3. (B) Electrophoretic mobility shift assay (EMSA) was performed using nuclear extracts derived from either chondrocytes [C] or fibroblasts [F]. The proteins were incubated with selected sequences (oligos 1–4) within the b2 enhancer in the presence of either dIdC or dGdC as nonspecific competitors. Two chondrocyte-specific complexes: a slow mobility complex (SMC) and a fast mobility complex (FMC) are shown by arrowheads. (C) The nucleotides critical for the interaction of the chondrocyte-specific complexes (SMC/FMC) with the DNA are displayed in blue. The common core motif ACAAA, present in each oligo, is outlined in the red box. (D) EMSA was performed using chondrocyte nuclear extracts incubated with either wild-type (W) oligos 1–4 or those containing mutations (M) in the ACAAA sequence, in the presence of dGdC or dIdC as indicated. (E) Schematic of wild-type and mutant b2-230-Luc reporter constructs with mutations of the ACAAA sequence located in oligos 1–4 as indicated (“X” represents site of mutation). Activity of these constructs in transfected chondrocytes is shown in either the absence or presence of cotransfected Smad1 and Runx2 expression vehicles plus BMP2 (S/R/B). Significance was calculated using Student's t test, ∗ denotes statistical significance at p ≤ 0.02. Error bars indicate standard error of the mean (SEM), where n = 3. See also Figures S1 and S2. Developmental Cell 2012 22, 927-939DOI: (10.1016/j.devcel.2012.03.011) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 The Slow Mobility Complex Contains Sox 5 (A) EMSA using in vitro-translated Sox5 and Sox9 incubated with oligo 1. (B) EMSA using chondrocyte nuclear extracts incubated with oligo 1 and specific anti-Sox family antibodies as indicated. Two antibodies made against different domains of the Sox5 protein (anti-Sox5a and anti-Sox5b) specifically super-shift the SMC but not the FMC gel shift. (C) Cotransfection of the b2-230-Collagen X Luciferase reporter together with Smad1, Runx2, and either Sox5, Sox6, or Sox9 expression vehicles in either chondrocytes or fibroblasts. Significance was calculated using Student's t test, ∗ denotes statistical significance at p ≤ 0.05. Error bars indicate standard error of the mean (SEM), where n = 3. (D) Immunostaining for Sox5 (green) or Collagen X (red) is displayed in either chondrocytes or fibroblasts. DAPI staining of DNA is shown in blue. Developmental Cell 2012 22, 927-939DOI: (10.1016/j.devcel.2012.03.011) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 The Fast Mobility Complex Contains FoxA2 (A) EMSA using in vitro-translated FoxA1 and FoxA2 incubated with oligos 1–4. (B) EMSA using chondrocyte (C) or fibroblast (F) nuclear extracts (NE) or in vitro- translated FoxA2 (IVT A2) and incubated with oligos 1–4 in the presence or absence of either anti-FoxA1 or anti-FoxA2 antibodies as indicated. (C) Cotransfection of the ABC-230-Collagen X Luciferase reporter together with Smad1, Runx2 and either FoxA1 or FoxA2 as indicated, in either chondrocytes or fibroblasts. Significance was calculated using Student's t test, there is not a significant statistical difference (p ≥ 0.05) between the luciferase activities of the collagen X luciferase reporter cotransfected with either FoxA1 or FoxA2 in chondrocytes versus in fibroblasts. Error bars indicate standard error of the mean (SEM) where n = 3. (D) EMSA of in vitro-translated FoxA2 with either wild-type (W) oligos 1–4 or those containing mutations (M) in the ACAAA sequence. (E) Schematic of wild-type and mutant b2-230-Luc reporter constructs with mutations of the ACAAA sequence located in oligos 1–4 as indicated (“X” represents the site of mutation). Activity of these constructs in transfected chondrocytes is shown in either the absence or presence of a cotransfected FoxA2 expression vehicle. Significance was calculated using Student's t test, ∗ denotes statistical significance at p ≤ 0.01. Error bars indicate standard error of the mean (SEM) where n = 3. Developmental Cell 2012 22, 927-939DOI: (10.1016/j.devcel.2012.03.011) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 Overexpression or Knockdown of FoxA2 Increases or Decreases Expression, Respectively, of Several Hypertrophic Chondrocyte Markers (A) Limb bud mesenchymal cells were cultured under high density micromass conditions for the indicated number of days. Gene expression was assayed by RT-qPCR. In both this and subsequent experiments, transcript levels were normalized to GAPDH levels. Significance was calculated using Student's t test, ∗ denotes statistical significance at p ≤ 0.05 when gene expression in high-density micromass cultures at various time points is compared with gene expression at the initial time of plating. Error bars indicate standard error of the mean (SEM), where n = 3. (B) FoxA1, FoxA2, and MEF2C are expressed in chondrocytes (C) but not in fibroblasts (F) as assayed by RT-qPCR. Significance was calculated using Student's t test, ∗ denotes statistical significance at p ≤ 0.01 when gene expression in chondrocyte cultures is compared with gene expression in fibroblast cultures. Error bars indicate standard error of the mean (SEM), where n = 3. (C) Either chondrocytes or fibroblasts were cotransfected with the b2-230-Luc reporter in either the absence or presence of exogenous FoxA2 or MEF2C, as indicated. Significance was calculated using Student's t test, ∗ denotes statistical significance at p ≤ 0.05 when luciferase activites from either FoxA2 or MEF2C cotransfected cells are compared to luciferase activities of control vehicle transfected cells. Error bars indicate standard error of the mean (SEM), where n = 3. (D) Overexpression of FoxA2 increases expression of several hypertrophic chondrocyte markers. Chondrocytes were infected with an avian retrovirus encoding either GFP (RCAS-GFP; black bars) or FoxA2 (RCAS-mFoxA2; gray bars). Cells were cultured in BMP2 for the indicated number of days and subsequently gene expression was assayed by quantitative real time PCR. Transcript levels relative to GAPDH are displayed. Significance was calculated using Student's t test, ∗ denotes statistical significance at p ≤ 0.05 when gene expression in chondrocyte cultures infected with RCAS-mFoxA2 is compared with gene expression in chondrocyte cultures infected with RCAS-GFP. Error bars indicate standard error of the mean (SEM), where n = 3. (E) Knockdown of FoxA2 decreases expression of several hypertrophic chondrocyte markers. Chondrocytes were infected with an avian retrovirus encoding either a scrambled shRNA (RCAS-sh-nonGFP; black bars) or shRNA targeting FoxA2 (RCAS-sh-cFoxA2; gray bars). Cells were cultured in BMP2 for the indicated number of days and subsequently gene expression was assayed by quantitative real time PCR. Transcript levels relative to GAPDH are displayed. Significance was calculated using Student's t test, ∗ denotes statistical significance at p ≤ 0.05 when gene expression in chondrocyte cultures infected with RCAS-sh-cFoxA2 is compared with gene expression in chondrocyte cultures infected with RCAS-sh-nonGFP. Error bars indicate standard error of the mean (SEM), where n = 3. Developmental Cell 2012 22, 927-939DOI: (10.1016/j.devcel.2012.03.011) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 Chondrocyte-Specific Knockout of FoxA2 and Deletion of FoxA3 in Mice Results in Delayed Hypertrophy and Postnatal Dwarfism (A) Whole mount X-gal staining of the skeleton of newborn FoxA3-Cre; ROSA26 mice as compared with control ROSA26 littermates (Aa–Ac). (B) X-gal/ Fast Red staining of tibial sections of newborn FoxA3-Cre; ROSA26 mice (panels 5–8) compared with control littermates (panels 1–4). (C) Hematoxylin and eosin (H&E) and X-gal staining on sections of either vertebrae, second sternebra, or forelimb digit from FoxA2-CreERT2; ROSA26 mice that were administered tamoxifen by intraperitoneal (IP) injection at either 7 or 14 dpc and harvested at the various times as indicated. (D) Alcian Blue/Alizarin Red skeletal preparations of either sternum, vertebral column, tail, or forelimb from E19.5 Col2-Cre; FoxA2flox/flox (2 null alleles; 2NA) mice embryos, Col2-Cre; FoxA2flox/flox; FoxA3−/− (4 null alleles; 4NA) mice embryos or their control littermates. Developmental Cell 2012 22, 927-939DOI: (10.1016/j.devcel.2012.03.011) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 6 Chondrocyte-Specific Knockout of FoxA2 and Deletion of FoxA3 in Mice Results in Delayed Hypertrophy, Loss of Alkaline Phosphatase Activity, and Delayed Mineralization (A–C) Sections of either E19.5 sternebrae (a, b), thoracic vertebrae (c, d), caudal vertebrae (e, f), femurs (g, h), or E16.5 femurs (i, j) taken from either control or Col2-Cre; FoxA2flox/flox; FoxA3−/− (4NA) embryos were stained with either H&E (A), for alkaline phosphatase activity (B), or with von Kossa stain (C). See also Figures S2 and S3. Developmental Cell 2012 22, 927-939DOI: (10.1016/j.devcel.2012.03.011) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 7 Chondrocyte-Specific Knockout of FoxA2 and Deletion of FoxA3 in Mice Results in Loss of Hypertrophic Chondrocyte Gene Expression in the Growth Plate (A) In situ hybridization analysis of tibial growth plates for the indicated genes is displayed for either E19.5 Col2-Cre; FoxA2flox/flox; FoxA3−/− (4NA) embryos or their control littermates. (B) Alcian Blue/Alizarin Red skeletal preparations of 2-week-old Col2-Cre; FoxA2flox/flox; FoxA3−/− (4NA) mice or their control littermates. (C) H&E staining of tibial epiphysis of either 2-week-old Col2-Cre; FoxA2flox/flox; FoxA3−/− (4NA) mice or control littermates. Quantification of the thickness of either the proliferative zone (PZ) or the hypertrophic zone (HZ) in the tibial growth plates is displayed. Significance was calculated using Student's t test, ∗ denotes statistical significance at p ≤ 0.001 when tibial growth plate zones from 2-week-old 4NA mutant mice are compared to the tibial growth plate zones from control animals. Error bars indicate standard error of the mean (SEM) where n = 6. (D) In situ hybridization analysis of tibial growth plates for the indicated genes is displayed for either 2-week-old Col2-Cre; FoxA2flox/flox; FoxA3−/− (4NA) mice or their control littermates. See also Figures S4 and S5. Developmental Cell 2012 22, 927-939DOI: (10.1016/j.devcel.2012.03.011) Copyright © 2012 Elsevier Inc. Terms and Conditions