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The transcription factor Sox2 is required for osteoblast self-renewal.

Basu-Roy U, Ambrosetti D, Favaro R, Nicolis SK, Mansukhani A, Basilico C - Cell Death Differ. (2010)

Bottom Line: In addition, expression of Sox2-specific shRNAs in independent osteoblastic cell lines suppressed their proliferative ability.Osteoblasts capable of forming 'osteospheres' are greatly enriched in Sox2 expression.These data identify a novel function for Sox2 in the maintenance of self-renewal in the osteoblastic lineage.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, NYU School of Medicine, New York, NY, USA.

ABSTRACT
The development and maintenance of most tissues and organs require the presence of multipotent and unipotent stem cells that have the ability of self-renewal as well as of generating committed, further differentiated cell types. The transcription factor Sox2 is essential for embryonic development and maintains pluripotency and self-renewal in embryonic stem cells. It is expressed in immature osteoblasts/osteoprogenitors in vitro and in vivo and is induced by fibroblast growth factor signaling, which stimulates osteoblast proliferation and inhibits differentiation. Sox2 overexpression can by itself inhibit osteoblast differentiation. To elucidate its function in the osteoblastic lineage, we generated mice with an osteoblast-specific, Cre-mediated knockout of Sox2. These mice are small and osteopenic, and mosaic for Sox2 inactivation. However, culturing calvarial osteoblasts from the mutant mice for 2-3 passages failed to yield any Sox2- cells. Inactivation of the Sox2 gene by Cre-mediated excision in cultured osteoblasts showed that Sox2- cells could not survive repeated passage in culture, could not form colonies, and arrested their growth with a senescent phenotype. In addition, expression of Sox2-specific shRNAs in independent osteoblastic cell lines suppressed their proliferative ability. Osteoblasts capable of forming 'osteospheres' are greatly enriched in Sox2 expression. These data identify a novel function for Sox2 in the maintenance of self-renewal in the osteoblastic lineage.

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Osteoblast-specific Sox2 conditional knock-out mice are smaller and have low bone density(A) Schematic of Sox2, Sox2geo (-), Sox2flox and Sox2del alleles. Map shows the location of diagnostic primer pairs in the Sox2 alleles. Beta geo is the beta geo gene in the Sox2  allele Sox2geo(-); green box is the neo gene; red triangles are the loxP sites. H3, HindIII; S1, SalI. The results obtained within the diagnostic PCR primer pairs depicted in this figure using DNA from Sox2flox/- osteoblasts or Sox2flox/- osteoblasts subjected to Cre excision are shown in Supplementary Figure 1A.(B) PCR analysis of calvarial DNA. DNA from littermates was isolated using standard techniques and PCR was performed with the indicated primers. The flox primer pair detects the deleted and undeleted flox alleles. The del flox primer pair detects only the deleted Sox2 allele after Cre recombination. 1. Sox2+/geo;Cre (control), 2. Sox2flox/geo;Cre (cko)(C) Four week old Sox2 cko (Sox2flox/-; Cre) and control (Sox2+/geo;Cre) littermate.(D) Reconstructed image from Micro cT analysis of 4-week-old distal femurs from Sox2 cko and control littermate.(E) Quantification of average bone mineral density, (F) bone volume per tissue volume (BV/TV), and (G) trabecular pattern factors obtained from micro-CT analysis of 4 and 8 week femurs of Sox2 cko and control littermate animals.
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Figure 1: Osteoblast-specific Sox2 conditional knock-out mice are smaller and have low bone density(A) Schematic of Sox2, Sox2geo (-), Sox2flox and Sox2del alleles. Map shows the location of diagnostic primer pairs in the Sox2 alleles. Beta geo is the beta geo gene in the Sox2 allele Sox2geo(-); green box is the neo gene; red triangles are the loxP sites. H3, HindIII; S1, SalI. The results obtained within the diagnostic PCR primer pairs depicted in this figure using DNA from Sox2flox/- osteoblasts or Sox2flox/- osteoblasts subjected to Cre excision are shown in Supplementary Figure 1A.(B) PCR analysis of calvarial DNA. DNA from littermates was isolated using standard techniques and PCR was performed with the indicated primers. The flox primer pair detects the deleted and undeleted flox alleles. The del flox primer pair detects only the deleted Sox2 allele after Cre recombination. 1. Sox2+/geo;Cre (control), 2. Sox2flox/geo;Cre (cko)(C) Four week old Sox2 cko (Sox2flox/-; Cre) and control (Sox2+/geo;Cre) littermate.(D) Reconstructed image from Micro cT analysis of 4-week-old distal femurs from Sox2 cko and control littermate.(E) Quantification of average bone mineral density, (F) bone volume per tissue volume (BV/TV), and (G) trabecular pattern factors obtained from micro-CT analysis of 4 and 8 week femurs of Sox2 cko and control littermate animals.

Mentions: Since the Sox2 KO is lethal in early embryonic development (10), to precisely define the role of Sox2 in osteoblast proliferation and differentiation we created a conditional knock-out (CKO) of the Sox2 gene in the osteoblastic lineage utilizing mice with a Sox2flox/geo genotype (hereafter referred to as Sox2flox/-). In these mice, one of the two Sox2 alleles, is bracketed by loxP sequences, providing a target for the Cre-recombinase, while the other is replaced by the β-gal (geo) gene, that inactivates the Sox2 gene (Fig. 1A) (20) These mice, which are phenotypically normal, were crossed with transgenic mice expressing the Cre-recombinase under the control of the 2.3kb collagen I promoter, which is specifically expressed in the osteoblastic lineage, to elicit Sox2 excision in Cre-bearing Sox2-floxed F2 offspring (Fig. 1B) (21). The resulting Sox2 CKO mice were initially difficult to study because they died at birth with esophageal atresia, most likely due to aberrant expression of the Cre-recombinase in the esophagus and trachea and the essential role of Sox2 in the development of the foregut (22). However this phenotype has low penetrance, and at least 50% of the pups survive to adulthood. These mice are smaller than their wildtype littermates and are mostly sterile (Fig.1C). MicroCT analysis reveals that mutant mice have reduced bone density with defects in trabecular bone. Bone mineral density and bone volume are reduced, while trabecular pattern factor, a parameter of micro-CT analysis that is increased in poor quality bone (23), is higher in the Sox2 CKO mice (Fig. 1D-G). Cortical bone thickness is also reduced as is the density of parietal bones in the skull (not shown). Interestingly, these effects are clearly detected even though these mice are highly mosaic for Sox2 inactivation. At birth, >50% of the calvarial osteoblasts appear to have maintained the Sox2 gene. While in principle this could be due to inefficient expression or activity of the Cre recombinase, this seemed unlikely, since, if anything, the collagen I Cre mice appear to express Cre more uniformly and less tissue specifically than expected. This finding suggested that Sox2 expressing cells have a growth advantage over Sox2 osteoblasts, a notion supported by the experiments described below.


The transcription factor Sox2 is required for osteoblast self-renewal.

Basu-Roy U, Ambrosetti D, Favaro R, Nicolis SK, Mansukhani A, Basilico C - Cell Death Differ. (2010)

Osteoblast-specific Sox2 conditional knock-out mice are smaller and have low bone density(A) Schematic of Sox2, Sox2geo (-), Sox2flox and Sox2del alleles. Map shows the location of diagnostic primer pairs in the Sox2 alleles. Beta geo is the beta geo gene in the Sox2  allele Sox2geo(-); green box is the neo gene; red triangles are the loxP sites. H3, HindIII; S1, SalI. The results obtained within the diagnostic PCR primer pairs depicted in this figure using DNA from Sox2flox/- osteoblasts or Sox2flox/- osteoblasts subjected to Cre excision are shown in Supplementary Figure 1A.(B) PCR analysis of calvarial DNA. DNA from littermates was isolated using standard techniques and PCR was performed with the indicated primers. The flox primer pair detects the deleted and undeleted flox alleles. The del flox primer pair detects only the deleted Sox2 allele after Cre recombination. 1. Sox2+/geo;Cre (control), 2. Sox2flox/geo;Cre (cko)(C) Four week old Sox2 cko (Sox2flox/-; Cre) and control (Sox2+/geo;Cre) littermate.(D) Reconstructed image from Micro cT analysis of 4-week-old distal femurs from Sox2 cko and control littermate.(E) Quantification of average bone mineral density, (F) bone volume per tissue volume (BV/TV), and (G) trabecular pattern factors obtained from micro-CT analysis of 4 and 8 week femurs of Sox2 cko and control littermate animals.
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Related In: Results  -  Collection

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Figure 1: Osteoblast-specific Sox2 conditional knock-out mice are smaller and have low bone density(A) Schematic of Sox2, Sox2geo (-), Sox2flox and Sox2del alleles. Map shows the location of diagnostic primer pairs in the Sox2 alleles. Beta geo is the beta geo gene in the Sox2 allele Sox2geo(-); green box is the neo gene; red triangles are the loxP sites. H3, HindIII; S1, SalI. The results obtained within the diagnostic PCR primer pairs depicted in this figure using DNA from Sox2flox/- osteoblasts or Sox2flox/- osteoblasts subjected to Cre excision are shown in Supplementary Figure 1A.(B) PCR analysis of calvarial DNA. DNA from littermates was isolated using standard techniques and PCR was performed with the indicated primers. The flox primer pair detects the deleted and undeleted flox alleles. The del flox primer pair detects only the deleted Sox2 allele after Cre recombination. 1. Sox2+/geo;Cre (control), 2. Sox2flox/geo;Cre (cko)(C) Four week old Sox2 cko (Sox2flox/-; Cre) and control (Sox2+/geo;Cre) littermate.(D) Reconstructed image from Micro cT analysis of 4-week-old distal femurs from Sox2 cko and control littermate.(E) Quantification of average bone mineral density, (F) bone volume per tissue volume (BV/TV), and (G) trabecular pattern factors obtained from micro-CT analysis of 4 and 8 week femurs of Sox2 cko and control littermate animals.
Mentions: Since the Sox2 KO is lethal in early embryonic development (10), to precisely define the role of Sox2 in osteoblast proliferation and differentiation we created a conditional knock-out (CKO) of the Sox2 gene in the osteoblastic lineage utilizing mice with a Sox2flox/geo genotype (hereafter referred to as Sox2flox/-). In these mice, one of the two Sox2 alleles, is bracketed by loxP sequences, providing a target for the Cre-recombinase, while the other is replaced by the β-gal (geo) gene, that inactivates the Sox2 gene (Fig. 1A) (20) These mice, which are phenotypically normal, were crossed with transgenic mice expressing the Cre-recombinase under the control of the 2.3kb collagen I promoter, which is specifically expressed in the osteoblastic lineage, to elicit Sox2 excision in Cre-bearing Sox2-floxed F2 offspring (Fig. 1B) (21). The resulting Sox2 CKO mice were initially difficult to study because they died at birth with esophageal atresia, most likely due to aberrant expression of the Cre-recombinase in the esophagus and trachea and the essential role of Sox2 in the development of the foregut (22). However this phenotype has low penetrance, and at least 50% of the pups survive to adulthood. These mice are smaller than their wildtype littermates and are mostly sterile (Fig.1C). MicroCT analysis reveals that mutant mice have reduced bone density with defects in trabecular bone. Bone mineral density and bone volume are reduced, while trabecular pattern factor, a parameter of micro-CT analysis that is increased in poor quality bone (23), is higher in the Sox2 CKO mice (Fig. 1D-G). Cortical bone thickness is also reduced as is the density of parietal bones in the skull (not shown). Interestingly, these effects are clearly detected even though these mice are highly mosaic for Sox2 inactivation. At birth, >50% of the calvarial osteoblasts appear to have maintained the Sox2 gene. While in principle this could be due to inefficient expression or activity of the Cre recombinase, this seemed unlikely, since, if anything, the collagen I Cre mice appear to express Cre more uniformly and less tissue specifically than expected. This finding suggested that Sox2 expressing cells have a growth advantage over Sox2 osteoblasts, a notion supported by the experiments described below.

Bottom Line: In addition, expression of Sox2-specific shRNAs in independent osteoblastic cell lines suppressed their proliferative ability.Osteoblasts capable of forming 'osteospheres' are greatly enriched in Sox2 expression.These data identify a novel function for Sox2 in the maintenance of self-renewal in the osteoblastic lineage.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, NYU School of Medicine, New York, NY, USA.

ABSTRACT
The development and maintenance of most tissues and organs require the presence of multipotent and unipotent stem cells that have the ability of self-renewal as well as of generating committed, further differentiated cell types. The transcription factor Sox2 is essential for embryonic development and maintains pluripotency and self-renewal in embryonic stem cells. It is expressed in immature osteoblasts/osteoprogenitors in vitro and in vivo and is induced by fibroblast growth factor signaling, which stimulates osteoblast proliferation and inhibits differentiation. Sox2 overexpression can by itself inhibit osteoblast differentiation. To elucidate its function in the osteoblastic lineage, we generated mice with an osteoblast-specific, Cre-mediated knockout of Sox2. These mice are small and osteopenic, and mosaic for Sox2 inactivation. However, culturing calvarial osteoblasts from the mutant mice for 2-3 passages failed to yield any Sox2- cells. Inactivation of the Sox2 gene by Cre-mediated excision in cultured osteoblasts showed that Sox2- cells could not survive repeated passage in culture, could not form colonies, and arrested their growth with a senescent phenotype. In addition, expression of Sox2-specific shRNAs in independent osteoblastic cell lines suppressed their proliferative ability. Osteoblasts capable of forming 'osteospheres' are greatly enriched in Sox2 expression. These data identify a novel function for Sox2 in the maintenance of self-renewal in the osteoblastic lineage.

Show MeSH