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Timed deletion of Twist1 in the limb bud reveals age-specific impacts on autopod and zeugopod patterning.

Loebel DA, Hor AC, Bildsoe HK, Tam PP - PLoS ONE (2014)

Bottom Line: Gene expression analysis revealed significant upregulation of Hoxd10, Hoxd11 and Grem1 in the anterior half of the forelimb bud at E11.5.The specific skeletal phenotypes, which include duplication of digits and distal zeugopods but no overt posteriorization, differ from those of other Twist1 conditional knockout mutants.This outcome may be attributed to the deferment of Twist1 ablation to a later time frame of limb morphogenesis, which leads to the ectopic activation of posterior genes in the anterior tissues after the establishment of anterior-posterior anatomical identities in the forelimb bud.

View Article: PubMed Central - PubMed

Affiliation: Embryology Unit, Children's Medical Research Institute, Westmead, New South Wales, Australia; Sydney Medical School, The University of Sydney, Sydney, New South Wales, Australia.

ABSTRACT
Twist1 encodes a transcription factor that plays a vital role in limb development. We have used a tamoxifen-inducible Cre transgene, Ubc-CreERT2, to generate time-specific deletions of Twist1 by inducing Cre activity in mouse embryos at different ages from embryonic (E) day 9.5 onwards. A novel forelimb phenotype of supernumerary pre-axial digits and enlargement or partial duplication of the distal radius was observed when Cre activity was induced at E9.5. Gene expression analysis revealed significant upregulation of Hoxd10, Hoxd11 and Grem1 in the anterior half of the forelimb bud at E11.5. There is also localized upregulation of Ptch1, Hand2 and Hoxd13 at the site of ectopic digit formation, indicating a posterior molecular identity for the supernumerary digits. The specific skeletal phenotypes, which include duplication of digits and distal zeugopods but no overt posteriorization, differ from those of other Twist1 conditional knockout mutants. This outcome may be attributed to the deferment of Twist1 ablation to a later time frame of limb morphogenesis, which leads to the ectopic activation of posterior genes in the anterior tissues after the establishment of anterior-posterior anatomical identities in the forelimb bud.

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Efficient ablation of Twist1 expression in conditional knockout embryos.(A) qRT-PCR analysis of RNA from anterior (A) and posterior (P) halves of forelimb buds collected E11.5 from mothers injected with tamoxifen at E9.5. n = 4 pairs of forelimb buds per genotype. (B–E, B′–E′.) Immunostaining with anti-Twist1 monoclonal antibody (green) of cryosectioned wild type (fl/wt) (B, C, B′, C′) and conditional knockout (CKO) (D, E, D′, E′) E11.5 forelimb buds of embryos harvested from mothers injected with tamoxifen at E10.5. (C, C′, E, E′) Merged images showing counterstaining with DAPI (blue). (B′–E′) Higher magnification images of the boxed regions in B–E.
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pone-0098945-g001: Efficient ablation of Twist1 expression in conditional knockout embryos.(A) qRT-PCR analysis of RNA from anterior (A) and posterior (P) halves of forelimb buds collected E11.5 from mothers injected with tamoxifen at E9.5. n = 4 pairs of forelimb buds per genotype. (B–E, B′–E′.) Immunostaining with anti-Twist1 monoclonal antibody (green) of cryosectioned wild type (fl/wt) (B, C, B′, C′) and conditional knockout (CKO) (D, E, D′, E′) E11.5 forelimb buds of embryos harvested from mothers injected with tamoxifen at E10.5. (C, C′, E, E′) Merged images showing counterstaining with DAPI (blue). (B′–E′) Higher magnification images of the boxed regions in B–E.

Mentions: We used qRT-PCR and immunofluorescent staining to determine the efficiency of depletion of Twist1 in limb buds. Quantitative RT-PCR analysis of RNA from dissected forelimb buds collected at E11.5, following tamoxifen injection at E9.5 revealed very low levels of Twist1 transcripts in conditional knockout (CKO) embryos (Fig. 1 A). Immunostaining of E11.5 limb buds for Twist1 protein revealed that, whereas Twist1 was detected widely in the limb bud mesenchyme of control embryos (Fig. 1 B, C, B′, C′), specific nuclear staining was almost undetectable in CKO limb bud tissues 24 hours after tamoxifen injection at E10.5 (Fig. 1 D, E, D′, E′). These results confirm that Ubc-CreERT2 activity can efficiently ablate Twist1 following tamoxifen treatment.


Timed deletion of Twist1 in the limb bud reveals age-specific impacts on autopod and zeugopod patterning.

Loebel DA, Hor AC, Bildsoe HK, Tam PP - PLoS ONE (2014)

Efficient ablation of Twist1 expression in conditional knockout embryos.(A) qRT-PCR analysis of RNA from anterior (A) and posterior (P) halves of forelimb buds collected E11.5 from mothers injected with tamoxifen at E9.5. n = 4 pairs of forelimb buds per genotype. (B–E, B′–E′.) Immunostaining with anti-Twist1 monoclonal antibody (green) of cryosectioned wild type (fl/wt) (B, C, B′, C′) and conditional knockout (CKO) (D, E, D′, E′) E11.5 forelimb buds of embryos harvested from mothers injected with tamoxifen at E10.5. (C, C′, E, E′) Merged images showing counterstaining with DAPI (blue). (B′–E′) Higher magnification images of the boxed regions in B–E.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4044014&req=5

pone-0098945-g001: Efficient ablation of Twist1 expression in conditional knockout embryos.(A) qRT-PCR analysis of RNA from anterior (A) and posterior (P) halves of forelimb buds collected E11.5 from mothers injected with tamoxifen at E9.5. n = 4 pairs of forelimb buds per genotype. (B–E, B′–E′.) Immunostaining with anti-Twist1 monoclonal antibody (green) of cryosectioned wild type (fl/wt) (B, C, B′, C′) and conditional knockout (CKO) (D, E, D′, E′) E11.5 forelimb buds of embryos harvested from mothers injected with tamoxifen at E10.5. (C, C′, E, E′) Merged images showing counterstaining with DAPI (blue). (B′–E′) Higher magnification images of the boxed regions in B–E.
Mentions: We used qRT-PCR and immunofluorescent staining to determine the efficiency of depletion of Twist1 in limb buds. Quantitative RT-PCR analysis of RNA from dissected forelimb buds collected at E11.5, following tamoxifen injection at E9.5 revealed very low levels of Twist1 transcripts in conditional knockout (CKO) embryos (Fig. 1 A). Immunostaining of E11.5 limb buds for Twist1 protein revealed that, whereas Twist1 was detected widely in the limb bud mesenchyme of control embryos (Fig. 1 B, C, B′, C′), specific nuclear staining was almost undetectable in CKO limb bud tissues 24 hours after tamoxifen injection at E10.5 (Fig. 1 D, E, D′, E′). These results confirm that Ubc-CreERT2 activity can efficiently ablate Twist1 following tamoxifen treatment.

Bottom Line: Gene expression analysis revealed significant upregulation of Hoxd10, Hoxd11 and Grem1 in the anterior half of the forelimb bud at E11.5.The specific skeletal phenotypes, which include duplication of digits and distal zeugopods but no overt posteriorization, differ from those of other Twist1 conditional knockout mutants.This outcome may be attributed to the deferment of Twist1 ablation to a later time frame of limb morphogenesis, which leads to the ectopic activation of posterior genes in the anterior tissues after the establishment of anterior-posterior anatomical identities in the forelimb bud.

View Article: PubMed Central - PubMed

Affiliation: Embryology Unit, Children's Medical Research Institute, Westmead, New South Wales, Australia; Sydney Medical School, The University of Sydney, Sydney, New South Wales, Australia.

ABSTRACT
Twist1 encodes a transcription factor that plays a vital role in limb development. We have used a tamoxifen-inducible Cre transgene, Ubc-CreERT2, to generate time-specific deletions of Twist1 by inducing Cre activity in mouse embryos at different ages from embryonic (E) day 9.5 onwards. A novel forelimb phenotype of supernumerary pre-axial digits and enlargement or partial duplication of the distal radius was observed when Cre activity was induced at E9.5. Gene expression analysis revealed significant upregulation of Hoxd10, Hoxd11 and Grem1 in the anterior half of the forelimb bud at E11.5. There is also localized upregulation of Ptch1, Hand2 and Hoxd13 at the site of ectopic digit formation, indicating a posterior molecular identity for the supernumerary digits. The specific skeletal phenotypes, which include duplication of digits and distal zeugopods but no overt posteriorization, differ from those of other Twist1 conditional knockout mutants. This outcome may be attributed to the deferment of Twist1 ablation to a later time frame of limb morphogenesis, which leads to the ectopic activation of posterior genes in the anterior tissues after the establishment of anterior-posterior anatomical identities in the forelimb bud.

Show MeSH
Related in: MedlinePlus