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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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Quantitative RT-PCR analysis of gene expression in forelimb buds.Pairs of forelimb buds dissected from control (fl/wt) or conditional knockout (cko) E11.5 embryos harvested from mothers injected with tamoxifen at E9.5 were dissected into anterior (A) and posterior (P) halves and qRT-PCR was performed for Hoxd10 (A), Hoxd11 (B), Hoxd13 (C), Hand2 (D), Grem1 (E) and Ptch1 (F). Asterisk indicates p<0.05 in two-tailed t-test between anterior halves of fl/wt and cko limb buds. Reference gene was Polr2a. n = 4 embryos for each tissue and genotype.
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pone-0098945-g004: Quantitative RT-PCR analysis of gene expression in forelimb buds.Pairs of forelimb buds dissected from control (fl/wt) or conditional knockout (cko) E11.5 embryos harvested from mothers injected with tamoxifen at E9.5 were dissected into anterior (A) and posterior (P) halves and qRT-PCR was performed for Hoxd10 (A), Hoxd11 (B), Hoxd13 (C), Hand2 (D), Grem1 (E) and Ptch1 (F). Asterisk indicates p<0.05 in two-tailed t-test between anterior halves of fl/wt and cko limb buds. Reference gene was Polr2a. n = 4 embryos for each tissue and genotype.

Mentions: To further investigate the forelimb autopod and zeugopod defects in TAM E9.5 embryos, we examined the expression of genes that are normally differentially expressed in anterior and posterior parts of the forelimb bud by qRT-PCR (n = 4 embryos, collected at E11.5, for each genotype and transcript measured). In control embryos, Twist1 is preferentially expressed in the anterior half of the forelimb bud, and in TAM E9.5 embryos its expression was reduced to very low levels in both anterior and posterior fragments (Fig. S1 A). Hoxd10 and Hoxd11 are normally expressed most strongly in the posterior part of forelimb bud. In forelimb buds from TAM E9.5 CKO embryos, both genes remained strongly expressed in the posterior half of the bud, but were significantly up-regulated in the anterior half of the limb bud (Fig 4 A, B). This is consistent with a previous observation that Hoxd11 is upregulated in compound Twist1ska10/- mutant limb buds [13]. In contrast, Hoxd13 was not significantly upregulated in CKO anterior limb bud tissues compared to controls (Fig 4 C). Hand2, encoding a bHLH transcription factor normally expressed in the posterior limb bud also showed no significant upregulation (Fig. 4 D). Grem1, encoding a BMP antagonist that depends on Hoxd activity to initiate its expression [10] showed significantly higher expression in CKO anterior forelimb bud tissue than in control forelimb buds (Fig. 4 E). The expression of Ptch1, a target of SHH signaling, was not increased in CKO anterior forelimb relative to controls (Fig. 4 F) but was reduced in the posterior tissue of some CKO specimens. This suggested that there was a globally reduced level of SHH signaling in some TAM E9.5 CKO limb buds, which was also encountered in Twist1- embryos [12].


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)

Quantitative RT-PCR analysis of gene expression in forelimb buds.Pairs of forelimb buds dissected from control (fl/wt) or conditional knockout (cko) E11.5 embryos harvested from mothers injected with tamoxifen at E9.5 were dissected into anterior (A) and posterior (P) halves and qRT-PCR was performed for Hoxd10 (A), Hoxd11 (B), Hoxd13 (C), Hand2 (D), Grem1 (E) and Ptch1 (F). Asterisk indicates p<0.05 in two-tailed t-test between anterior halves of fl/wt and cko limb buds. Reference gene was Polr2a. n = 4 embryos for each tissue and genotype.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098945-g004: Quantitative RT-PCR analysis of gene expression in forelimb buds.Pairs of forelimb buds dissected from control (fl/wt) or conditional knockout (cko) E11.5 embryos harvested from mothers injected with tamoxifen at E9.5 were dissected into anterior (A) and posterior (P) halves and qRT-PCR was performed for Hoxd10 (A), Hoxd11 (B), Hoxd13 (C), Hand2 (D), Grem1 (E) and Ptch1 (F). Asterisk indicates p<0.05 in two-tailed t-test between anterior halves of fl/wt and cko limb buds. Reference gene was Polr2a. n = 4 embryos for each tissue and genotype.
Mentions: To further investigate the forelimb autopod and zeugopod defects in TAM E9.5 embryos, we examined the expression of genes that are normally differentially expressed in anterior and posterior parts of the forelimb bud by qRT-PCR (n = 4 embryos, collected at E11.5, for each genotype and transcript measured). In control embryos, Twist1 is preferentially expressed in the anterior half of the forelimb bud, and in TAM E9.5 embryos its expression was reduced to very low levels in both anterior and posterior fragments (Fig. S1 A). Hoxd10 and Hoxd11 are normally expressed most strongly in the posterior part of forelimb bud. In forelimb buds from TAM E9.5 CKO embryos, both genes remained strongly expressed in the posterior half of the bud, but were significantly up-regulated in the anterior half of the limb bud (Fig 4 A, B). This is consistent with a previous observation that Hoxd11 is upregulated in compound Twist1ska10/- mutant limb buds [13]. In contrast, Hoxd13 was not significantly upregulated in CKO anterior limb bud tissues compared to controls (Fig 4 C). Hand2, encoding a bHLH transcription factor normally expressed in the posterior limb bud also showed no significant upregulation (Fig. 4 D). Grem1, encoding a BMP antagonist that depends on Hoxd activity to initiate its expression [10] showed significantly higher expression in CKO anterior forelimb bud tissue than in control forelimb buds (Fig. 4 E). The expression of Ptch1, a target of SHH signaling, was not increased in CKO anterior forelimb relative to controls (Fig. 4 F) but was reduced in the posterior tissue of some CKO specimens. This suggested that there was a globally reduced level of SHH signaling in some TAM E9.5 CKO limb buds, which was also encountered in Twist1- embryos [12].

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