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.
Highlights
The vertebrate limb is patterned by the interplay of the regionalized activities of secreted morphogens and transcription factors that drive tissue morphogenesis
Regulation of sonic hedgehog (SHH) signaling in the limb bud requires the activity of the BMP antagonist Grem1, and FGF signaling from the apical ectodermal ridge (AER)
Previous studies of Twist1 and limb development have used tissue-specific Cre transgenes to delete Twist1 in the newly formed anterior mesoderm, including the mesoderm that contributes to the head and anterior forelimb (Mesp1-Cre; [15,24]) or in the limb buds shortly after initiation of outgrowth (Prrx1-Cre; [13,14])
Summary
The vertebrate limb is patterned by the interplay of the regionalized activities of secreted morphogens and transcription factors that drive tissue morphogenesis. Together they provide the positional information defining tissue patterns in the proximaldistal (PD), dorsal-ventral (DV), and anterior-posterior (AP) axes. Regulation of SHH signaling in the limb bud requires the activity of the BMP antagonist Grem, and FGF signaling from the apical ectodermal ridge (AER). Hoxa and Hoxd genes are important for regulating the number and arrangement of digits, development of the radius and ulna [9], and for initiating the SHH-BMP-FGF signaling loop [10]. Hoxa/d genes may act combinatorially in the AP patterning of the limb
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