RÉSUMÉ
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.
Sujet(s)
Bourgeons de membre/métabolisme , Protéines nucléaires/métabolisme , Protéine-1 apparentée à Twist/métabolisme , Animaux , Facteurs de transcription à motif basique hélice-boucle-hélice/génétique , Facteurs de transcription à motif basique hélice-boucle-hélice/métabolisme , Plan d'organisation du corps , Embryon de mammifère/métabolisme , Développement embryonnaire , Femelle , Membre thoracique/croissance et développement , Membre thoracique/métabolisme , Régulation de l'expression des gènes au cours du développement , Protéines à homéodomaine/génétique , Protéines à homéodomaine/métabolisme , Protéines et peptides de signalisation intercellulaire/génétique , Protéines et peptides de signalisation intercellulaire/métabolisme , Souris , Souris knockout , Protéines nucléaires/déficit , Protéines nucléaires/génétique , Récepteurs patched , Récepteur Patched-1 , Phénotype , Récepteurs de surface cellulaire/génétique , Récepteurs de surface cellulaire/métabolisme , Facteurs de transcription/génétique , Facteurs de transcription/métabolisme , Protéine-1 apparentée à Twist/déficit , Protéine-1 apparentée à Twist/génétique , Régulation positiveRÉSUMÉ
The basic helix-loop-helix transcription factor Twist1 is a key regulator of craniofacial development. Twist1-null mouse embryos exhibit failure of cephalic neural tube closure and abnormal head development and die at E11.0. To dissect the function of Twist1 in the cranial mesoderm beyond mid-gestation, we used Mesp1-Cre to delete Twist1 in the anterior mesoderm, which includes the progenitors of the cranial mesoderm. Deletion of Twist1 in mesoderm cells resulted in loss and malformations of the cranial mesoderm-derived skeleton. Loss of Twist1 in the mesoderm also resulted in a failure to fully segregate the mesoderm and the neural crest cells, and the malformation of some cranial neural crest-derived tissues. The development of extraocular muscles was compromised whereas the differentiation of branchial arch muscles was not affected, indicating a differential requirement for Twist1 in these two types of craniofacial muscle. A striking effect of the loss of Twist1 was the inability of the mesodermal cells to maintain their mesenchymal characteristics, and the acquisition of an epithelial-like morphology. Our findings point to a role of Twist1 in maintaining the mesenchyme architecture and the progenitor state of the mesoderm, as well as mediating mesoderm-neural crest interactions in craniofacial development.
Sujet(s)
Embryon de mammifère/métabolisme , Mésoderme/métabolisme , Protéines nucléaires/génétique , Protéine-1 apparentée à Twist/génétique , Animaux , Apoptose/génétique , Facteurs de transcription à motif basique hélice-boucle-hélice/génétique , Facteurs de transcription à motif basique hélice-boucle-hélice/métabolisme , Malformations crâniofaciales/génétique , Malformations crâniofaciales/métabolisme , Malformations crâniofaciales/anatomopathologie , Embryon de mammifère/embryologie , Technique d'immunofluorescence , Régulation de l'expression des gènes au cours du développement , Hybridation in situ , Mésoderme/embryologie , Souris , Souris de lignée C57BL , Souris knockout , Souris transgéniques , Modèles anatomiques , Modèles génétiques , Crête neurale/cytologie , Crête neurale/embryologie , Crête neurale/métabolisme , Protéines nucléaires/déficit , RT-PCR , Crâne/embryologie , Crâne/métabolisme , Facteurs temps , Protéine-1 apparentée à Twist/déficitRÉSUMÉ
Development of the mouse forelimb bud depends on normal Twist1 activity. Global loss of Twist1 function before limb bud formation stops limb development and loss of Twist1 throughout the mesenchyme after limb bud initiation leads to polydactyly, the ulnarization or loss of the radius and malformations and reductions of the shoulder girdle. Here we show that conditional deletion of Twist1 by Mesp1-Cre in the mesoderm that migrates into the anterior-proximal part of the forelimb bud results in the development of supernumerary digits and carpals, the acquisition of ulna-like characteristics by the radius and malformations of the humerus and scapula. The mirror-like duplications and posteriorization of pre-axial tissues are preceded by disruptions to anterior-posterior Shh, Bmp and Fgf signaling gradients and dysregulation of transcription factors that regulate anterior-posterior limb patterning.