ABSTRACT
Msx1, a member of the Msx gene family, encodes a homeodomain transcription factor and plays critical roles during mouse development in numerous organs. By homologous recombination, we generated a new Msx1 allele (Msx1(CreERT2) ) in which the CreERT2 fusion protein is produced in place of the endogenous Msx1 protein. Using different reporter mouse strains and appropriate tamoxifen treatments, we show that, in mice bearing the Msx1(CrERT2) allele, CreERT2 is capable to induce loxP genomic recombination specifically in Msx1-expressing cells and that this can be obtained during embryonic development as well as after birth. These results show that this new mouse line can be used for lineage tracing of Msx1-expressing cells and their descendants and, combined with Cre-inducible Msx null alleles, for the analysis of Msx1 and/or Msx2 functions in the Msx1-expressing organs, in a time-dependant manner.
Subject(s)
Alleles , Gene Expression Regulation/drug effects , Gene Knock-In Techniques/methods , MSX1 Transcription Factor/genetics , Tamoxifen/pharmacology , Animals , Cloning, Molecular , Embryo, Mammalian/drug effects , Flow Cytometry , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/immunology , Green Fluorescent Proteins/metabolism , Heterozygote , MSX1 Transcription Factor/metabolism , Mice , Mice, TransgenicABSTRACT
In tetrapods, the anteroposterior (AP) patterning of the limb is under the control of the antagonistic activities of the secreted factor Sonic hedgehog (Shh) and Gli3R, the truncated repressor form of the transcription factor Gli3. In this report, we show that Msx1 and Msx2 are targets and downstream effectors of Gli3R. Consequently, in Shh null mutants, Msx genes are overexpressed and, furthermore, partially responsible for the limb phenotype. This is exemplified by the fact that reducing Msx activity in Shh mutants partially restores a normal limb development. Finally, we show that the main action of the Msx genes, in both normal and Shh(-/-) limb development, is to control cell death in the mesenchyme. We propose that, in the limb, Msx genes act downstream of the Shh/Gli3 pathway by transducing BMP signaling and that, in the absence of Shh signaling, their deregulation contributes to the extensive apoptosis that impairs limb development.
Subject(s)
Apoptosis/physiology , Extremities/physiology , Hedgehog Proteins/physiology , Homeodomain Proteins/physiology , Kruppel-Like Transcription Factors/physiology , MSX1 Transcription Factor/physiology , Mesoderm/physiology , Nerve Tissue Proteins/physiology , Animals , Bone Morphogenetic Proteins/physiology , Extremities/embryology , Hedgehog Proteins/genetics , Homeodomain Proteins/genetics , MSX1 Transcription Factor/genetics , Mesoderm/embryology , Mice , Mice, Knockout , Mutation , Zinc Finger Protein Gli3ABSTRACT
Msx1 and Msx2, two members of the Msx gene family, encode homeoprotein transcription factors and play critical roles during mouse development. Because of the redundancy between the two genes, many of these roles can only be studied in double Msx1; Msx2 mutants. However, these animals die around 14.5 dpc, which precludes analysis of Msx gene function beyond this stage. Moreover, the pleiotropic defects displayed by these embryos make phenotypic analysis difficult. To overcome these restrictions and study the double Msx mutant phenotype at later stages, we generated an Msx2 conditional null allele using Cre/loxP technology. The strategy consisted of flanking the Msx2 gene coding sequence with two loxP sites. In addition, a green fluorescent protein (GFP) reporter gene was placed under Msx2 regulatory sequences in the modified locus. Our results demonstrate that the Msx2-GFP conditional allele behaves as a normal one, whereas Cre-mediated recombination creates an Msx2 null allele. With either allele, expression patterns of the GFP reporter gene and the Msx2 endogenous gene are identical.
Subject(s)
Alleles , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Gene Transfer Techniques , Green Fluorescent Proteins/genetics , Homeodomain Proteins/genetics , Animals , Cells, Cultured , DNA-Binding Proteins/biosynthesis , Female , Green Fluorescent Proteins/biosynthesis , Homeodomain Proteins/biosynthesis , Male , Mice , Mice, Inbred C57BL , Mice, KnockoutABSTRACT
The homeobox-containing genes Msx1 and Msx2 are highly expressed in the limb field from the earliest stages of limb formation and, subsequently, in both the apical ectodermal ridge and underlying mesenchyme. However, mice homozygous for a null mutation in either Msx1 or Msx2 do not display abnormalities in limb development. By contrast, Msx1; Msx2 double mutants exhibit a severe limb phenotype. Our analysis indicates that these genes play a role in crucial processes during limb morphogenesis along all three axes. Double mutant limbs are shorter and lack anterior skeletal elements (radius/tibia, thumb/hallux). Gene expression analysis confirms that there is no formation of regions with anterior identity. This correlates with the absence of dorsoventral boundary specification in the anterior ectoderm, which precludes apical ectodermal ridge formation anteriorly. As a result, anterior mesenchyme is not maintained, leading to oligodactyly. Paradoxically, polydactyly is also frequent and appears to be associated with extended Fgf activity in the apical ectodermal ridge, which is maintained up to 14.5 dpc. This results in a major outgrowth of the mesenchyme anteriorly, which nevertheless maintains a posterior identity, and leads to formation of extra digits. These defects are interpreted in the context of an impairment of Bmp signalling.
