Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

Institut Pasteur

The dorsal midline of the neural tube has recently emerged as a major signaling center for dorsoventral patterning. Msx genes are expressed at the dorsal midline, although their function at this site remains unknown. Using Msx1(nlacZ) mutant mice, we show that the normal expression domain of Msx1 is interrupted in the pretectum of mutant embryos. Morphological and gene expression data further indicate that a functional midline is not maintained along the whole prosomere 1 in Msx1 mutant mice. This results in the downregulation of genes expressed laterally to the midline in prosomere 1, confirming the importance of the midline as a signaling center. Wnt1 is essential for dorsoventral patterning of the neural tube. In the Msx1 mutant, Wnt1 is downregulated before the midline disappears, suggesting that its expression depends on Msx1. Furthermore, electroporation in the chick embryo demonstrates that Msx1 can induce Wnt1 expression in the diencephalon neuroepithelium and in the lateral ectoderm. In double Msx1/Msx2 mutants, Wnt1 expression is completely abolished at the dorsal midline of the diencephalon and rostral mesencephalon. This indicates that Msx genes may regulate Wnt1 expression at the dorsal midline of the neural tube. Based on these results, we propose a model in which Msx genes are intermediary between Bmp and Wnt at this site.

Msx1 is required for dorsal diencephalon patterning.

Pluridigite ( Pdt) is a semi-dominant mutation obtained after a mutagenesis experiment with ethyl-nitroso-urea (ENU). The mutant exhibits abnormal skeletal pattern formation characterized by the formation of extra digits (polydactyly) in the preaxial (anterior) part of the hindlimbs. The phenotype shows incomplete penetrance, depending on the genetic background. In an F2 cross with C57BL/6, the phenotype could not be associated with a single locus. Strong linkage was observed with markers located on Chromosome (Chr) 12, in a 2-cM interval between D12Mit136 and D12Mit153. This region contains the Twist gene, and we show that the [Pdt] phenotype is dependent upon a new allele of Twist. We further identified that the whole Chr 4 is associated with the [Pdt] phenotype. The Pluridigite phenotype thus results from the combination of a Twist mutant allele and at least two additional loci.

A new mouse limb mutation identifies a Twist allele that requires interacting loci on chromosome 4 for its phenotypic expression.

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.

Analysis of Msx1; Msx2 double mutants reveals multiple roles for Msx genes in limb development.

The limb has been a privileged object of investigation and reflection for scientists over the past two centuries and continues to provide a heuristic framework to analyze vertebrate development. Recently, accumulation of new data has significantly changed our view on the mechanisms of limb patterning, in particular along the anterior-posterior axis. These data have led us to revisit the mode of action of the zone of polarizing activity. They shed light on the molecular and cellular mechanisms of patterning linked to the Shh-Gli3 signaling pathway and give insights into the mechanism of activation of these cardinal factors, as well as the consequences of their activity. These new data are in good part the result of systematic Application of tools used in contemporary mouse molecular genetics. These have extended the power of mouse genetics by introducing mutational strategies that allow fine-tuned modulation of gene expression, interchromosomal deletions and duplication. They have even made the mouse embryo amenable to cell lineage analysis that used to be the realm of chick embryos. In this review, we focus on the data acquired over the last five years from the analysis of mouse limb development and discuss new perspectives opened by these results.

Anteroposterior patterning in the limb and digit specification: contribution of mouse genetics.

Extra-toes is a semidominant mutation that affects the Gli3 gene and provokes limb and brain abnormalities. Among the different alleles of this mutation, Xt(H) is due to a deletion that has not yet been fully characterized. Using a PCR-based strategy, we undertook a high-resolution mapping of this deletion and confirmed that Xt(H) is a null allele of Gli3. We further designed a PCR test to identify unequivocally heterozygous and homozygous embryos from their wild-type littermates. Despite the length of the Xt(H) deletion, available data on the mouse genome indicate that no genes other than Gli3 are deleted in Xt(H) mutants. Thus, the Xt(H) mutation can be used as a model for studying the effects that absence of Gli3 function has during development.

High-resolution mapping of the Gli3 deletion in the mouse extra-toesH mutant.

The genetic mechanisms that control the establishment of early polarities and their link with embryonic axis specification and patterning seem to substantially diverge across vertebrates. In amphibians and teleosts, the establishment of an early dorso-ventral polarity determines both the site of axis formation and its rostro-caudal orientation. In contrast, amniotes retain a considerable plasticity for their site of axis formation until blastula stages and rely on signals secreted by extraembryonic tissues, which have no clear equivalents in the former, for the establishment of their rostro-caudal pattern. The rationale for these differences remains unknown. Through detailed expression analyses of key development genes in a chondrichthyan, the dogfish Scyliorhinus canicula, we have reconstructed the ancestral pattern of axis specification in jawed vertebrates. We show that the dogfish displays compelling similarities with amniotes at blastula and early gastrula stages, including the presence of clear homologs of the hypoblast and extraembryonic ectoderm. In the ancestral state, these territories are specified at opposite poles of an early axis of bilateral symmetry, homologous to the dorso-ventral axis of amphibians or teleosts, and aligned with the later forming embryonic axis, from head to tail. Comparisons with amniotes suggest that a dorsal expansion of extraembryonic ectoderm, resulting in an apparently radial symmetry at late blastula stages, has taken place in their lineage. The synthesis of these results with those of functional analyses in model organisms supports an evolutionary link between the dorso-ventral polarity of amphibians and teleosts and the embryonic-extraembryonic organisation of amniotes. It leads to a general model of axis specification in gnathostomes, which provides a comparative framework for a reassessment of conservations both among vertebrates and with more distant metazoans.

Evolution of axis specification mechanisms in jawed vertebrates: insights from a chondrichthyan.

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.

Generation of an Msx2-GFP conditional null allele.

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.

Msx genes are important apoptosis effectors downstream of the Shh/Gli3 pathway in the limb.

Msx1 and Msx2 encode homeodomain transcription factors that play a crucial role in limb development. However, the limb phenotype of the double Msx1(null/null) Msx2(null/null) mutant is difficult to analyze, particularly along the anteroposterior axis, because of the complex effects of the double mutation on both ectoderm- and mesoderm-derived structures. Namely, in the mutant, formation of the apical ectodermal ridge (AER) is impaired anteriorly and, consequently, the subjacent mesenchyme does not form. Using the Cre/loxP system, we investigated the respective roles of Msx genes in ectoderm and mesoderm by generating conditional mutant embryos with no Msx activity solely in the mesoderm. In these mutants, the integrity of the ectoderm-derived AER was maintained, allowing formation of the anterior mesenchyme. With this strategy, we demonstrate that mesenchymal expression of Msx1 and Msx2 is required for proper Shh and Bmp4 signaling to specify digit number and identity.

Msx1 and Msx2 in limb mesenchyme modulate digit number and identity.

Vessels are primarily formed from an inner endothelial layer that is secondarily covered by mural cells, namely vascular smooth muscle cells (VSMCs) in arteries and veins and pericytes in capillaries and veinules. We previously showed that, in the mouse embryo, Msx1(lacZ) and Msx2(lacZ) are expressed in mural cells and in a few endothelial cells. To unravel the role of Msx genes in vascular development, we have inactivated the two Msx genes specifically in mural cells by combining the Msx1(lacZ), Msx2(lox) and Sm22α-Cre alleles. Optical projection tomography demonstrated abnormal branching of the cephalic vessels in E11.5 mutant embryos. The carotid and vertebral arteries showed an increase in caliber that was related to reduced vascular smooth muscle coverage. Taking advantage of a newly constructed Msx1(CreERT2) allele, we demonstrated by lineage tracing that the primary defect lies in a population of VSMC precursors. The abnormal phenotype that ensues is a consequence of impaired BMP signaling in the VSMC precursors that leads to downregulation of the metalloprotease 2 (Mmp2) and Mmp9 genes, which are essential for cell migration and integration into the mural layer. Improper coverage by VSMCs secondarily leads to incomplete maturation of the endothelial layer. Our results demonstrate that both Msx1 and Msx2 are required for the recruitment of a population of neural crest-derived VSMCs.

Msx genes define a population of mural cell precursors required for head blood vessel maturation.

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.

Generation and characterization of a tamoxifen inducible Msx1(CreERT2) knock-in allele.

The Dlx and Msx homeodomain transcription factors play important roles in the control of limb development. The combined disruption of Msx1 and Msx2, as well as that of Dlx5 and Dlx6, lead to limb patterning defects with anomalies in digit number and shape. Msx1;Msx2 double mutants are characterized by the loss of derivatives of the anterior limb mesoderm which is not observed in either of the simple mutants. Dlx5;Dlx6 double mutants exhibit hindlimb ectrodactyly. While the morphogenetic action of Msx genes seems to involve the BMP molecules, the mode of action of Dlx genes still remains elusive. Here, examining the limb phenotypes of combined Dlx and Msx mutants we reveal a new Dlx-Msx regulatory loop directly involving BMPs. In Msx1;Dlx5;Dlx6 triple mutant mice (TKO), beside the expected ectrodactyly, we also observe the hallmark morphological anomalies of Msx1;Msx2 double mutants suggesting an epistatic role of Dlx5 and Dlx6 over Msx2. In Msx2;Dlx5;Dlx6 TKO mice we only observe an aggravation of the ectrodactyly defect without changes in the number of the individual components of the limb. Using a combination of qPCR, ChIP and bioinformatic analyses, we identify two Dlx/Msx regulatory pathways: 1) in the anterior limb mesoderm a non-cell autonomous Msx-Dlx regulatory loop involves BMP molecules through the AER and 2) in AER cells and, at later stages, in the limb mesoderm the regulation of Msx2 by Dlx5 and Dlx6 occurs also cell autonomously. These data bring new elements to decipher the complex AER-mesoderm dialogue that takes place during limb development and provide clues to understanding the etiology of congenital limb malformations.

Yvan Lallemand

Department of Developmental and Stem Cell Biology,
CNRS UMR3738,
Department of Immunology,
Department of Developmental and Stem Cell Biology, CNRS UMR3738, Department of Immunology

Institut Pasteur

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Yvan Lallemand

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Developmental and Stem Cell Biology,CNRS UMR3738,Department of Immunology,Department of Developmental and Stem Cell Biology, CNRS UMR3738, Department of Immunology

Institut Pasteur

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Department of Developmental and Stem Cell Biology,
CNRS UMR3738,
Department of Immunology,
Department of Developmental and Stem Cell Biology, CNRS UMR3738, Department of Immunology