Msx1creERT2 knock‐In allele: A useful tool to target embryonic and adult cardiac valves

Summary : Heart valve development begins with the endothelial‐to‐mesenchymal transition (EMT) of endocardial cells. Although lineage studies have demonstrated contributions from cardiac neural crest and epicardium to semilunar and atrioventricular (AV) valve formation, respectively, most valve mesenchyme derives from the endocardial EMT. Specific Cre mouse lines for fate‐mapping analyses of valve endocardial cells are limited. Msx1 displayed expression in AV canal endocardium and cushion mesenchyme between E9.5 and E11.5, when EMT is underway. Additionally, previous studies have demonstrated that deletion of Msx1 and its paralog Msx2 results in hypoplastic AV cushions and impaired endocardial signaling. A knock‐in tamoxifen‐inducible Cre line was recently generated (Msx1^CreERT2) and characterized during embryonic development and after birth, and was shown to recapitulate the endogenous Msx1 expression pattern. Here, we further analyze this knock‐in allele and track the Msx1‐expressing cells and their descendants during cardiac development with a particular focus on their contribution to the valves and their precursors. Thus, Msx1^CreERT2 mice represent a useful model for lineage tracing and conditional gene manipulation of endocardial and mesenchymal cushion cells essential to understand mechanisms of valve development and remodeling. genesis 53:337–345, 2015. © 2015 Wiley Periodicals, Inc.     

Msx1 is required for dorsal diencephalon patterning

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.     

Construction and characterization of a doxycycline‐inducible transgenic system in Msx2 expressing cells

Homeobox gene Msx2 is widely expressed during both embryogenesis and postnatal development and plays important roles during organogenesis. We developed an Msx2‐rtTA BAC transgenic line which can activate TetO‐Cre expression in Msx2‐expressing cells upon doxycycline (Dox) treatment. Using the Rosa26‐LacZ (R26R) reporter line, we show that rtTA is activated in Msx2‐expressing organs including the limb, heart, external genitalia, urogenital system, hair follicles and craniofacial regions. Moreover, we show that in body appendages, the transgene can be activated in different domains depending on the timing of Dox treatment. In addition, the transgene can also be effectively activated in adult tissues such as the hair follicle and the urogenital system. Taken together, this Msx2‐rtTA;TetO‐Cre system is a valuable tool for studying gene function in the development of the aforementioned organs in a temporal and spatially‐restricted manner, as well as for tissue lineage tracing of Msx2‐expressing cells. When induced postnatally, this system can also be used to study gene function in adult tissues without compromising normal development and patterning. genesis 47:352–359, 2009. © 2009 Wiley‐Liss, Inc.     

Neuroectodermal and endodermal expression of the ascidian Cdx gene is separated by metamorphosis

Ascidians are a group of invertebrate chordates that exhibit a biphasic life history, with chordate-specific structures developing during embryogenesis (dorsal neural tube and notochord) and metamorphosis (pharyngeal gill slits and endostyle). Here we characterize the expression of a caudal/Cdx gene homologue, Hec-Cdx, from the ascidian Herdmania curvata. Vertebrate Cdx genes are expressed at gastrulation and in the posterior of the developing neural tube and endoderm. Hec-Cdx expression is initiated at the earliest stages of gastrulation, with peaks in RNA abundance occurring first during neurulation and tailbud extension and then in 3- to 5-day-old juveniles. Hec-Cdx is expressed in a pair of cells in the anterior lip of the blastopore in the late gastrula which form the most posterior portion of the neural plate. During tailbud formation expression is maintained in and solely restricted to these cells. During metamorphosis expression is localized to the intestine of the juvenile. These data, along with data for the H. curvata Otx gene, suggest that the evolution of the novel ascidian biphasic body plan was not accompanied by a deployment of these genes into new pathways but by a temporal separation of tissue-specific expression. Received: 10 October 1999 / Accepted: 1 November 1999     

The expression of the homeobox gene Msx1 reveals two populations of dermal progenitor cells originating from the somites

Experimental manipulation in birds has shown that trunk dermis has a double origin: dorsally, it derives from the somite dermomyotome, while ventrally, it is formed by the somatopleure. Taking advantage of an nlacZ reporter gene integrated into the mouse Msx1 locus (Msx1(nlacZ) allele), we detected segmental expression of the Msx1 gene in cells of the dorsal mesenchyme of the trunk between embryonic days 11 and 14. Replacing somites from a chick host embryo by murine Msx1(nlacZ )somites allowed us to demonstrate that these Msx1-(beta)-galactosidase positive cells are of somitic origin. We propose that these cells are dermal progenitor cells that migrate from the somites and subsequently contribute to the dorsalmost dermis. By analysing Msx1(nlacZ) expression in a Splotch mutant, we observed that migration of these cells does not depend on Pax3, in contrast to other migratory populations such as limb muscle progenitor cells and neural crest cells. Msx1 expression was never detected in cells overlying the dermomyotome, although these cells are also of somitic origin. Therefore, we propose that two somite-derived populations of dermis progenitor cells can be distinguished. Cells expressing the Msx1 gene would migrate from the somite and contribute to the dermis of the dorsalmost trunk region. A second population of cells would disaggregate from the somite and contribute to the dermis overlying the dermomyotome. This population never expresses Msx1. Msx1 expression was investigated in the context of the onset of dermis formation monitored by the Dermo1 gene expression. The gene is downregulated prior to the onset of dermis differentiation, suggesting a role for Msx1 in the control of this process.     

Msx1 and Msx2 have shared essential functions in neural crest but may be dispensable in epidermis and axis formation in Xenopus

The homeodomain factors Msx1 and Msx2 are expressed in essentially identical patterns in the epidermis and neural crest of Xenopus embryos during neurula stages. Disruption of Msx1 and Msx2 RNA splicing with antisense morpholino oligonucleotides shows that both factors are also required for expression of the neural crest gene Slug. Loss of Msx1 can be compensated by overexpression of Msx2 and vice versa. Loss of Msx factors also leads to alterations in the expression boundaries for neural and epidermal genes, but does not prevent or reduce expression of epidermal keratin in ventrolateral ectoderm, nor is there a detectable effect on dorsal mesodermal marker gene expression. These results indicate that Msx1 and Msx2 are both essential for neural crest development, but that the two genes have the same function in this tissue. If Msx genes have important functions in epidermis or axial mesoderm induction, these functions must be shared with other regulatory proteins.     

Ascidian Wnt-7 gene is expressed exclusively in the tail neural tube of tailbud embryos

In vertebrate embryogenesis, many Wnt genes are expressed in the neural tube and play important roles in regional specifications. There are many subfamilies of Wnt, and each subfamily shows distinct expression patterns in the neural tube. Ascidian larvae have a dorsal hollow neural tube similar to that of vertebrates. To date, the degree of correspondence between regionality of the neural tubes of ascidians and vertebrates remains unclear. To compare cellular differences in neural tubes, Wnt genes can be used as molecular probes. We report here that a new member of the ascidian Wnt gene family, HrWnt-7, was expressed in the tail neural tube at the early tailbud stage. Moreover, in cross-section, HrWnt-7 was expressed in the dorsal and ventral ependymal cells. Received: 14 July 2000 / Accepted: 1 August 2000     

An amphioxus snail gene: Expression in paraxial mesoderm and neural plate suggests a conserved role in patterning the chordate embryo

 Homologs of the Drosophila snail gene have been characterized in several vertebrates. In addition to being expressed in mesoderm during gastrulation, vertebrate snail genes are also expressed in presumptive neural crest and/or its derivatives. Given that neural crest is unique to vertebrates and is considered to be of fundamental importance in their evolution, we have cloned and characterized the expression of a snail gene from amphioxus, a cephalochordate widely accepted as the sister group of the vertebrates. We show that, at the amino acid sequence level, the amphioxus snail gene is a clear phylogenetic outgroup to all the characterized vertebrate snail genes. During embryogenesis snail expression initially becomes restricted to the paraxial or presomitic mesoderm of amphioxus. Later, snail is expressed at high levels in the lateral neural plate, where it persists during neurulation. Our results indicate that an ancestral function of snail genes in the lineage leading to vertebrates is to define the paraxial mesoderm. Furthermore, our results indicate that a cell population homologous to the vertebrate neural crest may be present in amphioxus, thus providing an important link in the evolution of this key vertebrate tissue. Received: 11 May 1998 / Accepted: 2 August 1998     

Xgravin-like (Xgl), a novel putative a-kinase anchoring protein (AKAP) expressed during embryonic development in Xenopus

A-kinase anchoring proteins (AKAPs) are a heterogeneous family of scaffolding proteins that regulate the compartmentalization of signaling components, in particular that of the broad specificity kinase PKA. Here we describe the identification of a new member of this gene family, termed Xenopus gravin-like (Xgl), which encodes a highly acidic protein of 268 kDa that shares extensive homology with human Gravin and murine SSeCKS. Xgl is zygotically expressed in a highly dynamic fashion. During gastrulation Xgl is expressed in posterior mesoderm of the dorsal blastopore lip. During neurulation expression is transiently detected in the forebrain, two bilateral neuroectodermal stripes and the notochord. At tailbud stages expression commences in the mandibular neural crest and the roof of the spinal cord from where neural crest cells migrate into the intersomitic region. In addition expression is detected in the heart and the anterior aspect of the chordoneural hinge.     

Of mites and zen: expression studies in a chelicerate arthropod confirm zen is a divergent Hox gene

 We have cloned, from an oribatid mite, a gene homologous to the zerknült (zen) genes of insects and the Hox 3 genes of vertebrates. Hox genes specify cell fates in specific regions of the body in all metazoans studied and are expressed in antero-posteriorly restricted regions of the embryo. This is true of the vertebrate Hox 3 but not of the zen genes, the insect homologs, and it has been proposed that the zen genes have lost their Hox-like function in the ancestor of the insects. We studied expression of a mite Hox 3/zen homolog and found that it is expressed in a discrete antero-posterior region of the body with an anterior boundary coinciding with that of the chelicerate homolog of the Drosophila Hox gene, proboscipedia, and propose that its loss of Hox function in insects is due to functional redundancy due to this overlap with another Hox gene. Received: 23 April 1998 / Accepted: 25 August 1998     

A highly conserved Wnt-dependent TCF4 binding site within the proximal enhancer of the anti-myogenic Msx1 gene supports expression within Pax3-expressing limb bud muscle precursor cells

The product of the Msx1 gene is a potent inhibitor of muscle differentiation. Msx1 is expressed in muscle precursor cells of the limb bud that also express Pax3. It is thought that Msx1 may facilitate distal migration by delaying myogenesis in these cells. Despite the role played by Msx1 in inhibiting muscle differentiation, nothing is known of the mechanisms that support the expression of the Msx1 gene within limb bud muscle precursor cells. In the present study we have used a combination of comparative genomics, mouse transgenic analysis, in situ hybridisation and immunohistochemistry to identify a highly conserved and tissue-specific regulatory sub-domain within the previously characterised Msx1 gene proximal enhancer element that supports the expression of the Msx1 gene in Pax3-expressing mouse limb pre-muscle masses. Furthermore, using a combination of in situ hybridisation, in vivo ChIP assay and transgenic explant culture analysis we provide evidence that Msx1 expression in limb bud muscle precursor cells is dependent on the canonical Wnt/TCF signalling pathway that is important in muscle shape formation. The results of these studies provide evidence of a mechanistic link between the Wnt/TCF and the Msx1/Pax3/MyoD pathways within limb bud muscle precursor cells.     

Homeobox genes in axolotl lateral line placodes and neuromasts

 Gene expression has been studied in considerable detail in the developing vertebrate brain, neural crest, and some placode-derived organs. As a further investigation of vertebrate head morphogenesis, expression patterns of several homeobox-containing genes were examined using whole-mount in situ hybridization in a sensory system primitive for the vertebrate subphylum: the axolotl lateral lines and the placodes from which they develop. Axolotl Msx-2 and Dlx-3 are expressed in all of the lateral line placodes. Both genes are expressed throughout development of the lateral line system and their expression continues in the fully developed neuromasts. Expression within support cells is highly polarized. In contrast to most other observations of Msx genes in vertebrate organogenesis, expression of Msx-2 in developing lateral line organs is exclusively epithelial and is not associated with epithelial-mesenchymal interactions. A Hox-complex gene, Hoxb-3, is shown to be expressed in the embryonic hindbrain and in a lateral line placode at the same rostrocaudal level, but not in other placodes nor in mature lateral line organs. A Hox gene of a separate paralog group, Hoxa-4, is expressed in a more posterior hindbrain domain in the embryo, but is not expressed in the lateral line placode at that rostrocaudal level. These data provide the first test of the hypothesis that the neurogenic placodes develop in two rostrocaudal series aligned with the rhombomeric segments and patterned by combinations of Hox genes in parallel with the central nervous system. Received: 2 April 1997 / Accepted: 2 July 1997