The vertebrate homeobox genes Msx-1 and Msx-2 are related to the Drosophila msh gene and are expressed in a variety of tissues during embryogenesis. Msx-1 expression in the heart is apparent in a number of non-myocardial cell populations, including cells undergoing an epithelial to mesenchymal transformation regions involved in heart septation and valve formation. Msx-2 expression is restricted to a population of myocardial cells become the cardiac conduction system (Chan-Thomas, 1993).

Notochord grafted laterally to the neural tube enhances the differentiation of the vertebral cartilage at the expense of the derivatives of the dermomyotome. In contrast, the dorsomedial graft of a notochord inhibits cartilage differentiation of the dorsal part of the vertebra carrying the spinous process. Cartilage differentiation is preceded by the expression of Pax family (Pax1/Pax9, Drosophila homolog: Pox meso) transcription factors in the ventrolateral domain, and Msx family transcription factors in the dorsal domain. The proliferation and differentiation of Msx-expressing cells in the dorsal precartilaginous domain of the vertebra are stimulated by BMP4, which acts upstream of Msx genes. SHH protein arising from the notochord (and floor plate) is necessary for the survival and further development of Pax1/Pax9-expressing sclerotomal cells. Shh acts antagonistically to Bmp4. SHH-producing cells grafted dorsally to the neural tube at E2 inhibit expression of Bmp4 and Msx genes and also inhibit the differentiation of the spinous process (Watanabe, 1998).

In spite of the fact that vertebrae are formed by a single cell type, cartilage, their development involves different molecular pathways according to the vertebral region considered. The ventrolateral part of the vertebra (i.e. vertebral body and neural arches) develops from the ventral sclerotomal cells that express the transcription factor Pax1 before the onset of chondrogenesis. Previous work has shown that chondrogenesis of the ventrolateral part of the vertebra takes place under the influence of the notochord: a supernumerary notochord grafted dorsomedially to the somite extends the Pax1-expressing somitic domain dorsally, and subsequently its differentiation into cartilage takes place to the point that the development of the dorsal somitic derivatives (i.e. the dermomyotome) can be totally suppressed. The most dorsal part of the vertebra that closes the vertebral arch differentiates from mesenchymal cells of somitic origin. This occurs between two ectodermal layers: the superficial ectoderm and the roof plate. Thus, the unilateral graft of quail somites into chick embryos results in the formation of chimeric vertebrae with a hemivertebral body and hemispinous process, and a neural arch made up of donor cells on the operated side and host cells on the intact side. The limit between the host's and donor's territories corresponds strikingly to the sagittal plane of the embryo. Therefore, somitic cells with a chondrogenic fate must migrate medially in order to surround the neural tube and form the vertebral body ventrally and the spinous process dorsally. The cells that migrate dorsally from E3 onward fail to express Pax1 but start to express Msx1 and Msx2 as they become positioned between the superficial ectoderm and the roof plate, which produces BMP4. Moreover, the lateral graft of a roof plate or of cells producing BMP4 induces ectopic expression of Msx genes in the host somitic mesenchyme. Such an induction, however, can occur only if the inducer (e.g. the roof plate) is placed in close proximity to the superficial ectoderm. This supports the contention that bone formation in the subcutaneous site, where the spinous process is formed, is under the control of BMP4, and that Msx genes are involved in the pathway leading to chondrogenesis. This view was confirmed by the fact that overexpression of BMP4 (or of the closely related compound BMP2) dorsal to the neural tube results in the expansion of the Msx1- and Msx2-positive mesenchymal territory and subsequently in the enlargement of the spinous process. Duality in vertebral chondrogenesis was further underlined by the opposite effect of BMPs on the development of the ventrolateral part of the vertebra. Chondrogenesis was strongly inhibited by the graft of BMP2/4-producing cells in a ventrolateral position, with respect to the neural tube (Watanabe, 1998 and references).

These observations raised the question of the nature of the factor of notochord/floor plate origin that is responsible for chondrogenesis in the ventrolateral domain of the vertebra. The most obvious candidate was the protein SHH. Lateral grafts of SHH-producing cells do indeed enhance Pax1 expression in sclerotomal cells and induce the over-development of cartilage laterally at the level of the neural arches. The positive influence of SHH protein on Pax1 expression by somitic cells has already been demonstrated by in vitro experiments and in vivo by the use of retroviral vectors controlling Shh gene expression. This paper demonstrates that enhancement of the number of Pax1-expressing cells by SHH is followed in vivo by the increase in size of the ventrolateral part of the vertebral cartilage. In contrast, dorso-medial grafts of notochord and of SHH-QT6 cells inhibit the expression of the Bmp4 gene in dorsal ectoderm, dorsal mesenchyme and roof plate. Since Msx gene expression has been shown to be controlled by BMP signaling in several induction systems, it is probable that, under the experimental conditions described here, the inhibition of Bmp4 expression is primarily responsible for that of Msx1 and Msx2 and for the failure of chondrogenesis in the dorsal part of the vertebra. This leads to the identification of two molecular pathways in bone development. They concern cartilage and bone formation in 'deep' and 'subectodermal' positions, respectively. Ectoderm has previously been shown to reduce or inhibit chondrogenesis in somitic explant cultures. Such an inhibition is proposed to be relieved by the local production of BMP4 by the dorsal ectoderm and neural tube, thus allowing the formation of superficial bony structures from mesodermal (or mesectodermal) mesenchyme to take place. The deep vertebral cartilage that develops at a distance from the ectoderm and surrounds the notochord and the ventrolateral part of the neural tube requires SHH signaling to differentiate from the sclerotome (Watanabe, 1998 and references).

In mouse embryos, the muscle segment homeobox genes, Msx-1 and Msx-2 are expressed during critical stages of neural tube, neural crest, and craniofacial development, suggesting that these genes play important roles in organogenesis and cell differentiation. Although the patterns of expression are intriguing, little is known about the function of these genes in vertebrate embryonic development. Therefore, the expression of both genes, separately and together, was disrupted using antisense oligodeoxynucleotides and whole embryo culture techniques. Antisense attenuation of Msx-1 during early stages of neurulation produce hypoplasia of the maxillary, mandibular, and frontonasal prominences, eye anomalies, and somite and neural tube abnormalities. Eye defects consist of enlarged optic vesicles, which may ultimately result in micropthalmia similar to that observed in Small eye mice homozygous for mutations in the Pax-6 gene. Histological sections and SEM analysis reveal a thinning of the neuroepithelium in the diencephalon and optic vesicle and mesenchymal deficiencies in the craniofacial region. Injections of Msx-2 antisense oligodeoxynucleotides produce similar malformations as those targeting Msx-1, with the exception that there is an increase in number and severity of neural tube and somite defects. Embryos injected with the combination of Msx-1 + Msx-2 antisense oligodeoxynucleotides show no novel abnormalities, suggesting that the genes do not operate in a redundant manner (Foerst-Potts, 1997).

To understand how the complex embryonic expression pattern of the Msx1 gene is produced, a transgenic analysis of 13 kb of DNA around the Msx1 locus was carried out. Most of the extensive expression pattern of the Msx1 gene is reproduced in transgenics using the LacZ gene fused to 5 kb of Msx1 5' flanking DNA. Two enhancer domains are identified that produce this pattern. The distal element, located 4006 bp 5' from the Msx1 start site, produces expression in the first arch and the nasal epithelium and is restricted to 240 bp. However, the proximal element (PE), which produces expression in superficial nasal epithelium, dorsal and ventral myotome, limb mesenchyme, eye, ear, roof plate, second arch, genital ridge and epiphysis, is contained in only 78 bp. PE is an early enhancer in the eye and limb, driving expression in the limb bud mesenchyme from day 9 and persisting until the formation of the hand plate at day 10.5 (MacKenzie, 1997).

To dissect the cis-regulatory elements of the murine Msx-1 promoter, which lacks a conventional TATA element, a putative Msx-1 promoter DNA fragment (from -1282 to +106 base pairs [bp]) or its congeners containing site-specific alterations were fused to luciferase reporter and introduced into NIH3T3 and C2C12 cells; the expression of luciferase was assessed in transient expression assays. Multiple positive and negative regulatory elements participate in regulating transcription of the Msx-1 gene. The optimal expression of Msx-1 promoter in either NIH3T3 or C2C12 cells requires only 165 bp of the upstream sequence. 5'-flanking regions from -161 to -154 and from -26 to -13 of the Msx-1 promoter contain (respectively) an authentic E box (proximal E box), capable of binding a protein immunologically related to the upstream stimulating factor 1 (USF-1) and a GC-rich sequence motif which can bind to Sp1 (proximal Sp1). The promoter activation is seriously hampered if the proximal E box is removed or mutated, and the promoter activity is eliminated completely if the proximal Sp1 site is similarly altered. Absolute dependence of the Msx-1 minimal promoter on Sp1 can be demonstrated by transient expression assays in the Sp1-deficient Drosophila cell line cotransfected with Msx-1-luciferase and an Sp1 expression vector pPacSp1. The transgenic mice embryos containing -165/106-bp Msx-1 promoter-LacZ DNA in their genomes abundantly express beta-galactosidase in maxillae and mandibles and in the cellular primordia involved in the formation of the meninges and the bones of the skull. Thus, the truncated murine Msx-1 promoter can target expression of a heterologous gene in the craniofacial tissues of transgenic embryos known for high level of expression of the endogenous Msx-1 gene and found to be severely defective in the Msx-1 knock-out mice (Takahashi, 1997).

In myoblast cell cultures, the Msx1 protein is able to repress myogenesis and maintain cells in an undifferentiated and proliferative state. However, there has been no evidence that Msx1 is expressed in muscle or its precursors in vivo. Using mice with the nlacZ gene integrated into theMsx1 locus, it has been shown that the reporter gene is expressed in the lateral dermomyotome of brachial and thoracic somites. Cells from this region will subsequently contribute to forelimb and intercostal muscles. Using Pax3 gene transcripts as a marker of limb muscle progenitor cells as they migrate from the somites, the somitic origin and timing of cell migration from somites to limb buds has been precisely defined in the mouse. Differences in the timing of migration between chick and mouse are discussed. Somites that label for Msx1(nlacZ) transgene expression in the forelimb region partially overlap with those that contribute Pax3-expressing cells to the forelimb. In order to see whether Msx1 is expressed in this migrating population, somites from the forelimb level of Msx1(nlacZ) mouse embryos were grafted into a chick host embryo. Most cells migrating into the wing field express the Msx1(nlacZ) transgene, together with Pax3. In these experiments, Msx1 expression in the somite depends on the axial position of the graft. Wing mesenchyme is capable of inducing Msx1 transcription in somites that normally would not express the gene; chick hindlimb mesenchyme, while permissive for this expression, does not induce it. In the mouse limb bud, the Msx1(nlacZ) transgene is downregulated prior to the activation of the Myf5 gene, an early marker of myogenic differentiation. These observations are consistent with the proposal that Msx1 is involved in the repression of muscle differentiation in the lateral half of the somite and in limb muscle progenitor cells during their migration (Houzelstein, 1999).

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), segmental expression of the Msx1 gene was detected in cells of the dorsal mesenchyme underlying the ectoderm of the trunk between embryonic days 11 and 14. Replacing somites from a chick host embryo by murine Msx1^nlacZ somites allows for a demonstration that these Msx1-beta-galactosidase positive cells are of somitic origin. It is proposed 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, it was 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 is never detected in cells overlying the dermomyotome, although these cells are also of somitic origin. Therefore, it is proposed 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 are thought to 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 (coding for a bHLH transcription factor related to Twist) gene expression. The gene is downregulated prior to the onset of dermis differentiation, suggesting a role for Msx1 in the control of this process (Houzelstein, 2000).

The prospect of using cell replacement therapies has raised the key issue of whether elucidation of developmental pathways can facilitate the generation of therapeutically important cell types from stem cells. This study shows that the homeodomain proteins Lmx1a and Msx1 function as determinants of midbrain dopamine neurons, cells that degenerate in patients with Parkinson's disease. Lmx1a is sufficient and required to trigger dopamine cell differentiation. An early activity of Lmx1a is to induce the expression of Msx1, which complements Lmx1a by inducing the proneural protein Ngn2 and neuronal differentiation. Importantly, expression of Lmx1a in embryonic stem cells results in a robust generation of dopamine neurons with a 'correct' midbrain identity. These data establish that Lmx1a and Msx1 are critical intrinsic dopamine-neuron determinants in vivo and suggest that they may be essential tools in cell replacement strategies in Parkinson's disease (Andersson, 2006).

Neither the mechanisms that govern lip morphogenesis nor the cause of cleft lip are well understood. Four paired prominences give rise to the vertebrate face: the medial nasal (mnp), lateral nasal (lnp), maxillary (maxp) and mandibular (manp) prominences, which are derived from the frontonasal prominence and the first pharyngeal (or branchial) arch. Mutations of the Tgfbeta/Bmp and Fgf pathways can result in cleft palate by diminishing proliferation or increasing apoptosis in orofacial primordia. This study reports that genetic inactivation of Lrp6, a co-receptor of the Wnt/beta-catenin signaling pathway, leads to cleft lip with cleft palate. The activity of a Wnt signaling reporter is blocked in the orofacial primordia by Lrp6 deletion in mice. The morphological dynamic that is required for normal lip formation and fusion is disrupted in these mutants. The expression of the homeobox genes Msx1 and Msx2 is dramatically reduced in the mutants, which prevents the outgrowth of orofacial primordia, especially in the fusion site. that Msx1 and Msx2 (but not their potential regulator Bmp4) are the downstream targets of the Wnt/beta-catenin signaling pathway during lip formation and fusion. By contrast, a 'fusion-resistant' gene, Raldh3 (also known as Aldh1a3), that encodes a retinoic acid-synthesizing enzyme is ectopically expressed in the upper lip primordia of Lrp6-deficient embryos, indicating a region-specific role of the Wnt/beta-catenin signaling pathway in repressing retinoic acid signaling. Thus, the Lrp6-mediated Wnt signaling pathway is required for lip development by orchestrating two distinctively different morphogenetic movements (Song, 2009).

To understand the actions of morphogens, it is crucial to determine how they elicit different transcriptional responses in different cell types. A BMP-responsive enhancer of Msx2, an immediate early target of bone morphogenetic protein (BMP) signaling, has been identified. The BMP-responsive region of Msx2 consists of a core element, required generally for BMP-dependent expression, and ancillary elements that mediate signaling in diverse developmental settings. Analysis of the core element identified two classes of functional sites: GCCG sequences related to the consensus binding site of Mad/Smad-related BMP signal transducers, and a single TTAATT sequence, matching the consensus site for Antennapedia superclass homeodomain proteins. Chromatin immunoprecipitation and mutagenesis experiments indicate that the GCCG sites are direct targets of BMP restricted Smads. Intriguingly, however, these sites are not sufficient for BMP responsiveness in mouse embryos; the TTAATT sequence is also required. DNA sequence comparisons reveal this element is highly conserved in Msx2 promoters from mammalian orders but is not detectable in other vertebrates or non-vertebrates. Despite this lack of conservation outside mammals, the Msx2 BMP-responsive element (BMPRE) serves as an accurate readout of Dpp signaling in a distantly related bilaterian -- Drosophila. Strikingly, in Drosophila embryos, as in mice, both TTAATT and GCCG sequences are required for Dpp responsiveness, showing that a common cis-regulatory apparatus can mediate the transcriptional activation of BMP-regulated genes in widely divergent bilaterians (Brugger, 2004).

That the homeodomain and Smad consensus sites are required coordinately for the function of the Msx2 BMPRE in murine embryos, and that the sequence of the BMPRE is highly conserved among mammalian groups, raises the question of whether this combination of homeodomain and Smad consensus sites reflects an ancient mechanism for BMP-dependent transcriptional activation. Cis-regulatory elements can undergo mutational turnover yet maintain their function. Thus, despite the apparent lack of sequence conservation of the BMPRE outside mammals, the possibility remained that it might function in a more diverse group of organisms (Brugger, 2004).

To test this idea, it was asked whether the Msx2 BMPRE was capable of responding to Dpp signals in Drosophila. Drosophila was chosen because its distant relation to mouse would provide a stringent test of the hypothesis that the BMPRE can function over a large phylogenetic distance. In addition, a large number of mutants in components of the Dpp pathway are available, enabling a rigorous test of whether the murine element was responding appropriately to Dpp signals (Brugger, 2004).

Transgenic flies were produced bearing murine 560, 480, 220 and 52 bp sequences driving nuclear-localized lacZ. Expression was examined in imaginal discs and embryos, both in wild-type flies and in mutants in which Dpp signaling was perturbed. The 480bpMsx2-lacZ transgene was expressed in wing imaginal discs in a pattern that closely resembled that of vestigial, a known Dpp target. Ectopic activation of Dpp signaling with A9Gal4>TkvA resulted in a corresponding expansion of Msx2 transgene expression. In stage 13 embryos, the 220bpMsx2-lacZ transgene was expressed in parasegments (ps) 3 and 7 of embryo visceral mesoderm in a pattern closely matching that of dpp. This pattern was expanded throughout the gut in embryos in which dpp expression was driven ectopically by a heat shock promoter. In dpp (S11/S22) regulatory mutants, in which dpp signaling is lost specifically in ps3, the 220bpMsx2-lacZ transgene was also downregulated in ps3 (Brugger, 2004).

The 52 bp sequence drove expression on the dorsal side of early blastoderm embryos in a pattern that matched Dpp signaling. Expanded transgene expression was evident in dorsal mutant embryos, consistent with the finding that Dorsal represses dpp transcription in ventral cells, thus restricting Dpp signaling to the dorsal region of the embryo. Expression was also lost in screw mutant embryos, in which levels of Dpp signaling are reduced. Conversely, ectopic activation of Dpp signaling using Tub Gal4>UAS Dpp resulted in expansion of transgene expression in the dorsal half of the embryo. Similarly, embryos mutant for brinker, a repressor of Dpp signaling, exhibited expanded lacZ activity Brugger, 2004).

When the expression of 52bpMsx2-lacZ transgenes bearing the same GCCG or TTAATT site mutations used for transgenic mice was tested, each mutation resulted in a profound loss of transgene expression. Similar results were apparent in stage 13 embryos bearing these mutant transgenes . Together, these data show: (1) that sequences within the Msx2 promoter can respond to Dpp signaling in Drosophila embryos and wing imaginal discs, and (2) that both TTAATT and GCCG sites are crucial for expression of the Msx2 BMPRE in Drosophila embryos as they are in mouse embryos (Brugger, 2004).

A null allele of the mouse Msx1 homeobox gene has been generated by insertion of an nlacZ reporter gene into its homeobox. The sensitivity of beta-galactosidase detection permitted the revelation of Msx1 gene expression in heterozygous embryos, in particular in ectoderm and mesoderm during gastrulation, and in migrating neural crest cells. Homozygous mutant mice die at birth with facial defects. Msx1 deficient mice exhibit cleft palate and abnormalities of craniofacial and tooth development. To investigate the reason for this limited phenotype, the pattern of Msx1 expression was compared with that of the closely related Msx2 gene in wild type embryos and in Msx1-/- mutants. Whereas the expression of Msx1 and Msx2 overlap in the developing limb, this is notably not the case in the facial regions most affected in the mutant (Houzelstein, 1997).

The cause of autosomal dominant selective tooth agenesis in one humanfamily is a missense mutation resulting in an arginine-to-proline substitution in the homeodomain of MSX1. To determine whether the tooth agenesis phenotype may result from haploinsufficiency or a dominant-negative mechanism, biochemical and functional analyses of the mutant murine protein Msx1(R31P) was performed. Msx1(R31P) has perturbed structure and reduced thermostability, as compared with wild-type Msx1. As a consequence, the biochemical activities of Msx1(R31P) are severely impaired, since it exhibits little or no ability to interact with DNA or other protein factors or to function in transcriptional repression. Msx1(R31P) is inactive in vivo, since it does not display the activities of wild-type Msx1 in assays of ectopic expression in the limb. Furthermore, Msx1(R31P) does not antagonize the activity of wild-type Msx1 in any of these assays. Because Msx1(R31P) appears to be inactive and does not affect the action of wild-type Msx1, it is proposed that the phenotype of affected individuals with selective tooth agenesis is due to haploinsufficiency (Hu, 1998).

The Hmx homeobox gene family, represented in Drosophila by Muscle segment homeobox, is of ancient origin, being present in species as diverse as Drosophila, sea urchins and mammals. The three members of the murine Hmx family, designated Hmx1, Hmx2 and Hmx3, are expressed in tissues that suggest a common functional role in sensory organ development and pregnancy. Hmx3 is one of the earliest markers for vestibular inner ear development during embryogenesis, and is also upregulated in the myometrium of the uterus during pregnancy. Targeted disruption of the Hmx3 gene results in mice with abnormal circling behavior and severe vestibular defects owing to a depletion of sensory cells in the saccule and utricle, and a complete loss of the horizontal semicircular canal crista, as well as a fusion of the utricle and saccule endolymphatic spaces into a common utriculosaccular cavity. Both the sensory and secretory epithelium of the cochlear duct appear normal in the Hmx3 null animals. The majority of Hmx3 null females have a reproductive defect. Hmx3 null females can be fertilized and their embryos undergo normal preimplantation development, but the embryos fail to implant successfully in the Hmx3 null uterus and subsequently die. Transfer of preimplantation embryos from mutant Hmx3 uterine horns to wild-type pseudopregnant females results in successful pregnancy, indicating a failure of the Hmx3 null uterus to support normal post-implantation pregnancy. Molecular analysis reveals the perturbation of Hmx, Wnt and LIF gene expression in the Hmx3 null uterus. Interestingly, expression of both Hmx1 and Hmx2 is downregulated in the Hmx3 null uterus, suggesting a hierarchical relationship among the three Hmx genes during pregnancy (Wang, 1998).

Msx2-deficient mice exhibit progressive hair loss, starting at P14 and followed by successive cycles of wavelike regrowth and loss. During the hair cycle, Msx2 deficiency shortens anagen phase, but prolongs catagen and telogen. Msx2-deficient hair shafts are structurally abnormal. Molecular analyses suggest a Bmp4/Bmp2/Msx2/Foxn1 acidic hair keratin pathway is involved. These structurally abnormal hairs are easily dislodged in catagen implying a precocious exogen. Deficiency in Msx2 helps to reveal the distinctive skin domains on the same mouse. Each domain cycles asynchronously — although hairs within each skin domain cycle in synchronized waves. Thus, the combinatorial defects in hair cycling and differentiation, together with concealed skin domains, account for the cyclic alopecia phenotype (Ma, 2003).

What factors reside upstream of Msx2 in the matrix and precortex region? During hair differentiation, Bmp4 is expressed in hair matrix cells and in hair shaft cells in contact with the IRS. Bmp2 is specifically expressed in the precortex cells, while noggin is expressed in the dermal papilla. Ectopic expression of noggin in the hair matrix under a minimal Msx2 promoter disrupts hair differentiation, with the cells remaining in a highly proliferative state in the precortex and hair shaft regions. These results provides strong evidence that BMPs are required during hair differentiation. Msx2 expression is markedly reduced in these mice in which noggin expression is driven by Msx2 promoter. Conversely, Bmp4 expression is preserved in Msx2 mutant skin. In addition, since the defects in Msx2-deficient hair follicles are mainly restricted to the hair shaft and are less severe than those associated with abolition of Bmp signaling (which involves both IRS and hair shaft), Msx2 is likely to function downstream of Bmp genes during hair differentiation (Ma, 2003).

The dorsal midline of the neural tube is 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, it has been 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, a model is proposed in which Msx genes are intermediary between Bmp and Wnt at this site (Bach, 2003).

Patterning of the dorsal neural tube involves Bmp signaling, which results in activation of multiple pathways leading to the formation of neural crest, roof plate and dorsal interneuron cell types. Constitutive activation of Bmp signaling at early stages (HH10-12) of chick neural tube development induces roof-plate cell fate, accompanied by an increase of programmed cell death and a repression of neuronal differentiation. These activities are mimicked by the overexpression of the homeodomain transcription factor Msx1, a factor known to be induced by Bmp signaling. By contrast, the closely related factor, Msx3, does not have these activities. At later stages of neural tube development (HH14-16), dorsal progenitor cells lose their competence to generate roof-plate cells in response to Bmp signaling and instead generate dorsal interneurons. This aspect of Bmp signaling is phenocopied by the overexpression of Msx3 but not Msx1. Taken together, these results suggest that these two different Msx family members can mediate distinct aspects of Bmp signaling during neural tube development (Liu, 2004).

Two lines of evidence suggest that the neural bHLH genes that are crucial for neuronal differentiation might be direct transcriptional targets of Msx1. (1) In pursuit of factors that regulate the expression of the neural bHLH genes by yeast one-hybrid screening, Msx1 was identified to be potential regulator for both Math1 and Mash1 and several consensus sites for Msx1 binding are present in the enhancer regions of Math1/Cath1 and Mash1/Cash1. (2) Both Msx1 and Msx3 can bind to these consensus sites in vitro. However, because in vivo Msx1 represses the bHLH factor expression and Msx3 induces Cath1 expression, additional in vivo co-factors or chromatin properties that modulate these activities must be invoked (Liu, 2004).

The neural crest is a multipotent, migratory cell population that contributes to a variety of tissues and organs during vertebrate embryogenesis. This study focused on the function of Msx1 and Msx2, homeobox genes implicated in several disorders affecting craniofacial development in humans. Msx1/2 mutants exhibit profound deficiencies in the development of structures derived from the cranial and cardiac neural crest. These include hypoplastic and mispatterned cranial ganglia, dysmorphogenesis of pharyngeal arch derivatives and abnormal organization of conotruncal structures in the developing heart. The expression of the neural crest markers Ap-2alpha, Sox10 and cadherin 6 (cdh6) in Msx1/2 mutants revealed an apparent retardation in the migration of subpopulations of preotic and postotic neural crest cells, and a disorganization of neural crest cells paralleling patterning defects in cranial nerves. In addition, normally distinct subpopulations of migrating crest underwent mixing. The expression of the hindbrain markers Krox20 and Epha4 was altered in Msx1/2 mutants, suggesting that defects in neural crest populations may result, in part, from defects in rhombomere identity. Msx1/2 mutants also exhibited increased Bmp4 expression in migratory cranial neural crest and pharyngeal arches. Finally, proliferation of neural crest-derived mesenchyme was unchanged, but the number of apoptotic cells was increased substantially in neural crest-derived cells that contribute to the cranial ganglia and the first pharyngeal arch. This increase in apoptosis may contribute to the mispatterning of the cranial ganglia and the hypoplasia of the first arch (Ishii, 2005).

The mechanisms regulating germ line sex determination and meiosis initiation are poorly understood. This study provides evidence for the involvement of homeobox Msx transcription factors in foetal meiosis initiation in mammalian germ cells. Upon meiosis initiation, Msx1 and Msx2 genes are strongly expressed in the foetal ovary, possibly stimulated by soluble factors found there: bone morphogenetic proteins Bmp2 and Bmp4, and retinoic acid. Analysis of Msx1/Msx2 double mutant embryos revealed a majority of undifferentiated germ cells remaining in the ovary and, importantly, a decrease in the number of meiotic cells. In vivo, the Msx1/Msx2 double-null mutation prevented full activation of Stra8, a gene required for meiosis. In F9 cells, Msx1 can bind to Stra8 regulatory sequences and Msx1 overexpression stimulates Stra8 transcription. Collectively, these data demonstrate for the first time that some homeobox genes are required for meiosis initiation in the female germ line (Le Bouffant, 2011).