Interactive Fly, Drosophila

muscle segment homeobox


BMPs and Msx homologs

Patterning of the embryonic ectoderm is dependent upon the action of negative (antineural) and positive (neurogenic) transcriptional regulators. Msx1 and Dlx3 are two antineural genes for which the anterior epidermal-neural boundaries of expression differ, probably due to differential sensitivity to BMP signaling in the ectoderm. In the extreme anterior neural plate, Dlx3 is strongly expressed while Msx1 is silent. While both of these factors prevent the activation of genes specific to the nascent central nervous system, Msx1 inhibits anterior markers, including Otx2 and cement gland-specific genes. Dlx3 has little, if any, effect on these anterior neural plate genes, instead providing a permissive environment for their expression while repressing more panneural markers, including prepattern genes belonging to the Zic family and BF-1. Zic3 is activated by chordin and suppressed by BMP4; overexpression of this factor results in conversion of ectoderm to anterior neural tissue. The finding that Dlx3 is able to suppress the activation of Zic3 suggests that a Dlx3-mediated regulatory step might exist between the initial disruption of BMP signaling and activation of this gene. To test this hypothesis, truncated BMP-4 receptor, Dlx3 and Zic3 RNAs were injected in combinations, followed by animal cap excision, culture and RNA isolation for Northern blot analysis. Dlx3 blocks the activation of the panneural marker Nrp1 by truncated BMP-4 receptor. Addition of Zic3 RNA to the injection mixture restores Nrp1 expression to levels comparable to those of truncated BMP-4 induced caps. Based on these results, it is concluded that the inductive effects of Zic3 function downstream of the antineurogenic stem mediated by Dlx3. These properties define a molecular mechanism for translating the organizer-dependent morphogenic gradient of BMP activity into spatially restricted gene expression in the prospective anterior neural plate (Feledy, 1999).

There is a striking parallel between the expression patterns of the Bmp4 (Drosophila homolog: decapentaplegic), Msx1 and Msx2 genes in the lateral ridges of the neural plate before neural tube closure and later on, in the dorsal neural tube and superficial midline ectoderm. The spinous process of the vertebra is formed from Msx1- and 2-expressing mesenchyme and that the dorsal neural tube can induce the differentiation of subcutaneous cartilage from the somitic mesenchyme. Mouse BMP4- or human BMP2-producing cells grafted dorsally to the neural tube at E2 or E3 increase considerably the amount of Msx-expressing mesenchymal cells which are normally recruited from the somite to form the spinous process of the vertebra. Later on, the dorsal part of the vertebra is enlarged, resulting in vertebral fusion and, in some cases (e.g. grafts made at E3), in the formation of a 'giant' spinous process-like structure dorsally. In strong contrast, BMP-producing cells grafted laterally to the neural tube at E2 exerted a negative effect on the expression of Pax1 and Pax3 genes in the somitic mesenchyme, which then turned on Msx genes. Moreover, sclerotomal cell growth and differentiation into cartilage were then inhibited. Dorsalization of the neural tube, manifested by expression of Msx and Pax3 genes in the basal plate contacting the BMP-producing cells, was also observed. In conclusion, this study demonstrates that differentiation of the ventrolateral and dorsal parts of the vertebral cartilage is controlled by different molecular mechanisms. The former develops under the influence of signals arising from the floor plate-notochord complex. These signals inhibit the development of dorsal subcutaneous cartilage forming the spinous process, which requires the influence of BMP4 to differentiate (Monsoro-Burg, 1996).

The mouse homeobox-genes Msx-1 and Msx-2 are expressed in several areas of the developing embryo, including the neural tube, neural crest, facial processes and limb buds. A third mouse Msx gene, designated Msx-3, has been isolated. The embryonic expression of Msx-3 differs from that of Msx-1 and -2 in that it is confined to the dorsal neural tube. In embryos with 5-8 somites, a segmental pattern of expression is observed in the hindbrain, with rhombomeres 3 and 5 lacking Msx-3 while other rhombomeres express Msx-3. This pattern of rhombomere expression is, however, transient: in embryos with 18 or more somites, expression is continuous throughout the dorsal hindbrain and anterior dorsal spinal cord. Differentiation of dorsal cell types in the neural tube can be induced by the addition of members of the Tgf-beta family. Additionally, Msx-1 and -2 have been shown to be activated by the addition of the Tgf-beta family member Bmp-4. To determine if Bmp-4 can activate Msx-3, embryonic hindbrain explants were incubated with exogenous Bmp-4. The dorsal expression of Msx-3 is seen to expand into more ventral regions of the neurectoderm in Bmp-4-treated cultures, implying that Bmp-4 may be able to mimic an in vivo signal that induces Msx-3 (Shimeld, 1996).

The mechanisms by which programmed cell death is spatially regulated are not well characterized. Msx1 and Msx2 are two closely related homeobox-containing genes that are expressed at sites where cellular proliferation and programmed cell death occur, including the developing limb and the cephalic neural crest. Tissue interactions are necessary for the maintenance of Msx1 and Msx2 expression and programmed cell death. It has been demonstrated that BMP4 can regulate cell death at these same sites as well as induce Msx expression. These observations lead to the hypothesis that Msx2 is a key regulator of cell death in the BMP-mediated pathway. Embryonic stem (ES) cell lines will undergo processes typical of early embryogenesis upon aggregation and have recently been shown to provide a model system for programmed cell death. In contrast to ES cells, P19 cells do not undergo pronounced cell death upon aggregation; however, constitutive ectopic Msx2 expression in P19 cells results in a marked increase in apoptosis induced upon aggregation but has no effect when cells are grown as a monolayer. If aggregates are allowed to interact with a substrate, the process of programmed cell death is completely inhibited. Addition of BMP4 to aggregated P19 cells also results in cell death; however, BMP4 does not increase levels of cell death in Msx2-expressing cells. Addition of BMP4 to P19 cells results in an induction of Msx2 transcription consistent with Msx2's proposed role in cell death in the embryo. These data support a model by which BMP4 induces programmed cell death via an Msx2-mediated pathway and provide direct functional evidence that Msx2 expression is a regulator of this process (Marazzi, 1997).

The facial primordia initially consist of buds of undifferentiated mesenchyme, which give rise to a variety of tissues including cartilage, muscle and nerve. These must be arranged in a precise spatial order for correct function. The bone morphogenetic proteins Bmp-2 and Bmp-4 are expressed in discrete regions at the distal tips of the early facial primordia suggesting possible roles for BMP-2 and BMP-4 during chick facial development. Expression of Bmp-4 and Bmp-2 is correlated with the expression of Msx-1 and Msx-2; ectopic application of BMP-2 and BMP-4 can activate Msx-1 and Msx-2 gene expression in the developing facial primordia. This activation of gene expression is correlated with changes in skeletal development. For example, activation of Msx-1 gene expression across the distal tip of the mandibular primordium is associated with an extension of Fgf-4 expression in the epithelium and bifurcation of Meckel's cartilage. In the maxillary primordium, extension of the normal domain of Msx-1 gene expression is correlated with extended epithelial expression of Sonic Hedgehog and bifurcation of the palatine bone. Application of BMP-2 can increase cell proliferation of the mandibular primordia. This work suggests that BMP-2 and BMP-4 are part of a signaling cascade that controls outgrowth and patterning of the facial primordia (Barlow, 1997).

Growth and differentiation of postnatal hair follicles are controlled by reciprocal interactions between the dermal papilla and the surrounding epidermal hair precursors. The first visible sign of hair formation is the hair placode, a thickening of the embryonic ectoderm induced by the underlying mesoderm. A signal from the hair placode then causes the mesenchymal cells to condense. A second mesodermal signal in turn induces proliferation in the ectodermal placode, which starts to grow down into the mesenchyme. Eventually, the epithelial cells surround the mesodermal condensation, which forms the dermal papilla, a permanent structure at the base of the follicle thought to control growth and differentiation of the hair. Once the basic structure of the hair follicle is established, differentiation of the concentric layers of keratinocytes begins. The peripheral layer forms the outer root sheath (ORS), which is continuous with the basal layer of the epidermis. Within the hair follicle, two new layers, the inner root sheath (IRS) and the hair shaft, develop from proliferating precursor cells in the matrix region surrounding the dermal papilla. As cells move distally, they stop dividing and differentiate according to their medio-lateral position within the follicle. Cells positioned next to the ORS form the IRS while centrally located precursors give rise to the hair shaft, consisting of the outer cuticle, the cortex and the central medulla (Kulessa, 2000).

The molecular nature of these interactions is largely unknown, but they are likely to involve several families of signaling molecules, including Fgfs, Wnts and Bmps. To analyze the function of Bmp signaling in postnatal hair development, transgenic mice were generated expressing the Bmp inhibitor, Noggin, under the control of the proximal Msx2 promoter, which drives expression in proliferating hair matrix cells and differentiating hair precursor cells. Differentiation of the hair shaft but not the inner root sheath is severely impaired in Msx2-Noggin transgenic mice. In addition to hair keratins, the expression of several transcription factors implicated in hair development, including Foxn1 (HNF-3/forkhead homolog 11) and Hoxc13, is severely reduced in the transgenic hair follicles. Proliferating cells, which are normally restricted to the hair matrix surrounding the dermal papilla, are found in the precortex and hair shaft region. These results identify Bmps as key regulators of the genetic program controlling hair shaft differentiation in postnatal hair follicles (Kulessa, 2000).

The expression of four genes, Foxn1, Hoxc13, Msx1 and Msx2, is strongly reduced or absent in Msx2-Noggin transgenic hair follicles, suggesting that they lie in a genetic pathway directly controlled by Bmp signaling. Can the loss of any of these genes account for the phenotype of the Msx2-Noggin transgenic mice? Foxn1 mutant nude mice develop a normal number of hair follicles, but show incomplete differentiation of the hair shafts, which form a discernible hair cortex and medulla but rarely penetrate the skin. Although Foxn1, like Bmps, is thought to influence both the proliferation and differentiation of hair keratinocytes, the nude hair phenotype is much weaker than that of the Msx2-Noggin transgenic mice. The loss of Foxn1 expression in the Noggin transgenic hair follicles may therefore contribute to the observed phenotype, but does not fully explain it. Hoxc13 mutant mice develop hair follicles with hair shafts that do not protrude through the skin. The morphological abnormalities and the molecular nature of the defect in Hoxc13 mutant hair follicles have not been characterized extensively, so it is difficult to say how they compare to the Msx2-Noggin phenotype. Nevertheless, the co-expression of Bmp4 and Hoxc13 is very striking. It is not only seen in the hair follicle, but extends to other keratinized structures like the nails and the filiform papillae of the tongue, suggesting that Bmps and Hoxc13 form part of a more general genetic program directing ‘hard’ keratin expression. Msx1 and Msx2 have been proposed as Bmp target genes in a number of tissues, suggesting that they mediate a common rather than a tissue-specific function of Bmp signaling. Overexpression of Msx2 in the hair matrix reduces proliferation and induces premature differentiation, consistent with it mediating part of the growth regulatory functions of Bmps. Gene inactivation of Msx1 and 2 has shown that they function redundantly during hair development. The requirement of Msx function for hair follicle induction raises the possibility that Bmp activity is already necessary at early stages of hair follicle development to maintain expression of Msx1 and 2. None of the Bmp-regulated transcription factors identified here clearly mediates the entire function of Bmp signaling in the hair follicle revealed by this study. This implies that it is the coordinated regulation of multiple factors by Bmps that controls hair shaft differentiation rather than the activation of a single key regulator (Kulessa, 2000 and references therein).

The differentiation, survival, and proliferation of developing sympathetic neuroblasts are all coordinately promoted by neurotrophins. Bone morphogenetic protein 4 (BMP4), a factor known to be necessary for the differentiation of sympathetic neurons, conversely reduces both survival and proliferation of cultured E14 sympathetic neuroblasts. The anti-proliferative effects of BMP4 occur more rapidly than the pro-apoptotic actions and appear to involve different intracellular mechanisms. BMP4 treatment induces expression of the transcription factor Msx-2 and the cyclin-dependent kinase inhibitor p21CIP1/WAF1 (p21). Treatment of cells with oligonucleotides antisense to either of these genes prevents cell death after BMP4 treatment but does not significantly alter the anti-proliferative effects. Thus Msx-2 and p21 are necessary for BMP4-mediated cell death but not for promotion of exit from cell cycle. Although treatment of cultured E14 sympathetic neuroblasts with neurotrophins alone does not alter cell numbers, BMP4-induced cell death was prevented by co-treatment with either neurotrophin-3 (NT-3) or nerve growth factor (NGF). This suggests that BMP4 may also induce dependence of the cells on neurotrophins for survival. Thus, sympathetic neuron numbers may be determined in part by factors that inhibit the proliferation and survival of neuroblasts and make them dependent upon exogenous factors for survival (Gomes, 2001).

The mammary glands develop initially as buds arising from the ventral embryonic epidermis. Recent work has shed light on signaling pathways leading to the patterning and formation of the mammary placodes and buds in mouse embryos. Relatively little is known of the signaling pathways that initiate branching morphogenesis and the formation of the ducts from the embryonic buds. Previous studies have shown that parathyroid hormone-related protein (PTHrP; also known as parathyroid hormone-like peptide, Pthlh) is produced by mammary epithelial cells and acts on surrounding mesenchymal cells to promote their differentiation into a mammary-specific dense mesenchyme. As a result of PTHrP signaling, the mammary mesenchyme supports mammary epithelial cell fate, initiates ductal development and patterns the overlying nipple sheath. In this report, it is demonstrated that PTHrP acts, in part, by sensitizing mesenchymal cells to BMP signaling. PTHrP upregulates BMP receptor 1A expression in the mammary mesenchyme, enabling it to respond to BMP4, which is expressed within mesenchymal cells underlying the ventral epidermis during mammary bud formation. BMP signaling is important for outgrowth of normal mammary buds and BMP4 can rescue outgrowth of PTHrP-/- mammary buds. In addition, the combination of PTHrP and BMP signaling is responsible for upregulating Msx2 gene expression within the mammary mesenchyme, and disruption of the Msx2 gene rescues the induction of hair follicles on the ventral surface of mice overexpressing PTHrP in keratinocytes (K14-PTHrP). These data suggest that PTHrP signaling sensitizes the mammary mesenchyme to the actions of BMP4, triggering outgrowth of the mammary buds and inducing MSX2 expression, which, in turn, leads to lateral inhibition of hair follicle formation within the developing nipple sheath (Hens, 2007).

MSX protein interactions

The MSX-1 homeodomain protein is a potent transcriptional repressor. MSX-1 interacts directly with the TATA binding protein (TBP). This interaction is mediated by the MSX-1 homeodomain, specifically through residues in the N-terminal arm. These same N-terminal arm residues are required for repression by MSX-1, suggesting a functional relationship between TBP association and transcriptional repression. DNA binding activity is separable from both TBP interaction and repression (Zhang, 1996).

Msx2 is a homeobox gene with a regulatory role in inductive tissue interactions, including those that pattern the skull. Individuals affected with an autosomal dominant disorder of skull morphogenesis (craniosynostosis, Boston type) bear a mutated form of Msx2 in which a histidine is substituted for a highly conserved proline in position 7 of the N-terminal arm of the homeodomain (p148h). The mutation behaves as a dominant positive in transgenic mice. The location of the mutation in the N-terminal arm of the homeodomain, a region which in other homeodomain proteins plays a key part in protein-protein interactions, prompted the undertaking of a yeast two hybrid screen for Msx2-interacting proteins. One such protein, designated Miz1 (Msx-interacting-zinc finger) is a zinc finger-containing protein whose amino acid sequence closely resembles that of the yeast protein Nfi-1. Together these proteins define a new, highly conserved protein family. Analysis of Miz1 expression by Northern blot and in situ hybridization reveals a spatiotemporal pattern that overlaps that of Msx2. Miz1 is a sequence specific DNA binding protein, and it can function as a positive-acting transcription factor. Miz1 interacts directly with Msx2 in vitro and enhances the DNA binding affinity of Msx2 for a functionally important element in the rat osteocalcin promoter. The p148h mutation in Msx2 augments the Miz1 effect on Msx2 DNA binding, suggesting a reason why this mutation behaves in vivo as a dominant positive, and providing a potential explanation of the craniosynostosis phenotype (Wu, 1997).

Protein-protein interactions are known to be essential for specifying the transcriptional activities of homeoproteins. Representative members of the Msx and Dlx homeoprotein families are shown to form homo- and hetero-dimeric complexes. Dimerization by Msx and Dlx proteins is mediated through their homeodomains and the residues required for this interaction correspond to those necessary for DNA binding. Unlike most other known examples of homeoprotein interactions, association of Msx and Dlx proteins does not promote cooperative DNA binding; instead, dimerization and DNA binding are mutually exclusive activities. Msx and Dlx proteins interact independently and noncooperatively with homeodomain DNA binding sites and dimerization is specifically blocked by the presence of such DNA sites. The transcriptional properties of Msx and Dlx proteins display reciprocal inhibition. Specifically, Msx proteins act as transcriptional repressors and Dlx proteins act as activators, while in combination, Msx and Dlx proteins counteract each other's transcriptional activities. The expression patterns of representative Msx and Dlx genes (Msx1, Msx2, Dlx2, and Dlx5) overlap in mouse embryogenesis during limb bud and craniofacial development, consistent with the potential for their protein products to interact in vivo. Based on these observations, it is proposed that functional antagonism through heterodimer formation provides a mechanism for regulating the transcriptional actions of Msx and Dlx homeoproteins in vivo (Zhang, 1997).

Msx genes encode a family of homeoproteins that function as transcription repressors through protein-protein interactions. Lhx2, a LIM-type homeoprotein, is a protein partner for Msx1 in vitro and in cellular extracts. The interaction between Msx1 and Lhx2 is mediated through the homeodomain-containing regions of both proteins. Interestingly, the LIM domains, which serve as protein interaction domains for other partners of Lhx2, are not required for the Msx1-Lhx2 association. Msx1 and Lhx2 form a protein complex in the absence of DNA, and DNA binding by either protein alone can occur at the expense of protein complex formation. The significance of this protein-protein interaction is underscored by the expression patterns of Msx1 and Lhx2, which are partially overlapping during murine embryogenesis. The description of Lhx2 as a protein partner for Msx1 suggests that the functional specificity of homeoproteins in vivo is determined by a balance between their association with DNA and their protein partners (Bendall, 1998).

The regeneration of digit tips in mammals, including humans and rodents, represents a model for organ regeneration in higher vertebrates. Digit tip regeneration during fetal and neonatal stages of digit formation has been characterized in the mouse; regenerative capability correlates with the expression domain of the Msx1 gene. Using the stage 11 (E14.5) digit, digit tip regeneration is shown to occur in organ culture and Msx1, but not Msx2, mutant mice display a regeneration defect. Associated with this phenotype, it has been found that Bmp4 expression is downregulated in the Msx1 mutant digit and that mutant digit regeneration can be rescued in a dose-dependent manner by treatment with exogenous BMP4. Studies with the BMP-binding protein noggin show that wild-type digit regeneration is inhibited without inhibiting the expression of Msx1, Msx2 or Bmp4. These data identify a signaling pathway essential for digit regeneration, in which Msx1 functions to regulate BMP4 production. Evidence is provided that endogenous Bmp4 expression is regulated by the combined activity of Msx1 and Msx2 in the forming digit tip; however, a compensatory Msx2 response has been discovered that involves an expansion into the wild-type Msx1 domain. Thus, although both Msx1 and Msx2 function to regulate Bmp4 expression in the digit tip, the data are not consistent with a model in which Msx1 and Msx2 serve completely redundant functions in the regeneration response. These studies provide the first functional analysis of mammalian fetal digit regeneration and identify a new function for Msx1 and BMP4 as regulators of the regenerative response (Han, 2003).

MINT, the Msx2 interacting nuclear matrix target, enhances Runx2-dependent activation of the osteocalcin fibroblast growth factor response element

Msx2 promotes osteogenic lineage allocation from mesenchymal progenitors but inhibits terminal differentiation demarcated by osteocalcin (OC) gene expression. Msx2 inhibits OC expression by targeting the fibroblast growth factor responsive element (OCFRE), a 42-bp DNA domain in the OC gene bound by the Msx2 interacting nuclear target protein (MINT) and Runx2/Cbfa1. To better understand Msx2 regulation of the OCFRE, functional interactions between MINT and Runx2, a master regulator of osteoblast differentiation, were studied. In MC3T3E1 osteoblasts (with endogenous Runx2 and FGFR2), MINT augments transcription driven by the OCFRE that is further enhanced by FGF2 treatment. OCFRE regulation can be reconstituted in the naive CV1 fibroblast cell background. In CV1 cells, MINT synergizes with Runx2 to enhance OCFRE activity in the presence of activated FGFR2. The RNA recognition motif domain of MINT (which binds the OCFRE) is required. Runx2 structural studies reveal that synergy with MINT uniquely requires Runx2 activation domain 3. In confocal immunofluorescence microscopy, MINT adopts a reticular nuclear matrix distribution that overlaps transcriptionally active osteoblast chromatin, extensively co-localizing with the phosphorylated RNA polymerase II meshwork. MINT only partially co-localizes with Runx2; however, co-localization is enhanced 2.5-fold by FGF2 stimulation. Msx2 abrogates Runx2-MINT OCFRE activation, and MINT-directed RNA interference reduces endogenous OC expression. In chromatin immunoprecipitation assays, Msx2 selectively inhibits Runx2 binding to OC chromatin. Thus, MINT enhances Runx2 activation of multiprotein complexes assembled by the OCFRE. Msx2 targets this complex as a mechanism of transcriptional inhibition. In osteoblasts, MINT may serve as a nuclear matrix platform that organizes and integrates osteogenic transcriptional responses (Sierra, 2004).

MSX, BMPs and tooth development

Growth factor-mediated signaling has been implicated in the regulation of epithelial-mesenchymal interactions during organogenesis. Bone morphogenetic protein 4 (BMP-4), a member of the transforming growth factor beta superfamily, is expressed in the presumptive dental epithelium at the initiation of tooth development. Subsequently, epithelial signaling leads to mesenchymal induction of BMP-4 expression. To address the role of this factor, BMP-4-releasing agarose beads were added to dental mesenchyme in culture. These beads induce a translucent mesenchymal zone similar to that induced by dental epithelium. Three transcription factors (Msx-1, Msx-2, and Egr-1) whose expression is governed by epithelial signaling are induced in response to BMP-4. BMP-4 also induces its own mesenchymal expression. These findings support the hypothesis that BMP-4 mediates epithelial-mesenchymal interactions during early tooth development (Vainio, 1993).

There has been rapid progress recently in the identification of signaling pathways regulating tooth development. It has become apparent that signaling networks involved in Drosophila development and the development of structures such as limbs are also used in tooth development. Teeth are epithelial appendages formed in the oral region of vertebrates; their early developmental anatomy resembles that of other strucures, such as hairs and glands. The neural crest origin of tooth mesenchyme has been confirmed and recent evidence suggests that specific combinations of homeobox genes expressed in the neural crest cells may regulate the types of teeth and their patterning. Signaling molecules in the Shh, FGF, BMP and Wnt families appear to regulate the early steps of tooth morphogenesis. Certain transcription factors associated with these pathways have been shown to be necessary for tooth development. BMP-2 and BMP-4 as well as BMP-7 are expressed in the early dental epithelium: interestingly, the expression of BMP-4 shifts to the mesenchyme at the time when the instructive capacity shifts from the epithelium. BMP2 and BMP-4 stimulate expression of the homeobox-containing transcription factors MSX1 and MSX-2. FGF-3 expression is confined to dental papilla mesenchyme and is downregulated as morphogenesis advances. FGF-4, FGF-8 and FGF-9 are expressed exclusively in dental epithelial cells. Their respective receptors are present in both epithelial and mesenchymal tissues in the tooth. The FGF's also use cell surface heparan sulfate proteoglycans as receptors. Msx-1 also appears to participate in the FGF signaling pathway. Several FGFs upregulate Msx-1 expression in the dental mesenchyme when applied in vitro. FGFs are potent stimulators of cell proliferation: they stimulate cell division both in dental mesenchyme and epithelium at several stages in tooth morphogenesis. Several of the conserved signals are also transiently expressed in the enamel knots in the dental epithelium. The enamel knots are associated with the characteristic epithelial folding morphogenesis, which is responsible for the development of tooth shape. It is currently believed that the enamel knots function as signaling centers, regulating the development of tooth shape. Enamel knots constitute a specific ectodermal cell lineage; it has been proposed that enamel knots determine the site of the first cusp of teeth and that they regulate the formation of other cusps in molar teeth (Thesleff, 1997).

During early tooth development, multiple signaling molecules are expressed in the dental lamina epithelium; these molecular signals induce the dental mesenchyme. One signal, BMP4, has been shown to induce morphologic changes in dental mesenchyme and mesenchymal gene expression via Msx1, but BMP4 cannot substitute for all the inductive functions of the dental epithelium. To investigate the role of FGFs during early tooth development, the expression of epithelial and mesenchymal Fgfs was examined in wild-type and Msx1 mutant tooth germs and the ability of FGFs to induce Fgf3 and Bmp4 expression was tested in wild-type and Msx1 mutant dental mesenchymal explants. Fgf8 expression is preserved in Msx1 mutant epithelium while that of Fgf3 is not detected in Msx1 mutant dental mesenchyme. Moreover, dental epithelium as well as beads soaked in FGF1, FGF2 or FGF8 induce Fgf3 expression in dental mesenchyme in an Msx1-dependent manner. These results indicate that, like BMP4, FGF8 constitutes an epithelial inductive signal capable of inducing the expression of downstream signaling molecules in dental mesenchyme via Msx1. However, the BMP4 and FGF8 signaling pathways are distinct. BMP4 cannot induce Fgf3 nor can FGFs induce Bmp4 expression in dental mesenchyme, even though both signaling molecules can induce Msx1 and Msx1 is necessary for Fgf3 and Bmp4 expression in dental mesenchyme. In addition, the effects of FGFs and BMP4 were investigated on the distal-less homeobox genes Dlx1 and Dlx2 and the relationship between Msx and Dlx gene function was clarified in the developing tooth. Dlx1,Dlx2 double mutants exhibit a lamina stage arrest in maxillary molar tooth development. Although the maintenance of molar mesenchymal Dlx2 expression at the bud stage is Msx1-dependent, both the maintenance of Dlx1 expression and the initial activation of mesenchymal Dlx1 and Dlx2 expression during the lamina stage are not. Moreover, in contrast to the tooth bud stage arrest observed in Msx1 mutants, Msx1,Msx2 double mutants exhibit an earlier phenotype closely resembling the lamina stage arrest observed in Dlx1,Dlx2 double mutants. These results are consistent with functional redundancy between Msx1 and Msx2 in dental mesenchyme and support a model whereby Msx and Dlx genes function in parallel within the dental mesenchyme during tooth initiation. Indeed, as predicted by such a model, BMP4 and FGF8, epithelial signals that induce differential Msx1 and Msx2 expression in dental mesenchyme, also differentially induce Dlx1 and Dlx2 expression, and do so in an Msx1-independent manner. These results integrate Dlx1, Dlx2 and Fgf3 and Fgf8 into the odontogenic regulatory hierarchy along with Msx1, Msx2 and Bmp4, and provide a basis for interpreting tooth induction in terms of transcription factors which, individually, are necessary but not sufficient for the expression of downstream signals and therefore must act in specific combinations (Bei, 1998).

The development of many organs depends on sequential epithelial-mesenchymal interactions, and the developing tooth germ provides a powerful model for elucidating the nature of these inductive tissue interactions. In Msx1-deficient mice, tooth development arrests at the bud stage when Msx1 is required for the expression of Bmp4 and Fgf3 in the dental mesenchyme. To define the tissue requirements for Msx1 function, tissue recombinations were performed between wild-type and Msx1 mutant dental epithelium and mesenchyme. Through the E14.5 cap stage of tooth development, Msx1 is required in the dental mesenchyme for tooth formation. After the cap stage, however, tooth development becomes Msx1 independent, although there is a further late function of Msx1 in odontoblast and dental pulp survival. These results suggest that prior to the cap stage, the dental epithelium receives an Msx1-dependent signal from the dental mesenchyme that is necessary for tooth formation. To further test this hypothesis, Msx1 mutant tooth germs were first cultured with either BMP4 or with various FGFs for two days in vitro and then grown under the kidney capsule of syngeneic mice to permit completion of organogenesis and terminal differentiation. Using an in vitro culture system, it has been shown that BMP4 stimulates the growth of Msx1 mutant dental epithelium. Using the more powerful kidney capsule grafting procedure, it has now been shown that when added to explanted Msx1-deficient tooth germs prior to grafting, BMP4 rescues Msx1 mutant tooth germs all the way to definitive stages of enamel and dentin formation. Collectively, these results establish a transient functional requirement for Msx1 in the dental mesenchyme that is almost fully supplied by BMP4 alone, and not by FGFs. In addition, they formally prove the postulated downstream relationship of BMP4 with respect to Msx1, establish the non-cell-autonomous nature of Msx1 during odontogenesis, and disclose an additional late survival function for Msx1 in odontoblasts and dental pulp (Bei, 2000).

Tooth development is regulated by a reciprocal series of epithelial-mesenchymal interactions. Bmp4 has been identified as a candidate signaling molecule in these interactions, initially as an epithelial signal and then later at the bud stage as a mesenchymal signal. A target gene for Bmp4 signaling is the homeobox gene Msx-1, identified by the ability of recombinant Bmp4 protein to induce expression in mesenchyme. There is, however, no evidence that Bmp4 is the endogenous inducer of Msx-1 expression. Msx-1 and Bmp-4 show dynamic, interactive patterns of expression in oral epithelium and ectomesenchyme during the early stages of tooth development. The temporal and spatial expression of these two genes was compared to determine whether the changing expression patterns of these genes are consistent with interactions between the two molecules. Changes in Bmp-4 expression precede changes in Msx-1 expression. At embryonic day (E)10.5-E11.0, expression patterns are consistent with BMP4 from the epithelium, inducing or maintaining Msx-1 in underlying mesenchyme. At E11.5, Bmp-4 expression shifts from epithelium to mesenchyme and is rapidly followed by localized up-regulation of Msx-1 expression at the sites of Bmp-4 expression. Using cultured explants of developing mandibles, it was confirmed that exogenous BMP4 is capable of replacing the endogenous source in epithelium and inducing Msx-1 gene expression in mesenchyme. By using noggin, a BMP inhibitor, endogenous Msx-1 expression can be inhibited at E10.5 and E11.5, providing the first evidence that endogenous Bmp-4 from the epithelium is responsible for regulating the early spatial expression of Msx-1. The mesenchymal shift in Bmp-4 is responsible for up-regulating Msx-1 specifically at the sites of future tooth formation. Thus, a reciprocal series of interactions act to restrict expression of both genes to future sites of tooth formation, creating a positive feedback loop that maintains expression of both genes in tooth mesenchymal cells (Tucker, 1998).

The murine tooth development is governed by sequential and reciprocal epithelial-mesenchymal interactions. Multiple signaling proteins are expressed in the developing tooth germ and interact with one another to mediate the inductive tissue interactions. Among these proteins are Sonic hedgehog (SHH), Bone Morphogenetic Protein-2 (BMP2) and Bone Morphogenetic Protein-4 (BMP4). The interactions among these signaling proteins during early tooth development have been investigated. Bmp4 is expressed in dental epithelium at the initiation stage (E11.5) and then shifts to the dental mesenchyme shortly afterwards at the early bud stage (E12.5). This shift of Bmp4 expression pattern coincides with the shift in tooth developmental potential between tissue layers, indicating that BMP4 may constitute one component of the odontogenic potential. Indeed, BMP4 can induce in the dental mesenchyme the morphological changes and expression of a number of genes, including the transcription factors Msx1, Msx2, Lef1 and Bmp4 itself, mimicking the effect of the early dental epithelium. The mesenchymally expressed Bmp4 is believed to exert its function upon the dental epithelium as a feedback signal for further tooth development. The expression of Shh and Bmp2 is downregulated at E12.5 and E13.5 in the dental epithelium of the Msx1 mutant tooth germ and Bmp4 expression is significantly reduced in the dental mesenchyme. Inhibition of BMP4 activity by noggin results in repression of Shh and Bmp2 in wild-type dental epithelium. When implanted in the dental mesenchyme of Msx1 mutants, beads soaked with BMP4 protein are able to restore the expression of both Shh and Bmp2 in the Msx1 mutant epithelium. These results demonstrate that mesenchymal BMP4 represents one component of the signal acting on the epithelium to maintain Shh and Bmp2 expression. In contrast, BMP4- soaked beads repress Shh and Bmp2 expression in the wild-type dental epithelium. Ectopic expression of human Bmp4 in the dental mesenchyme driven by the mouse Msx1 promoter restores Shh expression in the Msx1 mutant dental epithelium but represses Shh in the wild-type tooth germ in vivo. This regulation of Shh expression by BMP4 is conserved in the mouse developing limb bud. In addition, Shh expression is unaffected in the developing limb buds of the transgenic mice in which a constitutively active Bmpr-IB is ectopically expressed in the forelimb posterior mesenchyme and throughout the hindlimb mesenchyme, suggesting that the repression of Shh expression by BMP4 may not be mediated by BMP receptor-IB. These results provide evidence for a new function of BMP4. BMP4 can act upstream to Shh by regulating Shh expression in mouse developing tooth germ and limb bud. Taken together, these data provide insight into a new regulatory mechanism for Shh expression, and suggest that this BMP4-mediated pathway in Shh regulation may have a general implication in vertebrate organogenesis (Zhang, 2000).

Calvarial bones (skull vault) form by direct ossification of mesenchyme. This requires condensation of mesenchymal cells which then proliferate and differentiate into osteoblasts. Congenital hydrocephalus (ch) mutant mice lack the forkhead/winged helix transcription factor Foxc1. In ch mutant mice, calvarial bones remain rudimentary at the sites of initial osteogenic condensations. In this study, the ossification defect in ch mutants has been localized to the calvarial mesenchyme, which lacks the expression of transcription factors Msx2 and Alx4. This lack of expression is associated with a reduction in the proliferation of osteoprogenitor cells. BMP induces Msx2 in calvarial mesenchyme. BMP also induces Alx4 in this tissue. BMP-induced expression of Msx2 and Alx4 requires Foxc1. It is therefore suggested that Foxc1 regulates BMP-mediated osteoprogenitor proliferation and that this regulation is required for the progression of osteogenesis beyond the initial condensations in calvarial bone development (Rice, 2003).

MSX and neural crest

Inductive interactions between the neural plate and epidermis can generate neural crest cells, since juxtaposition of these tissues at early stages results in the formation of neural crest cells at the interface. BMP4 and BMP7 are expressed in the epidermal ectoderm and both proteins mimic its inductive activity. BMP4 is subsequently expressed in neural cells. MSX, the vertebrate homolog of Drosophila Muscle segment homeobox is expressed in neural crest precursors and appears to be a target of BMP4. Sonic hedgehog signals from the notochord provides an opposing influence, repressing MSX in ventral neural plate, thus restricting MSX transcription to dorsal (neural crest progenitor) cells. Conversly, BMP-4 acts to suppress the differentiation of ventral cell types (Liem, 1995).

Much of the neural crest that forms the brachial arches arises from the hindbrain or rhombencephalon, which is segmented along the rostrocaudal axis into eight units, termed rhombdomeres. Neural crest is responsible for craniofacial patterning. Head muscle connective tissues derived from a specific rhombdomeric origin are always exclusively anchored to skeletal domains derived from the same rhombdomeric origin. The rhombdomeric origin of complex muscle-skeletal connections assures that muscles innervated by a fixed set of rhombdomere pairs are always properly connected to the complicated composite skeletal elements. Although rhombdomeres 1, 2, 4 and 6 are foci of crest production, rhombdomeres 3 and 5 are depleted in neural crest development. Lack of neural crest from r3 and r5 is due to apoptotic elimination of crest from the brachial arches. Expression of msx-2 displays a tight spatial and temporal correspondence with neural crest apoptosis in rhombdomeres r3 and r5. Bmp4 also has an expression pattern that is coincident with the pattern of neural crest apoptosis. Expression of this gene is downregulated when either r3 or r5 is freed from its cell death program, suggesting that Bmp4 is regulated by an interaction of odd numbered rhombdomeres with adjacent even-numbered rhombdomeres. When BMP4 protein is added to cultured r3 or r5 cells, msx-2 expression is maintained and the apoptotic program is executed. BMP signaling in the limb demonstrates an analgous function to signaling in the neural crest. A dominant negative BMP receptor construct, which blocks BMP signaling, results in a lack of interdigital cell death. Likewise msx-2 is implicated as a target of BMP in the interdigital mesencheme (Graham, 1996 and references).

Numerous human syndromes are the result of abnormal cranial neural crest development. One group of such defects, referred to as CATCH-22 (cardiac defects, abnormal facies, thymic hypoplasia, cleft palate, hypocalcemia, associated with chromosome 22 microdeletion) syndrome, exhibit craniofacial and cardiac defects resulting from abnormal development of the third and fourth neural crest-derived branchial arches and branchial arch arteries. Mice harboring a null mutation of the endothelin-1 gene (Edn1), which is expressed in the epithelial layer of the branchial arches and encodes for the endothelin-1 (ET-1) signaling peptide (known to signal through G-protein coupled receptors), have a phenotype similar to CATCH-22 syndrome: this includes aortic arch defects and craniofacial abnormalities. The basic helix-loop-helix transcription factor, dHAND (closest known Drosophila homolog: Twist), is shown to be expressed in the mesenchyme underlying the branchial arch epithelium. Further, dHAND and the related gene, eHAND, are downregulated in the branchial and aortic arches of Edn1-null embryos. In mice homozygous null for the dHAND gene, the first and second arches are hypoplastic secondary to programmed cell death and the third and fourth arches fail to form. Molecular analysis reveals that most markers of the neural-crest-derived components of the branchial arch are expressed in dHAND-null embryos, suggesting normal migration of neural crest cells. However, expression of the homeobox gene, Msx1, was undetectable in the mesenchyme of dHAND-null branchial arches but unaffected in the limb bud, consistent with the separable regulatory elements of Msx1. Together, these data suggest a model in which epithelial secretion of ET-1 stimulates mesenchymal expression of dHAND, which regulates Msx1 expression in the growing, distal branchial arch. Complete disruption of this molecular pathway results in growth failure of the branchial arches from apoptosis, while partial disruption leads to defects of branchial arch derivatives, similar to those seen in CATCH-22 syndrome (Thomas, 1998).

The neural crest plays a crucial part in cardiac development. Cells of the cardiac subpopulation of cranial neural crest migrate from the hindbrain into the outflow tract of the heart where they contribute to the septum that divides the pulmonary and aortic channels. In Splotch mutant mice, which lack a functional Pax3 gene, migration of cardiac neural crest is deficient and aorticopulmonary septation does not occur. Downstream genes through which Pax3 regulates cardiac neural crest development are unknown. The deficiency of cardiac neural crest development in the Splotch mutant is caused by upregulation of Msx2, a homeobox gene with a well-documented role as a regulator of BMP signaling. Evidence is provided that Pax3 represses Msx2 expression via a direct effect on a conserved Pax3 binding site in the Msx2 promoter. These results establish Msx2 as an effector of Pax3 in cardiac neural crest development (Kwang, 2002).

Three lines of evidence support the hypothesis that Pax3 regulates Msx2 through a direct effect on its promoter. (1) Msx2 lacZ transgenes are upregulated in the dorsal neural tube of Pax3Sp/Sp embryos in a manner similar to the endogenous Msx2 gene. (2) The 560 bp Pax3-responsive region of the Msx2 promoter includes a 520 bp stretch that is highly conserved (87%) in 5' flanking DNA of the human MSX2 gene. Within this stretch is a single conserved Pax3 consensus site that Pax3 binds with high affinity. (3) Mutation of this element, designated Pax site 1M, causes upregulation of Msx2 lacZ transgene expression in the dorsal neural tube. This upregulation is similar in spatial pattern to that of the Msx2 lacZ transgenes in the Splotch mutant background. These data strongly suggest that Pax3 regulates Msx2 lacZ transgenes through a direct interaction with Pax site 1. Whether Pax site 1 is functional in the context of the endogenous Msx2 promoter is unclear, though in situ hybridization data show that in the Splotch mutant background, the changes in the pattern of endogenous Msx2 expression are strikingly similar to those of the Delta4Msx2-hsplacZ transgene bearing a mutation in Pax site 1. An analysis of approximately 13 kb of genomic sequence flanking the Msx2 gene has thus far failed to identify additional elements capable of driving hsp68-lacZ expression in the neural tube and neural crest; thus Pax site 1 may be of crucial importance in the context of the endogenous Msx2 promoter (Kwang. 2002).

Cleft palate, the most frequent congenital craniofacial birth defects in humans, arises from genetic or environmental perturbations in the multi-step process of palate development. Mutations in the MSX1 homeobox gene are associated with non-syndromic cleft palate and tooth agenesis in humans. Msx1-deficient mice have been used as a model system that exhibits severe craniofacial abnormalities, including cleft secondary palate and lack of teeth, to study the genetic regulation of mammalian palatogenesis. Msx1 expression is restricted to the anterior of the first upper molar site in the palatal mesenchyme and Msx1 is required for the expression of Bmp4 and Bmp2 in the mesenchyme and Shh in the medial edge epithelium (MEE) in the same region of developing palate. In vivo and in vitro analyses indicate that the cleft palate seen in Msx1 mutants results from a defect in cell proliferation in the anterior palatal mesenchyme rather than a failure in palatal fusion. Transgenic expression of human Bmp4 driven by the mouse Msx1 promoter in the Msx1–/– palatal mesenchyme rescues the cleft palate phenotype and neonatal lethality. Associated with the rescue of the cleft palate is a restoration of Shh and Bmp2 expression, as well as a return of cell proliferation to the normal levels. Ectopic Bmp4 appears to bypass the requirement for Msx1 and functions upstream of Shh and Bmp2 to support palatal development. Further in vitro assays indicate that Shh (normally expressed in the MEE) activates Bmp2 expression in the palatal mesenchyme, which in turn acts as a mitogen to stimulate cell division. Msx1 thus controls a genetic hierarchy involving BMP and Shh signals that regulates the growth of the anterior region of palate during mammalian palatogenesis. These findings provide insights into the cellular and molecular etiology of the non-syndromic clefting associated with Msx1 mutations (Zhang, 2002).

There is evidence in Xenopus and zebrafish embryos that the neural crest/neural folds are specified at the border of the neural plate by a precise threshold concentration of a Bmp gradient. In order to understand the molecular mechanism by which a gradient of Bmp is able to specify the neural crest, a study was carried out of how the expression of Bmp targets, the Msx genes, is regulated, and the role that Msx genes has in neural crest specification was examined. Since Msx genes are directly downstream of Bmp, Msx gene expression was analyzed after experimental modification in the level of Bmp activity (1) by grafting a bead soaked with noggin into Xenopus embryos, (2) by expressing in the ectoderm a dominant-negative Bmp4 or Bmp receptor in Xenopus and zebrafish embryos, and (3) by examining Bmp pathway component mutants in the zebrafish. All the results show that a reduction in the level of Bmp activity leads to an increase in the expression of Msx genes in the neural plate border. Interestingly, by reaching different levels of Bmp activity in animal cap ectoderm, it has been shown that a specific concentration of Bmp induces msx1 expression to a level similar to that required to induce neural crest. These results indicate that an intermediate level of Bmp activity specifies the expression of Msx genes in the neural fold region. In addition, the role that msx1 plays on neural crest specification was examined. Since msx1 has a role in dorsoventral pattering, conditional gain- and loss-of-function experiments were carried out using different msx1 constructs fused to a glucocorticoid receptor element to avoid an early effect of this factor. msx1 expression is able to induce all other early neural crest markers tested (snail, slug, foxd3) at the time of neural crest specification. Furthermore, the expression of a dominant negative of Msx genes leads to the inhibition of all the neural crest markers analyzed. snail is one of the earliest genes acting in the neural crest genetic cascade. In order to study the hierarchical relationship between msx1 and snail/slug several rescue experiments were performed using dominant negatives for these genes. The rescuing activity by snail and slug on neural crest development of the msx1 dominant negative, together with the inability of msx1 to rescue the dominant negatives of slug and snail strongly argue that msx1 is upstream of snail and slug in the genetic cascade that specifies the neural crest in the ectoderm. A model is proposed where a gradient of Bmp activity specifies the expression of Msx genes in the neural folds; it is proposed that this expression is essential for the early specification of the neural crest (Tríbulo, 2004).

The flat bones of the vertebrate skull vault develop from two migratory mesenchymal cell populations, the cranial neural crest and paraxial mesoderm. At the onset of skull vault development, these mesenchymal cells emigrate from their sites of origin to positions between the ectoderm and the developing cerebral hemispheres. There they combine, proliferate and differentiate along an osteogenic pathway. Anomalies in skull vault development are relatively common in humans. One such anomaly is familial calvarial foramina, persistent unossified areas within the skull vault. Mutations in MSX2 and TWIST are known to cause calvarial foramina in humans. Little is known of the cellular and developmental processes underlying this defect. Neither is it known whether MSX2 and TWIST function in the same or distinct pathways. The origin of the calvarial foramen defect in Msx2 mutant mice was traced to a group of skeletogenic mesenchyme cells that compose the frontal bone rudiment. This cell population is reduced not because of apoptosis or deficient migration of neural crest-derived precursor cells, but because of defects in its differentiation and proliferation. In addition heterozygous loss of Twist function causes a foramen in the skull vault similar to that caused by loss of Msx2 function. Both the quantity and proliferation of the frontal bone skeletogenic mesenchyme are reduced in Msx2-Twist double mutants compared with individual mutants. Thus Msx2 and Twist cooperate in the control of the differentiation and proliferation of skeletogenic mesenchyme. Molecular epistasis analysis suggests that Msx2 and Twist do not act in tandem to control osteoblast differentiation, but function at the same epistatic level (Ishii, 2003).

The pattern of programmed cell death was studied in the neural crest and how it is controlled by the activity of the transcription factors Slug and msx1 was examined. The results indicate that apoptosis is more prevalent in the neural folds than in the rest of the neural ectoderm. Through gain- and loss-of-function experiments with inducible forms of both Slug and msx1 genes, it was shown that Slug acts as an anti-apoptotic factor whereas msx1 promotes cell death, either in the neural folds of the whole embryos, in isolated or induced neural crest and in animal cap assays. The protective effect of expressing Slug can be reversed by expressing the apoptotic factor Bax, while the apoptosis promoted by msx1 can be abolished by expressing the Xenopus homologue of Bcl2 (XR11). Furthermore, Slug and msx1 control the transcription of XR11 and several caspases required for programmed cell death. In addition, expression of Bax or Bcl2 produced similar effects on the survival of the neural crest and on the development of its derivatives as those produced by altering the activity of Slug or msx1. Finally, it was shown that in the neural crest, the region of the neural folds where Slug is expressed, cells undergo less apoptosis, than in the region where the msx1 gene is expressed; this region corresponds to cells adjacent to the neural crest. The expression of Slug and msx1 controls cell death in certain areas of the neural folds, and how this equilibrium is necessary to generate sharp boundaries in the neural crest territory and to precisely control cell number among neural crest derivatives is discussed (Tribulo, 2004).

FGF, WNT, and BMP signaling promote neural crest formation at the neural plate boundary in vertebrate embryos. To understand how these signals are integrated, the role of the transcription factors Msx1 and Pax3 was analyzed. Using a combination of overexpression and morpholino-mediated knockdown strategies in Xenopus, it was shown that Msx1 and Pax3 are both required for neural crest formation; they display overlapping but nonidentical activities, and Pax3 acts downstream of Msx1. In neuralized ectoderm, Msx1 is sufficient to induce multiple early neural crest genes. Msx1 induces Pax3 and ZicR1 cell autonomously, in turn, Pax3 combined with ZicR1 activates Slug in a WNT-dependent manner. Upstream of this, WNTs initiate Slug induction through Pax3 activity, whereas FGF8 induces neural crest through both Msx1 and Pax3 activities. Thus, WNT and FGF8 signals act in parallel at the neural border and converge on Pax3 activity during neural crest induction (Monsoro-Burq, 2005).

The homeobox genes Msx1 and Msx2 function as transcriptional regulators that control cellular proliferation and differentiation during embryonic development. Mutations in the Msx1 and Msx2 genes in mice disrupt tissue-tissue interactions and cause multiple craniofacial malformations. Although Msx1 and Msx2 are both expressed throughout the entire development of the frontal bone, the frontal bone defect in Msx1 or Msx2 null mutants is rather mild, suggesting the possibility of functional compensation between Msx1 and Msx2 during early frontal bone development. To investigate this hypothesis, Msx1−/−;Msx2−/− mice were generated. These double mutant embryos died at E17 to E18 with no formation of the frontal bone. There was no apparent defect in CNC migration into the presumptive frontal bone primordium, but differentiation of the frontal mesenchyme and establishment of the frontal primordium was defective, indicating that Msx1 and Msx2 genes are specifically required for osteogenesis in the cranial neural crest lineage within the frontal bone primordium. Mechanistically, these data suggest that Msx genes are critical for the expression of Runx2 in the frontonasal subpopulation of cranial neural crest cells and for differentiation of the osteogenic lineage. This early function of the Msx genes is likely independent of the Bmp signaling pathway (Han, 2007).

Wnt signalling is required for neural crest (NC) induction; however, the direct targets of the Wnt pathway during NC induction remain unknown. This study shows that the homeobox gene Gbx2 is essential in this process and is directly activated by Wnt/beta-catenin signalling. By ChIP and transgenesis analysis it was shown that Gbx2 regulatory elements that drive expression in the NC respond directly to Wnt/beta-catenin signalling. Gbx2 has previously been implicated in posteriorization of the neural plate. This study unveils a new role for this gene in neural fold patterning. Loss-of-function experiments using antisense morpholinos against Gbx2 inhibit NC and expand the preplacodal domain, whereas Gbx2 overexpression leads to transformation of the preplacodal domain into NC cells. The NC specifier activity of Gbx2 is dependent on the interaction with Zic1 and the inhibition of preplacodal genes such as Six1. In addition, that Gbx2 is upstream of the neural fold specifiers Pax3 and Msx1. These results place Gbx2 as the earliest factor in the NC genetic cascade being directly regulated by the inductive molecules, and support the notion that posteriorization of the neural folds is an essential step in NC specification. A new genetic cascade is proposed that operates in the distinction between anterior placodal and NC territories (Li, 2009).

MSX and limb development

Dorsoventral (DV) patterning of the vertebrate limb requires the function of the transcription factor Engrailed 1 (EN1) in the ventral ectoderm. EN1 restricts, to the dorsal half of the limb, the expression of the two genes known to specify dorsal pattern. Limb growth along the proximodistal (PD) axis is controlled by the apical ectodermal ridge (AER), a specialized epithelium that forms at the distal junction between dorsal and ventral ectoderm. Using retroviral-mediated misexpression of the bone morphogenetic protein (BMP) antagonist Noggin or an activated form of the BMP receptor in the chick limb, it has been demonstrated that BMP plays a key role in both DV patterning and AER induction. Thus, the DV and PD axes are linked by a common signal. Loss and gain of BMP function experiments show that BMP signaling is both necessary and sufficient to regulate EN1 expression, and consequently DV patterning. These results also indicate that BMPs are required during induction of the AER. Manipulation of BMP signaling results in either disruptions in the endogenous AER, leading to absent or severely truncated limbs or the formation of ectopic AERs that can direct outgrowth. Moreover, BMP controls the expression of the MSX transcription factors, and the results suggest that MSX acts downstream of BMP in AER induction. It is proposed that the BMP signal bifurcates at the level of EN1 and MSX to mediate differentially DV patterning and AER induction, respectively (Pizette, 2001).

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. This 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. In the absence of Msx proteins, Bmp signalling is affected along the whole apex of the limb. Anteriorly, this results in loss of the AER, agenesis of mesoderm and lack of skeletal elements, but posteriorly only to incomplete AER maturation. Posteriorly, a deficit in Bmp signalling may be of limited effect or, conversely, other genes may fulfil the role of Msx (Lallemand, 2005).

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

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. In the mouse embryo, Msx1lacZ and Msx2lacZ are expressed in mural cells and in a few endothelial cells. To unravel the role of Msx genes in vascular development, the two Msx genes were inactivated specifically in mural cells by combining the Msx1lacZ, Msx2lox 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 Msx1CreERT2 allele, it was 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. These results demonstrate that both Msx1 and Msx2 are required for the recruitment of a population of neural crest-derived VSMCs (Lopes, 2011).

Msx1 and Msx2 act as essential activators of Atoh1 expression in the murine spinal cord

Dorsal spinal neurogenesis is orchestrated by the combined action of signals secreted from the roof plate organizer and a downstream transcriptional cascade. Within this cascade, Msx1 and Msx2, two homeodomain transcription factors (TFs), are induced earlier than bHLH neuralizing TFs. Whereas bHLH TFs have been shown to specify neuronal cell fate, the function of Msx genes remains poorly defined. This study describes dramatic alterations of neuronal patterning in Msx1/Msx2 double-mutant mouse embryos. The most dorsal spinal progenitor pool fails to express the bHLH neuralizing TF Atoh1, which results in a lack of Lhx2-positive and Barhl2-positive dI1 interneurons. Neurog1 and Ascl1 expression territories are dorsalized, leading to ectopic dorsal differentiation of dI2 and dI3 interneurons. In proportion, the amount of Neurog1-expressing progenitors appears unaffected, whereas the number of Ascl1-positive cells is increased. These defects occur while BMP signaling is still active in the Msx1/Msx2 mutant embryos. Cell lineage analysis and co-immunolabeling demonstrate that Atoh1-positive cells derive from progenitors expressing both Msx1 and Msx2. In vitro, Msx1 and Msx2 proteins activate Atoh1 transcription by specifically interacting with several homeodomain binding sites in the Atoh1 3' enhancer. In vivo, Msx1 and Msx2 are required for Atoh1 3' enhancer activity and ChIP experiments confirm Msx1 binding to this regulatory sequence. These data support a novel function of Msx1 and Msx2 as transcriptional activators. This study provides new insights into the transcriptional control of spinal cord patterning by BMP signaling, with Msx1 and Msx2 acting upstream of Atoh1 (Duval, 2014).

MSX and differentiation

The migration of myogenic precursors to the vertebrate limb exemplifies a common problem in development -- namely, how migratory cells that are committed to a specific lineage postpone terminal differentiation until they reach their destination. In chicken embryos, expression of the Msx1 homeobox gene overlaps with Pax3 in migrating limb muscle precursors, which are committed myoblasts that do not express myogenic differentiation genes such as MyoD. Ectopic expression of Msx1 in the forelimb and somites of chicken embryos inhibits MyoD expression as well as muscle differentiation. Conversely, ectopic expression of Pax3 activates MyoD expression, while co-ectopic expression of Msx1 and Pax3 neutralizes one another's effects on MyoD. Msx1 represses and Pax3 activates MyoD regulatory elements in cell culture, while in combination, Msx1 and Pax3 oppose one another's trancriptional actions on MyoD. The Msx1 protein interacts with Pax3 in vitro, thereby inhibiting DNA binding by Pax3. Thus, it is proposed that Msx1 antagonizes the myogenic activity of Pax3 in migrating limb muscle precursors via direct protein-protein interaction. These results implicate functional antagonism through competitive protein-protein interactions as a mechanism for regulating the differentiation state of migrating cells (Bendall, 1999).

Throughout its complex morphogenesis, the vertebrate skull must at once protect the brain and expand to accommodate its growth. A key structural adaptation that allows this dual role is the separation of the bony plates of the skull with sutures, fibrous joints that serve as growth centers and allow the calvarial bones to expand as the brain enlarges. Craniosynostosis, the premature fusion of one or more calvarial bones with consequent abnormalities in skull shape, is a common developmental anomaly that disrupts this process. A single amino acid substitution in the homeodomain of the human MSX2 gene is associated with the autosomal dominant disorder craniosynostosis, Boston type. This mutation enhances the affinity of Msx2 for its target sequence, suggesting that the mutation acts by a dominant positive mechanism. Consistent with this prediction, general overexpression of Msx2 under the control of the broadly expressed CMV promoter causes the calvarial bones to invade the sagittal suture. Tissue-specific overexpression of Msx2 within the calvarial sutures was used to address the developmental mechanisms of craniosynostosis and skull morphogenesis. A segment of the Msx2 promoter directs reporter gene expression to subsets of cells within the sutures. In late embryonic and neonatal stages, this promoter is expressed in undifferentiated mesenchymal cells medial to the growing bone. By P4, promoter activity is reduced in the suture, exhibiting a punctate pattern in undifferentiated osteoblastic cells in the outer margin of the osteogenic front. Overexpression of Msx2 under the control of this promoter is sufficient to enhance parietal bone growth into the sagittal suture by P6. This phenotype is preceded by an increase in both the number and the BrdU labeling of osteoblastic cells in the osteogenic fronts of the calvarial bones. These findings suggest that an important early event in MSX2-mediated craniosynostosis in humans is a transient retardation of osteogenic cell differentiation in the suture and a consequent increase in the pool of osteogenic cells (Liu, 1999).

Msx2 is believed to play a role in regulating bone development, particularly at the sutures of cranial bone. The effects of retroviral-mediated overexpression of Msx2 mRNA, in both sense and antisense orientations, have been investigated on primary cultured chick calvarial osteoblasts. Unregulated overexpression of sense mRNA produces high levels of Msx2 protein throughout the culture period, preventing the expected decline in Msx2 protein as the cells differentiate. The continued high expression of Msx2 prevents osteoblastic differentiation and mineralization of the extracellular matrix. In contrast, expression of antisense Msx2 RNA decreases proliferation and accelerates differentiation. In other studies, the Msx2 promoter has been shown to be widely expressed during the proliferative phase of mouse calvarial osteoblast cultures but is preferentially downregulated in osteoblastic nodules. These results support a model in which Msx2 prevents differentiation and stimulates proliferation of cells at the extreme ends of the osteogenic fronts of the calvariae, facilitating expansion of the skull and closure of the suture. Downregulation of Msx2 is necessary for the progression of cells further into the osteoblastic lineage (Dodig, 1999).

The process of cellular differentiation culminating in terminally differentiated mammalian cells is thought to be irreversible. Evidence is presented that terminally differentiated murine myotubes can be induced to dedifferentiate. Ectopic expression of msx1 in C2C12 myotubes reduces the nuclear muscle proteins MyoD, myogenin, MRF4, and p21 to undetectable levels in 20%-50% of the myotubes. Approximately 9% of the myotubes cleave to produce either smaller multinucleated myotubes or proliferating, mononucleated cells. Finally, clonal populations of the myotube-derived mononucleated cells can be induced to redifferentiate into cells expressing chondrogenic, adipogenic, myogenic, and osteogenic markers. These results suggest that terminally differentiated mammalian myotubes can dedifferentiate when stimulated with the appropriate signals and that msx1 can contribute to the dedifferentiation process (Odelberg, 2000).

During development, patterning and morphogenesis of tissues are intimately coordinated through control of cellular proliferation and differentiation. A mechanism is described by which vertebrate Msx homeobox genes inhibit cellular differentiation by regulation of the cell cycle. Misexpression of Msx1 via retroviral gene transfer inhibits differentiation of multiple mesenchymal and epithelial progenitor cell types in culture. This activity of Msx1 is associated with its ability to upregulate cyclin D1 expression and Cdk4 activity, while Msx1 has minimal effects on cellular proliferation. Transgenic mice that express Msx1 under the control of the mouse mammary tumor virus long terminal repeat (MMTV LTR) display impaired differentiation of the mammary epithelium during pregnancy; this is accompanied by elevated levels of cyclin D1 expression. It is proposed that Msx1 gene expression maintains cyclin D1 expression and prevents exit from the cell cycle, thereby inhibiting terminal differentiation of progenitor cells. This model provides a framework for reconciling the mutant phenotypes of Msx and other homeobox genes with their functions as regulators of cellular proliferation and differentiation during embryogenesis (Hu, 2001).

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