Msx1 cooperates with Runx1 for inhibiting myoblast differentiation.

Myogenesis is an important and complicated biological process, especially during the process of embryonic development. The homeoprotein Msx1 is a crucial transcriptional repressor of myogenesis and maintains myogenic precursor cells in an undifferentiated, proliferative state. However, the molecular mechanism through which Msx1 coordinates myogenesis remains to be elucidated. Here, we determine the interacting partner proteins of Msx1 in myoblast cells by a proteomic screening method. Msx1 is found to interact with 55 proteins, among which our data demonstrate that the cooperation of Runt-related transcription factor 1 (Runx1) with Msx1 is required for myoblast cell differentiation. Our findings provide important insights into the mechanistic roles of Msx1 in myoblast cell differentiation, and lays foundation for the myogenic differentiation process.

MSX1-A Potential Marker for Uterus-Preserving Therapy of Endometrial Carcinomas.

Prognostic factors are of great interest in patients with endometrial cancer. One potential factor could be the protein MSX1, a transcription repressor, that has an inhibitory effect on the cell cycle. For this study, endometrioid endometrial carcinomas (n = 53), clear cell endometrial carcinomas (n = 6), endometrioid ovarian carcinomas (n = 19), and clear cell ovarian carcinomas (n = 11) were immunochemically stained for the protein MSX1 and evaluated using the immunoreactive score (IRS). A significant stronger expression of MSX1 was found in endometrioid endometrial carcinomas (p < 0.001), in grading 2 (moderate differentiation) (p = 0.001), and in tumor material of patients with no involvement of lymph nodes (p = 0.031). Correlations were found between MSX1 expression and the expression of ß-Catenin, p21, p53, and the steroid receptors ERα, ERß, PRα, and PRß. A significant (p = 0.023) better survival for patients with an MSX1 expression in more than 10% of the tumor cells was observed for endometrioid endometrial carcinomas (21.3 years median survival (MSX1-positive) versus 17.3 years (MSX1-negative)). Although there is evidence that MSX1 expression correlates with improved long-term survival, further studies are necessary to evaluate if MSX1 can be used as a prognostic marker.

Homeobox protein MSX-1 inhibits expression of bone morphogenetic protein 2, bone morphogenetic protein 4, and lymphoid enhancer-binding factor 1 via Wnt/ß-catenin signaling to prevent differentiation of dental mesenchymal cells during the late bell stage.

Homeobox protein MSX-1 (hereafter referred to as MSX-1) is essential for early tooth-germ development. Tooth-germ development is arrested at bud stage in Msx1 knockout mice, which prompted us to study the functions of MSX-1 beyond this stage. Here, we investigated the roles of MSX-1 during late bell stage. Mesenchymal cells of the mandibular first molar were isolated from mice at embryonic day (E)17.5 and cultured in vitro. We determined the expression levels of ß-catenin, bone morphogenetic protein 2 (Bmp2), Bmp4, and lymphoid enhancer-binding factor 1 (Lef1) after knockdown or overexpression of Msx1. Our findings suggest that knockdown of Msx1 promoted expression of Bmp2, Bmp4, and Lef1, resulting in elevated differentiation of odontoblasts, which was rescued by blocking the expression of these genes. In contrast, overexpression of Msx1 decreased the expression of Bmp2, Bmp4, and Lef1, leading to a reduction in odontoblast differentiation. The regulation of Bmp2, Bmp4, and Lef1 by Msx1 was mediated by the Wnt/ß-catenin signaling pathway. Additionally, knockdown of Msx1 impaired cell proliferation and slowed S-phase progression, while overexpression of Msx1 also impaired cell proliferation and prolonged G1-phase progression. We therefore conclude that MSX-1 maintains cell proliferation by regulating transition of cells from G1-phase to S-phase and prevents odontoblast differentiation by inhibiting expression of Bmp2, Bmp4, and Lef1 at the late bell stage via the Wnt/ß-catenin signaling pathway.

Muscle Segment Homeobox Genes Direct Embryonic Diapause by Limiting Inflammation in the Uterus.

Embryonic diapause is a reproductive strategy widespread in the animal kingdom. This phenomenon is defined by a temporary arrest in blastocyst growth and metabolic activity within a quiescent uterus without implantation until the environmental and maternal milieu become favorable for pregnancy to progress. We found that uterine Msx expression persists during diapause across species; their inactivation in the mouse uterus results in termination of diapause with the development of implantation-like responses ("pseudoimplantation") that ultimately succumbed to resorption. To understand the cause of this failure, we compared proteome profiles between floxed and Msx-deleted uteri. In deleted uteri, several functional networks, including transcription/translation, ubiquitin-proteasome, inflammation, and endoplasmic reticulum stress, were dysregulated. Computational modeling predicted intersection of these pathways on an enhanced inflammatory signature. Further studies showed that this signature was reflected in increased phosphorylated IκB levels and nuclear NFκB in deleted uteri. This was associated with enhanced proteasome activity and endoplasmic reticulum stress. Interestingly, treatment with anti-inflammatory glucocorticoid (dexamethasone) reduced the inflammatory signature with improvement of the diapause phenotype. These findings highlight an unexpected role of uterine Msx in limiting aberrant inflammatory responses to maintain embryonic diapause.

Msx1 and Msx2 promote meiosis initiation.

The mechanisms regulating germ line sex determination and meiosis initiation are poorly understood. Here, we provide 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, our data demonstrate for the first time that some homeobox genes are required for meiosis initiation in the female germ line.

DNAJB6 governs a novel regulatory loop determining Wnt/ß-catenin signalling activity.

DKK1 (dickkopf 1 homologue) is a secreted inhibitor of the Wnt signalling pathway and a critical modulator of tumour promotion and the tumour microenvironment. However, mechanisms regulating DKK1 expression are understudied. DNAJB6 {DnaJ [HSP40 (heat-shock protein 40 kDa)] homologue, subfamily B, member 6} is an HSP40 family member whose expression is compromised during progression of breast cancer and melanoma. Inhibition of the Wnt/ß-catenin signalling pathway by up-regulation of DKK1 is one of the key mechanisms by which DNAJB6 suppresses tumour metastasis and EMT (epithelial-mesenchymal transition). Analysis of the DKK1 promoter to define the cis-site responsible for its up-regulation by DNAJB6 revealed the presence of two binding sites for a transcriptional repressor, MSX1 (muscle segment homeobox 1). Our investigations showed that MSX1 binds the DKK1 promoter and inhibits DKK1 transcription. Interestingly, silencing DNAJB6 resulted in up-regulation of MSX1 concomitant with increased stabilization of ß-catenin. ChIP (chromatin immunoprecipitation) studies revealed that ß-catenin binds the MSX1 promoter and stabilization of ß-catenin elevates MSX1 transcription, indicating that ß-catenin works as a transcription co-activator for MSX1. Functionally, exogenous expression of MSX1 in DNAJB6-expressing cells promotes the mesenchymal phenotype by suppression of DKK1. Thus we have identified a novel regulatory mechanism of DNAJB6-mediated DKK1 transcriptional up-regulation that can influence EMT. DKK1 is a feedback regulator of ß-catenin levels and thus our studies also define an additional negative control of this ß-catenin/DKK1 feedback loop by MSX1, which may potentially contribute to excessive stabilization of ß-catenin.

Osr2 acts downstream of Pax9 and interacts with both Msx1 and Pax9 to pattern the tooth developmental field.

Mammalian tooth development depends on activation of odontogenic potential in the presumptive dental mesenchyme by the Msx1 and Pax9 transcription factors. We recently reported that the zinc finger transcription factor Osr2 was expressed in a lingual-to-buccal gradient pattern surrounding the developing mouse molar tooth germs and mice lacking Osr2 developed supernumerary teeth lingual to their molars. We report here generation of a gene-targeted mouse strain that allows conditional inactivation of Pax9 and subsequent activation of expression of Osr2 in the developing tooth mesenchyme from the Pax9 locus. Expression of Osr2 from one copy of the Pax9 gene did not disrupt normal tooth development but was sufficient to suppress supernumerary tooth formation in the Osr2(-/-) mutant mice. We found that endogenous Osr2 mRNA expression was significantly downregulated in the developing tooth mesenchyme in Pax9(del/del) mice. Mice lacking both Osr2 and Pax9 exhibited early tooth developmental arrest with significantly reduced Bmp4 and Msx1 mRNA expression in the developing tooth mesenchyme, similar to that in Pax9(del/del) mutants but in contrast to the rescue of tooth morphogenesis in Msx1(-/-)Osr2(-/-) double mutant mice. Furthermore, we found that Osr2 formed stable protein complexes with the Msx1 protein and interacted weakly with the Pax9 protein in co-transfected cells. These data indicate that Osr2 acts downstream of Pax9 and patterns the mesenchymal odontogenic field through protein-protein interactions with Msx1 and Pax9 during early tooth development.

Genetic interactions between Pax9 and Msx1 regulate lip development and several stages of tooth morphogenesis.

Developmental abnormalities of craniofacial structures and teeth often occur sporadically and the underlying genetic defects are not well understood, in part due to unknown gene-gene interactions. Pax9 and Msx1 are co-expressed during craniofacial development, and mice that are single homozygous mutant for either gene exhibit cleft palate and an early arrest of tooth formation. Whereas in vitro assays have demonstrated that protein-protein interactions between Pax9 and Msx1 can occur, it is unclear if Pax9 and Msx1 interact genetically in vivo during development. To address this question, we compounded the Pax9 and Msx1 mutations and observed that double homozygous mutants exhibit an incompletely penetrant cleft lip phenotype. Moreover, in double heterozygous mutants, the lower incisors were consistently missing and we find that transgenic BMP4 expression partly rescues this phenotype. Reduced expression of Shh and Bmp2 indicates that a smaller "incisor field" forms in Pax9(+/-);Msx1(+/-) mutants, and dental epithelial growth is substantially reduced after the bud to cap stage transition. This defect is preceded by drastically reduced mesenchymal expression of Fgf3 and Fgf10, two genes that encode known stimulators of epithelial growth during odontogenesis. Consistent with this result, cell proliferation is reduced in both the dental epithelium and mesenchyme of double heterozygous mutants. Furthermore, the developing incisors lack mesenchymal Notch1 expression at the bud stage and exhibit abnormal ameloblast differentiation on both labial and lingual surfaces. Thus, Msx1 and Pax9 interact synergistically throughout lower incisor development and affect multiple signaling pathways that influence incisor size and symmetry. The data also suggest that a combined reduction of PAX9 and MSX1 gene dosage in humans may increase the risk for orofacial clefting and oligodontia.

Msx1 and Dlx5 function synergistically to regulate frontal bone development.

The Msx and Dlx families of homeobox proteins are important regulators for embryogenesis. Loss of Msx1 in mice results in multiple developmental defects including craniofacial malformations. Although Dlx5 is widely expressed during embryonic development, targeted null mutation of Dlx5 mainly affects the development of craniofacial bones. Msx1 and Dlx5 show overlapping expression patterns during frontal bone development. To investigate the functional significance of Msx1/Dlx5 interaction in regulating frontal bone development, we generated Msx1 and Dlx5 double null mutant mice. In Msx1(-/-) ;Dlx5(-/-) mice, the frontal bones defect was more severe than that of either Msx1(-/-) or Dlx5(-/-) mice. This aggravated frontal bone defect suggests that Msx1 and Dlx5 function synergistically to regulate osteogenesis. This synergistic effect of Msx1 and Dlx5 on the frontal bone represents a tissue specific mode of interaction of the Msx and Dlx genes. Furthermore, Dlx5 requires Msx1 for its expression in the context of frontal bone development. Our study shows that Msx1/Dlx5 interaction is crucial for osteogenic induction during frontal bone development.

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

In tetrapods, the anteroposterior (AP) patterning of the limb is under the control of the antagonistic activities of the secreted factor Sonic hedgehog (Shh) and Gli3R, the truncated repressor form of the transcription factor Gli3. In this report, we show that Msx1 and Msx2 are targets and downstream effectors of Gli3R. Consequently, in Shh null mutants, Msx genes are overexpressed and, furthermore, partially responsible for the limb phenotype. This is exemplified by the fact that reducing Msx activity in Shh mutants partially restores a normal limb development. Finally, we show that the main action of the Msx genes, in both normal and Shh(-/-) limb development, is to control cell death in the mesenchyme. We propose that, in the limb, Msx genes act downstream of the Shh/Gli3 pathway by transducing BMP signaling and that, in the absence of Shh signaling, their deregulation contributes to the extensive apoptosis that impairs limb development.

Suppression of the transcription factor MSX1 gene delays bovine preimplantation embryo development in vitro.

This study was conducted to investigate the effect of suppressing transcription factor gene MSX1 on the development of in vitro produced bovine oocytes and embryos, and identify its potential target genes regulated by this gene. Injection of long double-stranded RNA (LdsRNA) and small interfering RNA (siRNA) at germinal vesicle stage oocyte reduced MSX1 mRNA expression by 73 and 37% respectively at metaphase II stage compared with non-injected controls. Similarly, injection of the same anti-sense oligomers at zygote stage reduced MSX1 mRNA expression by 52 and 33% at 8-cell stage compared with non-injected controls. Protein expression was also reduced in LdsRNA- and siRNA-injected groups compared with non-injected controls at both stages. Blastocysts rates were 33, 28, 20 and 18% in non-injected control, scrambled RNA (scRNA), LdsRNA- and siRNA-injected groups respectively. Cleavage rates were also significantly reduced in Smartpool siRNA (SpsiRNA)-injected group (53.76%) compared with scRNA-injected group (57.76%) and non-injected control group (61%). Large-scale gene expression analysis showed that 135 genes were differentially regulated in SpsiRNA-injected group compared with non-injected controls, of which 54 and 81 were down- and up-regulated respectively due to suppression of MSX1. Additionally, sequence homology mapping and gene enrichment analysis with known human pathway information identified several functional modules that were affected due to suppression of MSX1. In conclusion, suppression of MSX1 affects oocyte maturation, embryo cleavage rate and the expression of several genes, suggesting its potential role in the development of bovine preimplantation embryos.

Bone morphogenetic protein-induced MSX1 and MSX2 inhibit myocardin-dependent smooth muscle gene transcription.

During the onset and progression of atherosclerosis, the vascular smooth muscle cell (VSMC) phenotype changes from differentiated to dedifferentiated, and in some cases, this change is accompanied by osteogenic transition, resulting in vascular calcification. One characteristic of dedifferentiated VSMCs is the down-regulation of smooth muscle cell (SMC) marker gene expression. Bone morphogenetic proteins (BMPs), which are involved in the induction of osteogenic gene expression, are detected in calcified vasculature. In this study, we found that the BMP2-, BMP4-, and BMP6-induced expression of Msx transcription factors (Msx1 and Msx2) preceded the down-regulation of SMC marker expression in cultured differentiated VSMCs. Either Msx1 or Msx2 markedly reduced the myocardin-dependent promoter activities of SMC marker genes (SM22alpha and caldesmon). We further investigated interactions between Msx1 and myocardin/serum response factor (SRF)/CArG-box motif (cis element for SRF) using coimmunoprecipitation, gel-shift, and chromatin immunoprecipitation assays. Our results showed that Msx1 or Msx2 formed a ternary complex with SRF and myocardin and inhibited the binding of SRF or SRF/myocardin to the CArG-box motif, resulting in inhibition of their transcription.

msh/Msx gene family in neural development.

The involvement of Msx homeobox genes in skull and tooth formation has received a great deal of attention. Recent studies also indicate a role for the msh/Msx gene family in development of the nervous system. In this article, we discuss the functions of these transcription factors in neural-tissue organogenesis. We will deal mainly with the interactions of the Drosophila muscle segment homeobox (msh) gene with other homeobox genes and the repressive cascade that leads to neuroectoderm patterning; the role of Msx genes in neural-crest induction, focusing especially on the differences between lower and higher vertebrates; their implication in patterning of the vertebrate neural tube, particularly in diencephalon midline formation. Finally, we will examine the distinct activities of Msx1, Msx2 and Msx3 genes during neurogenesis, taking into account their relationships with signalling molecules such as BMP.

Concerted action of Msx1 and Msx2 in regulating cranial neural crest cell differentiation during frontal bone development.

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, we generated Msx1(-/-);Msx2(-/-) mice. 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, our 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.

Distal cis-regulatory elements are required for tissue-specific expression of enamelin (Enam).

Enamel formation is orchestrated by the sequential expression of genes encoding enamel matrix proteins; however, the mechanisms sustaining the spatio-temporal order of gene transcription during amelogenesis are poorly understood. The aim of this study was to characterize the cis-regulatory sequences necessary for normal expression of enamelin (Enam). Several enamelin transcription regulatory regions, showing high sequence homology among species, were identified. DNA constructs containing 5.2 or 3.9 kb regions upstream of the enamelin translation initiation site were linked to a LacZ reporter and used to generate transgenic mice. Only the 5.2-Enam-LacZ construct was sufficient to recapitulate the endogenous pattern of enamelin tooth-specific expression. The 3.9-Enam-LacZ transgenic lines showed no expression in dental cells, but ectopic beta-galactosidase activity was detected in osteoblasts. Potential transcription factor-binding sites were identified that may be important in controlling enamelin basal promoter activity and in conferring enamelin tissue-specific expression. Our study provides new insights into regulatory mechanisms governing enamelin expression.

MSX1-Induced Neural Crest-Like Reprogramming Promotes Melanoma Progression.

Melanoma cells share many biological properties with neural crest stem cells. Here we show that the homeodomain transcription factor MSX1, which is significantly correlated with melanoma disease progression, reprograms melanocytes and melanoma cells toward a neural crest precursor-like state. MSX1-reprogrammed normal human melanocytes express the neural crest marker p75 and become multipotent. MSX1 induces a phenotypic switch in melanoma, which is characterized by an oncogenic transition from an E-cadherin-high nonmigratory state toward a ZEB1-high invasive state. ZEB1 up-regulation is responsible for the MSX1-induced migratory phenotype in melanoma cells. Depletion of MSX1 significantly inhibits melanoma metastasis in vivo. These results show that neural crest-like reprogramming achieved by a single factor is a critical process for melanoma progression.

Temporal requirement for bone morphogenetic proteins in regeneration of the tail and limb of Xenopus tadpoles.

Bone morphogenetic protein (BMP) signalling is necessary for both the development of the tail bud and for tail regeneration in Xenopus laevis tadpoles. Using a stable transgenic line in which expression of the soluble BMP inhibitor noggin is under the control of the temperature inducible hsp70 promoter, we have investigated the timing of the requirement for BMP signalling during tail regeneration. If noggin expression is induced followed by partial amputation of the tail, then wound closure and the formation of the neural ampulla occur normally but outgrowth of the regeneration bud is inhibited. Furthermore, we show that BMP signalling is also necessary for limb bud regeneration, which occurs in Xenopus tadpoles prior to differentiation. When noggin expression is induced, limb bud regeneration fails at an early stage and a stump is formed. The situation appears similar to the tail, with formation of the limb bud blastema occurring but renewed outgrowth inhibited. The transcriptional repressor Msx1, a direct target of BMP signalling with known roles in vertebrate appendage regeneration, fails to be re-expressed in both tail and limb in the presence of noggin. DNA labelling studies show that proliferation in the notochord and spinal cord of the tail, and of the blastema in the limb bud, is significantly inhibited by noggin induction, suggesting that in the context of these regenerating appendages BMP is mainly required, directly or indirectly, as a mitogenic factor.

Msx1 and Dlx5 act independently in development of craniofacial skeleton, but converge on the regulation of Bmp signaling in palate formation.

Msx and Dlx homeoproteins control the morphogenesis and organization of craniofacial skeletal structures, specifically those derived from the pharyngeal arches. In vitro Msx and Dlx proteins have opposing transcriptional properties and form heterodimeric complexes via their homeodomain with reciprocal functional repression. In this report we examine the skeletal phenotype of Msx1; Dlx5 double knock-out (DKO) mice in relationship with their expression territories during craniofacial development. Co-expression of Dlx5 and Msx1 is only observed in embryonic tissues in which these genes have independent functions, and thus direct protein interactions are unlikely to control morphogenesis of the cranium. The DKO craniofacial phenotypes indicate a complex interplay between these genes, acting independently (mandible and middle ear), synergistically (deposition of bone tissue) or converging on the same morphogenetic process (palate growth and closure). In the latter case, the absence of Dlx5 rescues in part the Msx1-dependent defects in palate growth and elevation. At the basis of this effect, our data implicate the Bmp (Bmp7, Bmp4)/Bmp antagonist (Follistatin) signal: in the Dlx5(-/-) palate changes in the expression level of Bmp7 and Follistatin counteract the reduced Bmp4 expression. These results highlight the importance of precise spatial and temporal regulation of the Bmp/Bmp antagonist system during palate closure.

The regenerative plasticity of isolated urodele myofibers and its dependence on MSX1.

The conversion of multinucleate postmitotic muscle fibers to dividing mononucleate progeny cells (cellularisation) occurs during limb regeneration in salamanders, but the cellular events and molecular regulation underlying this remarkable process are not understood. The homeobox gene Msx1 has been studied as an antagonist of muscle differentiation, and its expression in cultured mouse myotubes induces about 5% of the cells to undergo cellularisation and viable fragmentation, but its relevance for the endogenous programme of salamander regeneration is unknown. We dissociated muscle fibers from the limb of larval salamanders and plated them in culture. Most of the fibers were activated by dissociation to mobilise their nuclei and undergo cellularisation or breakage into viable multinucleate fragments. This was followed by microinjection of a lineage tracer into single fibers and analysis of the labelled progeny cells, as well as by time-lapse microscopy. The fibers showing morphological plasticity selectively expressed Msx1 mRNA and protein. The uptake of morpholino antisense oligonucleotides directed to Msx1 led to a specific decrease in expression of Msx1 protein in myonuclei and marked inhibition of cellularisation and fragmentation. Myofibers of the salamander respond to dissociation by activation of an endogenous programme of cellularisation and fragmentation. Lineage tracing demonstrates that cycling mononucleate progeny cells are derived from a single myofiber. The induction of Msx1 expression is required to activate this programme. Our understanding of the regulation of plasticity in postmitotic salamander cells should inform strategies to promote regeneration in other contexts.

Regulation of proliferation in developing human tooth germs by MSX homeodomain proteins and cyclin-dependent kinase inhibitor p19INK4d.

Before the secretion of hard dental tissues, tooth germs undergo several distinctive stages of development (dental lamina, bud, cap and bell). Every stage is characterized by specific proliferation patterns, which is regulated by various morphogens, growth factors and homeodomain proteins. The role of MSX homeodomain proteins in odontogenesis is rather complex. Expression domains of genes encoding for murine Msx1/2 during development are observed in tissues containing highly proliferative progenitor cells. Arrest of tooth development in Msx knockout mice can be attributed to impaired proliferation of progenitor cells. In Msx1 knockout mice, these progenitor cells start to differentiate prematurely as they strongly express cyclin-dependent kinase inhibitor p19INK4d. p19INK4d induces terminal differentiation of cells by blocking the cell cycle in mitogen-responsive G1 phase. Direct suppression of p19INK4d by Msx1 protein is, therefore, important for maintaining proliferation of progenitor cells at levels required for the normal progression of tooth development. In this study, we examined the expression patterns of MSX1, MSX2 and p19INK4d in human incisor tooth germs during the bud, cap and early bell stages of development. The distribution of expression domains of p19INK4d throughout the investigated period indicates that p19INK4d plays active role during human tooth development. Furthermore, comparison of expression domains of p19INK4d with those of MSX1, MSX2 and proliferation markers Ki67, Cyclin A2 and pRb, indicates that MSX-mediated regulation of proliferation in human tooth germs might not be executed by the mechanism similar to one described in developing tooth germs of wild-type mouse.