MSX1 Research Articles

Changes of distal-less homeobox genes 5 and Msh homeobox 1 in a rat model of bisphosphonate related osteonecrosis of the jaw

Objective: To further study the effects of distal-less homeobox gene 5 (Dlx-5) and Msh homeobox 1 (Msx-1) in the pathogenic mechanism of bisphosphonate related osteonecrosis of the jaw (BRONJ) . Methods: Twenty-four SD rats were divided into two groups, the experimental group was injected intraperitoneally with zoledronic acid for 12 weeks (0.2 mg/kg, three times a week), and the control group was injected with saline solution for 12 weeks. The first mandibular molars were extracted after 12 weeks. All of the animals were sacrificed eight weeks after teeth extraction. The BRONJ was diagnosed by gross observation, X-ray examination and histopathlolgical examination. Through real-time PCR, the expression level of Dlx-5 and Msx-1 were detected in the mandible of BRONJ samples and normal samples. Results: X-ray examination and histopathlolgical analysis showed the presence of BRONJ. The results of real-time PCR showed that the expression levels of Dlx-5 were increased (P=0.001) and the expression level of Msx-1 was decreased (P=0.001) in the experimental group compared with the control group. Conclusions: Dlx-5 and Msx-1 genes play roles in the pathogenic mechanism of BRONJ.

Lens-specific deletion of the Msx2 gene increased apoptosis by enhancing the caspase-3/caspase-8 signaling pathway.

ObjectiveTo investigate the influence of Msx2 conditional gene knockout during lens development in mice.MethodsLens-specific Msx2 knockout mice were generated using the Cre-loxP system. The eyes of Msx2 conditional knockout (Msx2CKO) and wild-type (Msx2WT) mice were examined during embryonic and early postnatal periods using histological, immunofluorescence, in situ hybridization, cell proliferation, apoptosis, and mRNA microarray analyses.ResultsMsx2CKO mice exhibited small lens formation and microphthalmia after birth, while Msx2CKO embryos exhibited a persistent lens stalk, small lens formation, and microphthalmia. Conditional deletion of Msx2 also led to an increased apoptosis rate, a significant reduction in FoxE3 expression, and an upregulation of Prox1 expression in the lens vesicle during the early embryonic period. Microarray comparison of Msx2CKO and Msx2WT lens transcriptomes identified a large number of differentially expressed genes. Real-time PCR showed that Casp8 and Casp3 expression was upregulated in Msx2CKO mice at post-natal day 1.ConclusionThe activation of apoptosis through the caspase-8/caspase-3 signaling pathway, together with the downregulation of FoxE3 expression, appeared to account for the smaller lens formation in Msx2CKO mice.

A novel chemo-resistant gene MSX2 discovered by establishment of two pancreatic cancer drug resistant cell lines JF305/CDDP and PANC-1/GEM

Objective: To explore new multidrug resistant genes of pancreatic cancer by establishment and characterization of chemo-resistant cell lines. Methods: The cisplatin-resistant cell line JF305/CDDP and the gemcitabine-resistant cell line PANC-1/GEM were induced by high-dose intermittent treatment. CCK-8 assay was used to detect the 50% inhibiting concentration (IC(50)), drug resistance index (R), cross-resistance, and growth difference of different cells. The changes of cell cycle and migration ability of drug-resistant cells were determined by flow cytometry and transwell assay, respectively. And then real-time fluorescence quantitative PCR was used to detect the expression of multidrug resistance-related genes. Results: The drug resistance indexes of JF305/CDDP and PANC-1/GEM were 15.3 and 27.31, respectively, and there was cross-resistance. Compared with the parental cells, the proliferation rate of JF305/CDDP was decreased by 40% on the fourth day (P<0.05); the proportion of S phase was decreased from (45±2)% to (30±2)% (P<0.05), and the migration ability was enhanced from (32 ±1) cells per field to (158±5) cells per field (P<0.01). The expression of multidrug resistance-related genes MRP2, MDR1, LRP and MSX2 was increased in JF305/CDDP cells (P<0.05). Knockdown of MSX2 in JF305 cells reduced the expression of MRP2, whereas overexpression of MSX2 in PANC-1 cells upregulated MRP2 level (P<0.05). Conclusions: Two stable multidrug resistant cell lines of pancreatic cancer, JF305/CDDP and PANC-1/GEM, were successfully established. MSX2 might be a new drug resistance related gene in pancreatic cancer cells by up-regulation of MRP2 expression.

MSH homeobox 1 polymorphisms and the risk of non-syndromic orofacial clefts: a meta-analysis.

MSH homebox 1 (MSX1) is a susceptibility gene for non-syndromic orofacial clefts (NSOCs). Here, a meta-analysis was conducted to assess their associations. A systematic search of PubMed to 1 September 2017, was performed to retrieve all eligible studies. Odds ratios (ORs) were used to calculate the associations. The stability of the results was evaluated by sensitivity analysis. Publication bias was assessed using Begg's funnel plots and the Egger test. In silico Msx1 expression during early mouse craniofacial development was evaluated by the Gene Expression Omnibus. In the overall analysis, MSX1 rs12532 (G>A) contributed to a decreased risk of NSOC. In an analysis stratified according to disease type, rs12532 was associated with the risk of cleft palate only (CPO) but not with the risk of cleft lip with or without cleft palate (CL/P). The association of rs12532 with the occurrence of NSOC in Asian and Caucasian populations but not South American populations was observed in an analysis stratified according to ethnicity. However, no significant associations were detected between any of the other MSX1 SNPs and the risk of NSOC in either the overall or subgroup analysis. The Msx1 gene was widely expressed in mouse craniofacial structures from embryonic day (E)8.5-E10.5. Taken together, the study indicates that MSX1 rs12532 is associated with the risk of NSOC.

Homeobox genes for embryo implantation: From mouse to human.

The proper development of uterus to a state of receptivity and the attainment of implantation competency for blastocyst are 2 indispensable aspects for implantation, which is considered to be a critical event for successful pregnancy. Like many developmental processes, a large number of transcription factors, such as homeobox genes, have been shown to orchestrate this complicated but highly organized physiological process during implantation. In this review, we focus on progress in studies of the role of homeobox genes, especially the Hox and Msx gene families, during implantation, together with subsequent development of post‐implantation uterus and related reproductive defects in both mouse models and humans, that have led to better understanding of how implantation is precisely regulated and provide new insights into infertility.

Growth factors FGF8 and FGF2 and their receptor FGFR1, transcriptional factors Msx-1 and MSX-2, and apoptotic factors p19 and RIP5 participate in the early human limb development

The expression pattern of fibroblast growth factors FGF8 and FGF2 and their receptor FGFR1, transcription factors MSX-1 and MSX-2, as well as cell proliferation (Ki-67) and cell death associated caspase-3, p19 and RIP5 factors were analyzed in histological sections of eight 4th-9th-weeks developing human limbs by immunohistochemistry and semi-thin sectioning. Increasing expression of all analyzed factors (except FGF8) characterized both the multilayered human apical ectodermal ridge (AER), sub-ridge mesenchyme (progress zone) and chondrocytes in developing human limbs. While cytoplasmic co-expression of MSX-1 and MSX-2 was observed in both limb epithelium and mesenchyme, p19 displayed strong cytoplasmic expression in non-proliferating cells. Nuclear expression of Ki-67 proliferating cells, and partly of MSX-1 and MSX-2 was detected in the whole limb primordium. Strong expression of factors p19 and RIP5, both in the AER and mesenchyme of human developing limbs indicates their possible involvement in control of cell senescence and cell death. In contrast to animal studies, expression of FGFR1 in the surface ectoderm and p19 in the whole limb primordium might reflect interspecies differences in limb morphology. Expression of FGF2 and downstream RIP5 gene, and transcription factors Msx-1 and MSX-2 did not show human-specific changes in expression pattern. Based on their spatio-temporal expression during human limb development, our study indicates role of FGFs and Msx genes in stimulation of cell proliferation, limb outgrowth, digit elongation and separation, and additionally MSX-2 in control of vasculogenesis. The cascade of orchestrated gene expressions, including the analyzed developmental factors, jointly contribute to the complex human limb development.

Transcription profile analysis of the endometrium revealed molecular markers of the personalized ‘window of implantation’ during in vitro fertilization

To determine the most informative markers for assessing the functional state of endometrium during the ‘window of implantation’ and creating a model for assessment of the readiness of endometrium for embryo implantation.Forty-seven women with tubal infertility and a successful IVF pregnancy participated in the study. Pipelle endometrial sample was performed during the supposed ‘window of implantation’ in natural cycle with subsequent histological study, and transcriptional profile of genes GPX3, PAEP, DPP4, TAGLN, HABP2, IMPA2, AQP3, HLA-DOB, MSX1, POSTN determined by real-time quantitative polymerase chain reaction (qRT-PCR).Differences in the level of mRNA expression of all the studied genes in the receptive endometrium were found in comparison to the prereceptive one, which allowed us to classify two functional states of the endometrium. The results of histological examination responded to the stage of maturation of the endometrium in 78.7% of cases. Receptive endometrial status can be determined based on the integral evaluation of mRNA expression level of 4 PAEP, DPP4, MSX1, and HLA-DOB genes. The model for determining a personalized `window implantation' is offered for practical application in ART.

Characterization of PAX9 variant P20L identified in a Japanese family with tooth agenesis.

Transcription factors PAX9 and MSX1 play crucial roles in the development of permanent teeth at the bud stage, and their loss-of-function variants have been associated with congenital tooth agenesis. We sequenced the coding regions of the PAX9 and MSX1 genes from nine patients with non-syndromic tooth agenesis, and identified a missense mutation, P20L, of PAX9 in a single familial case involving three patients in two generations. Identical mutation was previously reported by other authors, but has not been characterized in detail. The mutation was located in a highly conserved N-terminal subdomain of the paired domain and co-segregated as a heterozygote with tooth agenesis. The patients showed defects primarily in the first and second molars, which is typical for cases attributable to PAX9 mutation. Luciferase reporter assay using the 2.3-kb promoter region of BMP4 and electrophoretic mobility shift assay using the CD19-2(A-ins) sequence revealed that P20L substitution eliminated most of the transactivation activity and specific DNA binding activity of PAX9 under the experimental conditions we employed, while some residual activity of the mutant was evident in the former assay. The hypomorphic nature of the variant may explain the relatively mild phenotype in this case, as compared with other PAX9 pathogenic variants such as R26W.

A review on non-syndromic tooth agenesis associated with PAX9 mutations.

SummaryTooth agenesis in the reduction of tooth number which includes hypodontia, oligodontia and anodontia is caused by disturbances and gene mutations that occur during odontogenesis. To date, several genetic mutations that unlock the causes of non-syndromic tooth agenesis are being discovered; these have been associated with certain illnesses because tooth development involves the interaction of several genes for tooth epithelium and mesenchyme odontogenesis. Mutation of candidate genes PAX9 and MSX1 have been identified as the main causes of hypodontia and oligodontia; meanwhile, AXIN2 mutation is associated with anodontia. Previous study using animal models reported that PAX9-deficient knockout mice exhibit missing molars due to an arrest of tooth development at the bud stage. PAX9 frameshift, missense and nonsense mutations are reported to be responsible; however, the most severe condition showed by the phenotype is caused by haploinsufficiency. This suggests that PAX9 is dosage-sensitive. Understanding the mechanism of genetic mutations will benefit clinicians and human geneticists in future alternative treatment investigations.

Activity of msx-1 type genes during craniofacial development

Summary: MSX-1 gene is a member of the MSX homeoboxgene family and it has different functions during the organogenesis process. This homeobox gene isexpressed in sites where epithelium-mesenchyme interactions are required. It also seems to have animportant activity controlling dental and craniofacial development, as investigated in human andmurine models. The following article reviews severasl studies in order to identify MSX.1relationships with dental and craniofacial development. Key words: Odontogenesis. MSX-1.Craniofacial development. Cleft palate. Tooth agenesis.

A novel PAX9 mutation causing oligodontia

IntroductionAn extended family presenting with several members affected by developmentally missing teeth was investigated by analysis of the MSX1 and PAX9 genes. Materials and methodsSaliva samples were collected and DNA extracted. Primers were designed to span the exons and intron-exon junctions of the MSX1 and PAX9 genes. These primers were optimised using gradient Polymerase Chain Reaction. The amplified fragments were sent for Sanger sequencing Resultsa novel heterozygote missense mutation in exon 3 of PAX9 (c.296G > C, p.A99P), was found in two severely affected members of the family as well as a potentially pathogenic heterozygote variant (c.119C > G, p.A40G) in exon 1 of the MSX1 gene. ConclusionThe PAX9 A99P mutation is in the DNA binding domain and is predicted to be pathogenic.

MSX1 Gene Mutations in South Indian Population: A Genetic Research

MSX1 Gene Mutations in South Indian Population: A Genetic Research

Role of MSX1 Gene in Orofacial Clefting : A Systematic Review

Introduction Orofacial clefting has always been the bane of the human race. It affects the overall wellbeing of the affected individual both phycological as well as physical. The Incidence of orofacial clefts have been around 1:500.4 Orofacial clefts can occur from an isolated cleft palate to a

Nine Novel PAX9 Mutations and a Distinct Tooth Agenesis Genotype-Phenotype

Tooth agenesis is one of the most common developmental anomalies affecting function and esthetics. The paired-domain transcription factor, Pax9, is critical for patterning and morphogenesis of tooth and taste buds. Mutations of PAX9 have been identified in patients with tooth agenesis. Despite significant progress in the genetics of tooth agenesis, many gaps in knowledge exist in refining the genotype-phenotype correlation between PAX9 and tooth agenesis. In the present study, we complete genetic and phenotypic characterization of multiplex Chinese families with nonsyndromic (NS) tooth agenesis. Direct sequencing of polymerase chain reaction products revealed 9 novel (c.140G>C, c.167T>A, c.332G>C, c.194C>A, c.271A>T, c.146delC, c.185_189dup, c.256_262dup, and c.592delG) and 2 known heterozygous mutations in the PAX9 gene among 120 probands. Subsequently, pedigrees were extended, and we confirmed that the mutations co-segregated with the tooth agenesis phenotype (with exception of families in which DNA analysis was not available). In 1 family (n = 6), 2 individuals harbored both the PAX9 c.592delG mutation and a heterozygous missense mutation (c.739C>T) in the MSX1 gene. Clinical characterization of families segregating a PAX9 mutation reveal that all affected individuals were missing the mandibular second molar and their maxillary central incisors are most susceptible to microdontia. A significant reduction of bitter taste perception was documented in individuals harboring PAX9 mutations (n = 3). Functional studies revealed that PAX9 haploinsufficiency or a loss of function of the PAX9 protein underlies tooth agenesis.

Top-Down Inhibition of BMP Signaling Enables Robust Induction of hPSCs Into Neural Crest in Fully Defined, Xeno-free Conditions.

SummaryDefects in neural crest development have been implicated in many human disorders, but information about human neural crest formation mostly depends on extrapolation from model organisms. Human pluripotent stem cells (hPSCs) can be differentiated into in vitro counterparts of the neural crest, and some of the signals known to induce neural crest formation in vivo are required during this process. However, the protocols in current use tend to produce variable results, and there is no consensus as to the precise signals required for optimal neural crest differentiation. Using a fully defined culture system, we have now found that the efficient differentiation of hPSCs to neural crest depends on precise levels of BMP signaling, which are vulnerable to fluctuations in endogenous BMP production. We present a method that controls for this phenomenon and could be applied to other systems where endogenous signaling can also affect the outcome of differentiation protocols.

Electrophysiological and gene expression characterization of the ontogeny of nestin-expressing cells in the adult mouse midbrain

The birth of new neurons, or neurogenesis, in the adult midbrain is important for progressing dopamine cell-replacement therapies for Parkinson's disease. Most studies suggest newborn cells remain undifferentiated or differentiate into glia within the adult midbrain. However, some studies suggest nestin+neural precursor cells (NPCs) have a propensity to generate new neurons here. We sought to confirm this by administering tamoxifen to adult NesCreERT2/R26eYFP transgenic mice, which permanently labelled adult nestin-expressing cells and their progeny with enhanced yellow fluorescent protein (eYFP). eYFP+ midbrain cells were then characterized 1–32weeks later in acutely prepared brain slices using whole-cell patch clamp electrophysiology combined with single-cell RT-qPCR. Most eYFP+ cells exhibited a mature neuronal phenotype with large amplitude fast action potentials (APs), spontaneous post-synaptic currents (sPSCs), and expression of ‘mature’ neuronal genes (NeuN, Gad1, Gad2 and/or VGLUT2). This was the case even at the earliest time-point following tamoxifen (i.e. 1week). In comparison to neighboring eYFP− (control) cells, eYFP+ cells discharged more APs per unit current injection, and had faster AP time-to-peak, hyperpolarized resting membrane potential, smaller membrane capacitance and shorter duration sPSCs. eYFP+ cells were also differentiated from eYFP− cells by increased expression of ‘immature’ pro-neuronal genes (Pax6, Ngn2 and/or Msx1). However, further analyses failed to reveal evidence of a place of birth, neuronal differentiation, maturation and integration indicative of classical neurogenesis. Thus our findings do not support the notion that nestin+NPCs in the adult SNc and midbrain generate new neurons via classical neurogenesis. Rather, they raise the possibility that mature neurons express nestin under unknown circumstances, and that this is associated with altered physiology and gene expression.

NKL homeobox gene MSX1 acts like a tumor suppressor in NK-cell leukemia.

NKL homeobox gene MSX1 is physiologically expressed in lymphoid progenitors and subsequently downregulated in developing T- and B-cells. In contrast, elevated expression levels of MSX1 persist in mature natural killer (NK)-cells, indicating a functional role in this compartment. While T-cell acute lymphoblastic leukemia (T-ALL) subsets exhibit aberrant overexpression of MSX1, we show here that in malignant NK-cells the level of MSX1 transcripts is aberrantly downregulated. Chromosomal deletions at 4p16 hosting the MSX1 locus have been described in NK-cell leukemia patients. However, NK-cell lines analyzed here showed normal MSX1 gene configurations, indicating that this aberration might be uncommon. To identify alternative MSX1 regulatory mechanisms we compared expression profiling data of primary normal NK-cells and malignant NK-cell lines. This procedure revealed several deregulated genes including overexpressed IRF4, MIR155HG and MIR17HG and downregulated AUTS2, EP300, GATA3 and HHEX. As shown recently, chromatin-modulator AUTS2 is overexpressed in T-ALL subsets where it mediates aberrant transcriptional activation of MSX1. Here, our data demonstrate that in malignant NK-cell lines AUTS2 performed MSX1 activation as well, but in accordance with downregulated MSX1 transcription therein we detected reduced AUTS2 expression, a small genomic deletion at 7q11 removing exons 3 and 4, and truncating mutations in exon 1. Moreover, genomic profiling and chromosomal analyses of NK-cell lines demonstrated amplification of IRF4 at 6p25 and deletion of PRDM1 at 6q21, highlighting their potential oncogenic impact. Functional analyses performed via knockdown or forced expression of these genes revealed regulatory network disturbances effecting downregulation of MSX1 which may underlie malignant development in NK-cells.

Msh homeobox 1 (Msx1)- and Msx2-overexpressing bone marrow-derived mesenchymal stem cells resemble blastema cells and enhance regeneration in mice

Amputation of the proximal region in mammals is not followed by regeneration because blastema cells (BCs) and expression of regenerative genes, such as Msh homeobox (Msx) genes, are absent in this animal group. The lack of BCs and positional information in other cells is therefore the main obstacle to therapeutic approaches for limb regeneration. Hence, this study aimed to create blastema-like cells (BlCs) by overexpressing Msx1 and Msx2 genes in mouse bone marrow-derived mesenchymal stem cells (mBMSCs) to regenerate a proximally amputated digit tip. We transduced mBMSCs with Msx1 and Msx2 genes and compared osteogenic activity and expression levels of several Msx-regulated genes (Bmp4, Fgf8, and keratin 14 (K14)) in BlC groups, including MSX1, MSX2, and MSX1/2 (in a 1:1 ratio) with those in mBMSCs and BCs in vitro and in vivo following injection into the amputation site. We found that Msx gene overexpression increased expression of specific blastemal markers and enhanced the proliferation rate and osteogenesis of BlCs compared with mBMSCs and BCs via activation of Fgf8 and Bmp4 Histological analyses indicated full regrowth of digit tips in the Msx-overexpressing groups, particularly in MSX1/2, through endochondral ossification 6 weeks post-injection. In contrast, mBMSCs and BCs formed abnormal bone and nail. Full digit tip was regenerated only in the MSX1/2 group and was related to boosted Bmp4, Fgf8, and K14 gene expression and to limb-patterning properties resulting from Msx1 and Msx2 overexpression. We propose that Msx-transduced cells that can regenerate epithelial and mesenchymal tissues may potentially be utilized in limb regeneration.

Establishment of a congenital tooth agenesis related gene MSX1 knockout human embryonic stem cell lines by CRISPR-Cas9 technology

Human MSX1 gene is mapped to chromosome 4 and encodes a 303aa homeobox protein MSX1. MSX1 expression appears during early tooth development of vertebrate embryogenesis. Mutations in this protein are related to human tooth anomalie, cleft lip and palate and congenital ectodermal dysplasia syndrome. Most of the confirmed pathogenic mutations are located in exon2 encoded homeobox domain. Here, we report the establishment of MSX1 gene knockout human embryonic stem (hES) cell lines by CRISPR-Cas9 technology. These cell lines provide good materials for further studies of the roles MSX1 plays in human tooth development and congenital tooth agenesis.

Can Valproic Acid Regulate Neurogenesis from Nestin+ Cells in the Adult Midbrain?

Degeneration of dopamine (DA) neurons in the substantia nigra pars compacta (SNc) causes the motor symptoms (e.g. tremor, muscle rigidity, bradykinesia, postural instability) of Parkinson's disease (PD). It is generally agreed that replacing these neurons will provide better motor symptom relief and fewer side effects than current pharmacotherapies. One potential approach to this is up-regulating endogenous DA neurogenesis in SNc. In the present study, we conducted bioinformatics analyses to identify signalling pathways that control expression of Pax6 and Msx1 genes, which have been identified as potentially important neurogenic regulators in the adult midbrain. From this Valproic acid (VPA) was identified as a regulator of these pathways, and we tested VPA for its ability to regulate midbrain neurogenesis in adult mice. VPA was infused directly into the midbrain of adult NesCreERT2/R26eYFP mice using osmotic pumps attached to implanted cannula. These mice enable permanent eYFP+ labelling of adult Nestin-expressing neural precursor cells and their progeny/ontogeny. VPA did not affect the number of eYFP+ midbrain cells, but significantly reduced the number of Pax6+, Pax6+/NeuN+, eYFP+/NeuN+ and eYFP-/NeuN+ cells. However, this reduction in NeuN expression was probably via VPA's Histone de-acetylase inhibitory properties rather than reduced neuronal differentiation by eYFP + cells. We conclude that Pax6 and Msx1 are not viable targets for regulating neurogenesis in the adult midbrain.