MSX1 Research Articles

Abstract 413: Targeting Fundamental Mechanisms Of Aging To Attenuate Molecular Drivers Of Vascular Calcification

Increases in vascular calcification (VC) and fibrosis are a near ubiquitous finding with aging. Here, we sought to test the hypothesis that Metformin (MET)—an AMPK activator entering large clinical trials that is thought to attenuate multimorbidity associated with aging—attenuates key molecular processes known to drive vascular calcification (VC) and fibrogenic signaling in vascular smooth muscle cells (VSMCs) in vitro. Given recent reports highlighting the importance of mitochondria and sirtuins with aging, we also tested the hypothesis that MET-driven reductions in osteogenic signaling are associated with upregulation of SIRT3. We used exogenous BMP2 (100 ng/ml) and TGFβ1 (10 ng/ml) as osteogenic and fibrogenic stimuli, respectively. Consistent with prior work from our lab, treatment with BMP2 for 24 hours robustly increased expression of the osteogenic genes Msx2 (3.9±1.5 fold-change, FC), Runx2 (3.5±1.2 FC), and osterix (17.2±5.2 FC) in VSMCs. Critically, co-treatment with Metformin dramatically reduced expression of these genes by 68 ± 22%, 58 ±20%, and 63 ± 4%, respectively. Subsequent experiments treating cells with exogenous TGFβ1 resulted in significant induction of the fibrogenic marker/known TGFβ1 target versican (1.7±0.4 FC), an effect that was largely attenuated by co-treatment with MET (58±3%). Treatment with MET did not, however, impact TGFβ1-induced increases in CTGF or fibronectin. Treatment with either BMP2 or TGFβ1 resulted in significant increases in mRNA levels of SIRT3 (2.5 ±0.11 and 1.4 ±0.17 FC, respectively), whereas treatment with MET resulted in modest and less consistent increases in SIRT3 mRNA (1.3 ± 0.2 FC). Interestingly, however, co-treatment with BMP2+MET or TGFβ1+MET resulted in uniform suppression of BMP2/ TGFβ1-induced increases in SIRT3 (54±19% and 25±12%, respectively). Collectively, these data suggest that treatment with MET may be a viable strategy to attenuate age-associated increases in osteogenic and fibrogenic signaling in conduit vessels, and suggest further in vivo investigation into therapeutic efficacy is warranted.

Rehabilitation of congenitally missing teeth with precision attachment and fixed PFM bridge: Case reports

The definition of hypodontia is obvious and unambiguous: “one or additional congenitally absent teeth” it’s a genetic disease that denotes a stronger clinical expression of hypodontia, includes a wide and non-specific. To define oligodontia. The “absence of the many teeth, sometimes related to little size of the prevailing teeth and alternative anomalies”. The prevalence of genetic disease has been reported as zero.3%. It affects females the maximum amount as males, with a gender quantitative relation of 3:2.3. Genes accountable for non syndromic genetic disease area unit found to be MSX1 and PAX9 genes. Within the current edition of the glossary of dentistry Terms, the definition reads “the formation of less than full complete teeth. Several such teeth area unit smaller than normal”. Current article describe a case of genetic disease treated with jaw unilateral exactitude attachment on distal side of 11 and bar & clip maintained overdenture for the second quadrant with a 1 piece forged denture support of anterioposterior-palatal strap and mandibular complete arch rehabilitation with metal ceramic crowns.

Generation of a MSX1 knockout human embryonic stem cell line using CRISPR/Cas9 technology

The MSX1 gene encodes a transcriptional repressor and plays important roles in limb-pattern formation, craniofacial development, and odontogenesis during vertebrate embryogenesis. Previous studies demonstrated that human MSX1 mutations are associated with tooth agenesis, orofacial clefting, and nail dysplasia. Here, we generated a MSX1 knockout cell line from human embryonic stem cell (hESC) line (H9) by CRISPR/cas9-mediated gene targeting. This cell line may serve as a valuable in vitro cell model for MSX1 mutation-related diseases and help to gain more insight into the biological function of MSX1.

Induction of Salivary Gland-Like Tissue by Induced Pluripotent Stem Cells In Vitro.

To investigate the in vitro induction of salivary gland-like tissue by ips cells in an interferon regulatory factor 6 (IRF6) overexpression and parotid conditioned medium environment. Urine-derived ips cells were isolated, identified, transfected with IRF6 and cultured in parotid conditioned medium to induce ips cells into salivary gland differentiation, morphological changes of ips cells were observed, CCK-8 was used to determine the cell proliferation efficiency and transcriptome sequencing was used to detect the expression of genes related to parotid gland formation. Immunofluorescence staining showed that the isolated ips cells were positive for NANOG, SSEA4 and OCT4 and had embryonic-like stem cell characteristics; CCK-8 showed that there was no statistical difference in the proliferation efficiency between the IRF6+ induced group and the simple induced group after induction of ips cells into salivary glands. The results of transcriptome sequencing showed that there were a total of 643 differentially expressed genes, including 365 up-regulated genes and 278 down-regulated genes in the IRF6+ induced group compared to the blank control group, and the salivary gland related genes HAPLN1, CCL2, MSX2, ANXA1, CYP11A1, HES1 and LUM were all highly expressed in the IRF6+ induced group. IRF6 promotes salivary gland differentiation in urine-derived iPSCs, and its mechanism of promoting differentiation may be that IRF6 upregulates the expression of HAPLN1, CCL2, MSX2, ANXA1, CYP11A1, HES1 and LUM to promote epithelial differentiation.

Developmental Transcriptome Profiling of the Tibial Reveals the Underlying Molecular Basis for Why Newly Hatched Quails Can Walk While Newly Hatched Pigeons Cannot.

Birds can be classified into altricial and precocial species. The hatchlings of altricial birds cannot stand, whereas precocial birds can walk and run soon after hatching. It might be owing to the development of the hindlimb bones in the embryo stage, but the molecular regulatory basis underlying the divergence is unclear. To address this issue, we chose the altricial pigeon and the precocial Japanese quail as model animals. The data of tibia weight rate, embryonic skeletal staining, and tibia tissues paraffin section during the embryonic stage showed that the Japanese quail and pigeon have similar skeletal development patterns, but the former had a faster calcification rate. We utilized the comparative transcriptome approach to screen the genes and pathways related to this heterochronism. We separately analyzed the gene expression of tibia tissues of quail and pigeon at two consecutive time points from an inability to stand to be able to stand. There were 2910 differentially expressed genes (DEGs) of quail, and 1635 DEGs of pigeon, respectively. A total of 409 DEGs in common in the quail and pigeon. On the other hand, we compared the gene expression profiles of pigeons and quails at four time points, and screened out eight pairs of expression profiles with similar expression trends but delayed expression in pigeons. By screening the common genes in each pair of expression profiles, we obtained a gene set consisting of 152 genes. A total of 79 genes were shared by the 409 DEGs and the 152 genes. Gene Ontology analysis of these common genes showed that 21 genes including the COL gene family (COL11A1, COL9A3, COL9A1), IHH, MSX2, SFRP1, ATP6V1B1, SRGN, CTHRC1, NOG, and GDF5 involved in the process of endochondral ossification. These genes were the candidate genes for the difference of tibial development between pigeon and quail. This is the first known study on the embryo skeletal staining in pigeon. It provides some new insights for studying skeletal development mechanisms and locomotor ability of altricial and precocial bird species.

Hypermethylation of PDX1, EN2, and MSX1 predicts the prognosis of colorectal cancer

Despite numerous observations regarding the relationship between DNA methylation changes and cancer progression, only a few genes have been verified as diagnostic biomarkers of colorectal cancer (CRC). To more practically detect methylation changes, we performed targeted bisulfite sequencing. Through co-analysis of RNA-seq, we identified cohort-specific DNA methylation markers: CpG islands of the intragenic regions of PDX1, EN2, and MSX1. We validated that these genes have oncogenic features in CRC and that their expression levels are increased in correlation with the hypermethylation of intragenic regions. The reliable depth of the targeted bisulfite sequencing data enabled us to design highly optimized quantitative methylation-specific PCR primer sets that can successfully detect subtle changes in the methylation levels of candidate regions. Furthermore, these methylation levels can divide CRC patients into two groups denoting good and poor prognoses. In this study, we present a streamlined workflow for screening clinically significant differentially methylated regions. Our discovery of methylation markers in the PDX1, EN2, and MSX1 genes suggests their promising performance as prognostic markers and their clinical application in CRC patients.

Abstract 11412: Methyltransferase Smyd2 Mediate Atherosclerotic Plaque Calcification

Introduction: Clinical manifestation of atherosclerosis is marked by calcification of vasculature, including the expression of mineralization regulating proteins (Sox9, Msx2, and Runx2) by vascular smooth muscle cells (VSMCs). This pathogenic remodeling of VSMCs into osteochondrogenic cell types is accompanied by increased proliferation, migration, and secretion of extracellular matrix proteins. This study aimed to determine the spatiotemporal role of SMYD2 in atherosclerotic plaque progression and calcification. Methods: SMC-specific SMYD2 knockout was generated in ApoE -/- mice (SMYD2 ΔSMC ApoE -/- ). Age and body weight-matched mice were fed with Western diet (WD) for 18 or 26 weeks. The distribution of atherosclerotic plaque was examined by En-face Oil-Red-O staining (ORO) of freshly collected aorta. Atherosclerotic plaque burden was determined by Hematoxylin & Eosin (H&E) staining. Collagen content and calcification of atherosclerotic plaque were determined by Masson's trichrome staining and von Kossa staining, respectively. The calcification-regulatory genes (MGP1, AGG and transcription factors SOX9, MSX2, BMP2, and RUNX2) in the aorta were analyzed by RT-qPCR. Results: SMC-specific deletion of SMYD2 protected mice from atherosclerotic plaque calcification. En-face ORO and H&E analysis of Brachiocephalic artery demonstrated a significant decrease in ORO positive area, total plaque size, and necrotic core area in SMYD2 ΔSMC ApoE -/- mice as compared to ApoE-/- mice, with no change in the distribution of collagen fibers. Von Kossa staining showed hydroxyapatite depots at the medial lining of the vasculature (microcalcification) in ApoE -/- mice fed with 18-week Western diet, while a smaller depot of macrocalcification was observed in SMYD2 ΔSMC ApoE -/- mice compared with ApoE -/- mice when fed with 26-week WD. mRNA analysis of key calcification transcription factors showed significant suppression of SOX9 and MSX2 genes in aortas of SMYD2 ΔSMC ApoE -/- mice compared with ApoE -/- mice, with downward trends of BMP2 and RUNX2 in both 18- or 26-week WD. Conclusion: SMYD2 ablation attenuates atherosclerotic plaque formation and calcification, suggests targeting SMYD2 can be a potential therapeutic strategy for atherosclerosis.

Novel MSX1 frameshift mutation in a Japanese family with nonsyndromic oligodontia

Congenital tooth agenesis is a common anomaly in humans. We investigated the etiology of human tooth agenesis by exome analysis in Japanese patients, and found a previously undescribed heterozygous deletion (NM_002448.3(MSX1_v001):c.433_449del) in the first exon of the MSX1 gene. The deletion leads to a frameshift and generates a premature termination codon. The truncated form of MSX1, namely, p.(Trp145Leufs*24) lacks the homeodomain, which is crucial for transcription factor function.

Quantitative label-free optical technique to analyze the ultrastructure changes and spatiotemporal relationship of enamel induced by Msx2 deletion.

New advances in the molecular mechanism of enamel mineralization reveal the practical significance of regenerative medicine in clinical transformation. Muscle segment homeobox 2 (MSX2), a transcription factor, is recently reported to be closely associated with the amelogenesis imperfecta (AI). To elucidate the biomineralization framework of AI enamel, herein, Msx2 gene mutant mice are investigated by dual-mode noninvasive spectroscopic analytical techniques for the first time. Optical coherence tomography (OCT) records the depth-resolved structural information of mice teeth, where a dramatic decrease in enamel thickness and quality occurred in Msx2 deficient (Msx2-/- ) enamel. And it has the advantages of fast, noninvasive and low cost. Raman spectroscopy, a powerful molecular fingerprint tool, further witnesses an imbalance of inorganic and organic contents in Msx2-/- enamel. In addition, abnormal expression of MSX2 also influences the spatial distribution of phosphate in enamel according to the Raman spectral imaging. Therefore, OCT integrated with Raman spectroscopy provides the quantitative label-free optical parameters of both the physical structure and chemical component in mice enamel, which strengthens the understanding of the biomineralization process underlying the Msx2-related amelogenesis imperfect.

The absence of muscle segment homeobox 2 leads to the pyroptosis of ameloblasts by inducing squamous epithelial hyperplasia in the enamel organ.

Muscle segment homeobox 2 (MSX2) has been confirmed to be involved in the regulation of early tooth development. However, the role of MSX2 has not been fully elucidated in enamel development. To research the functions of MSX2 in enamel formation, we used a Msx2 −/− (KO) mouse model with no full Msx2 gene. In the present study, the dental appearance and enamel microstructure were detected by scanning electron microscopy and micro‐computed tomography. The results showed that the absence of Msx2 resulted in enamel defects, leading to severe tooth wear in KO mice. To further investigate the mechanism behind the phenotype, we performed detailed histological analyses of the enamel organ in KO mice. We discovered that ameloblasts without Msx2 could secrete a small amount of enamel matrix protein in the early stage. However, the enamel epithelium occurred squamous epithelial hyperplasia and partial keratinization in the enamel organ during subsequent developmental stages. Ameloblasts depolarized and underwent pyroptosis. Overall, during the development of enamel, MSX2 affects the formation of enamel by regulating the function of epithelial cells in the enamel organ.

Association of Polymorphic and Haplotype Variants of the MSX1 Gene and the Impacted Teeth Phenomenon.

It is known that genetic factors determine odontogenesis; furthermore, studies have revealed that various genes in humans can regulate the development of different types and generations of teeth. In this study it has been assumed that tooth impaction—at least to some extent—also depends on the presence of specific genetic markers, especially allelic variants of the MSX1 gene. The primary objective of the study was to evaluate the suitability of selected molecular markers located within the MSX1 gene for the determination of the risk of tooth impaction in particular patients. The study participants were divided into two groups: (1) the study group—at least one secondary tooth was impacted in the jaws; (2) the control group—no impacted tooth in the jaws. Real-Time PCR and TaqMan probes were used to detect selected polymorphisms in the analyzed genes. Two single nucleotide polymorphisms of MSX1 were analyzed. After the two subgroups of patients were distinguished in the study group based on the number of impacted teeth, statistically significant differences in the frequency of genotypes described for rs12532 in the MSX1 gene were found.

Foramina parietalia permagna: descrizione di un caso clinico e revisione della letteratura

The purpose of the present work is to expose the defects of parietal bone ossification and to identify the criteria for differential diagnosis and brain changes related to the condition, with particular attention to the venous developmental anomalies and the pathological features associated. Foramina parietalia permagna (FPP) are caused by an insufficient intramembranous ossification around the parietal notch that is normally obliterated in the fifth month of normal foetal development. During the first few years of life as calvarial growth continues, cranium bifidum tends to resolve into two distinct, large parietal foramina. Most people with FPP have a positive family history as the condition is inherited in an autosomal dominant fashion with high, but incomplete penetrance. Mutations of either MSX2 or ALX4 genes are associated with enlarged parietal foramina. Meningeal cortical vascular malformation of the straight sinus and persistent falcine sinus have also been reported in the literature as possible associated anomalies.

Disorders of sex development in Wolf\u2013Hirschhorn syndrome: a genotype\u2013phenotype correlation and MSX1 as candidate gene

BackgroundWolf–Hirschhorn (WHS) is a set of congenital physical anomalies and mental retardation associated with a partial deletion of the short arm of chromosome 4. To establish a genotype–phenotype correlation; we carried out a molecular cytogenetic analysis on two Tunisian WHS patients. Patient 1 was a boy of 1-year-old, presented a typical WHS phenotype while patient 2, is a boy of 2 days presented an hypospadias, a micropenis and a cryptorchidie in addition to the typical WHS phenotype. Both the array comparative genomic hybridization and fluorescence in situ hybridization techniques were used.ResultsResults of the analysis showed that patient 2 had a greater deletion size (4.8 Mb) of chromosome 4 than patient 1 (3.4 Mb). Here, we notice that the larger the deletion, the more genes are likely to be involved, and the more severe the phenotype is likely to be. If we analyze the uncommon deleted region between patient1 and patient 2 we found that the Muscle Segment Homeobox (MSX1) gene is included in this region. MSX1 is a critical transcriptional repressor factor, expressed in the ventral side of the developing anterior pituitary and implicated in gonadotrope differentiation. Msx1 acts as a negative regulatory pituitary development by repressing the gonadotropin releasing hormone (GnRH) genes during embryogenesis. We hypothesized that the deletion of MSX1 in our patient may deregulate the androgen synthesis.ConclusionBased on the MSX1 gene function, its absence might be indirectly responsible for the hypospadias phenotype by contributing to the spatiotemporal regulation of GnRH transcription during development.

Tooth agenesis: What do we know and is there a connection to cancer?

Like all developmental processes, odontogenesis is highly complex and dynamically regulated, with hundreds of genes co-expressed in reciprocal networks. Tooth agenesis (missing one or more/all teeth) is a common human craniofacial anomaly and may be caused by genetic variations and/or environmental factors. Variants in PAX9, MSX1, AXIN2, EDA, EDAR, and WNT10A genes are associated with tooth agenesis. Currently, variants in ATF1, DUSP10, CASC8, IRF6, KDF1, GREM2, LTBP3, and components and regulators of WNT signaling WNT10B, LRP6, DKK, and KREMEN1 are at the forefront of interest. Due to the interconnectedness of the signaling pathways of carcinogenesis and odontogenesis, tooth agenesis could be a suitable marker for early detection of cancer predisposition. Variants in genes associated with tooth agenesis could serve as prognostic or therapeutic targets in cancer. This review aims to summarize existing knowledge of development and clinical genetics of teeth. Concurrently, the review proposes possible approaches for future research in this area, with particular attention to roles in monitoring, early diagnosis and therapy of tumors associated with defective tooth development.

Novel MSX1 variants identified in families with nonsyndromic oligodontia

The goal of this study was to identify MSX1 gene variants in multiple Chinese families with nonsyndromic oligodontia and analyse the functional influence of these variants. Whole-exome sequencing (WES) and Sanger sequencing were performed to identify the causal gene variants in five families with nonsyndromic oligodontia, and a series of bioinformatics databases were used for variant confirmation and functional prediction. Phenotypic characterization of the members of these families was described, and an in vitro analysis was performed for functional evaluation. Five novel MSX1 heterozygous variants were identified: three missense variants [c.662A>C (p.Q221P), c.670C>T (p.R224C), and c.809C>T (p.S270L)], one nonsense variant [c.364G>T (p.G122*)], and one frameshift variant [c.277delG (p.A93Rfs*67)]. Preliminary in vitro studies demonstrated that the subcellular localization of MSX1 was abnormal with the p.Q221P, p.R224C, p.G122*, and p.A93Rfs*67 variants compared to the wild type. Three variants (p.Q221P, p.G122*, and p.A93Rfs*67) were classified as pathogenic or likely pathogenic, while p.S270L and p.R224C were of uncertain significance in the current data. Moreover, we summarized and analysed the MSX1-related tooth agenesis positions and found that the type and variant locus were not related to the severity of tooth loss. Our results expand the variant spectrum of nonsyndromic oligodontia and provide valuable information for genetic counselling.

Diagnostic Outcomes for Genetic Testing of 54 Genes in Pregnancy Loss Using Array CGH Method: A Two-Year Retrospective Study

Objective: The current study aimed to retrospectively evaluate different diag- nostic approaches for the array genetic analysis of the cases from all trimester fetal loss in the medical genetics clinic between 2016 and 2017. The Quantitati- ve Fluorescent Polymerase Chain Reaction(QFPCR) test was performed on 50 samples, and aneuploidy was detected in 11 samples as a result of the test, and the array-CGH was performed when 39 QF-PCR resulted in normal test results. Under this purpose, we aimed to analyze and determine the possible copy num- ber variation(CNV), gene deletions, and/or duplications involved in embryonic cell division, tissue differentiation, intended.Materials and methods: DNA isolation from cases of this retrospective study was completed using the PureLink Genomic DNA isolation kit. DNA samples were then genoty - ped for molecular etiological reasons by oligonücleotid microarray -CGH method (aCGH , 60 K ISCA design , Agilent , Germany ). Hybridized probe correlations of the case and reference DNAs were evaluated with databases (Database of Genomic Variants Analysis ) used in genomic variation analysis in terms of 54 functional genes CNVs associated with intrauterine losses.Results: CNV was detected in 30 (77%) of 39 fetal samples analyzed within the scope of the research. Fifty-five percent of CNVs were found to be duplication (55%) and forty-five percent were deletions (45%). As a result of the evaluation, deletion was detected in 19 (35%) of 54 genes, duplication was detected in 26 (48%), while in 3 (6%) both deletion and duplication were detected. Although CNV detected in autosomal chromosomes (chromosome 1, 2, 3, 4, 5, 7, 8, 10, 12, 13, 14, 15 and 20), CNV was established the most common in X chromosome. In our study, CNVs associated with COX7B, ZIC1, MECP2, FMR1, HOXD13, JAG1, MSX2, NEXN, and SIX3 genes were found to be more frequent in terms of fetal loss etiology.Conclusions: Based on our experience, the array-CGH method can be used to investigate the etiology of the normal results of QF-PCR in cases of fetal loss. The array CGH method will be preferred more and more due to the ease of application and the data obtained. When we look at the literature, it is seen that there is not enough research on array CGH about fetal loss and more studies are needed to increase the experience in this field.

An Msx2-Sp6-Follistatin Pathway Operates During Late Stages of Tooth Development to Control Amelogenesis.

BackgroundAmeloblasts are epithelially derived cells responsible for enamel formation through a process known as amelogenesis. Amongst the several transcription factors that are expressed during amelogenesis, both Msx2 and Sp6 transcription factors play important role. Msx2 and Sp6 mouse mutants, exhibit similar amelogenesis defects, namely enamel hypoplasia, while humans with amelogenesis imperfecta (AI) carry mutations in the human homologues of MSX2 or SP6 genes. These across species similarities in function indicate that these two transcription factors may reside in the same developmental pathway. In this paper, we test whether they work in a coordinated manner to exert their effect during amelogenesis.MethodsTwo different dental epithelial cell lines, the mouse LS8 and the rat G5 were used for either overexpression or silencing of Msx2 or Sp6 or both. Msx2 mutant mouse embryos or pups were used for in vivo studies. In situ hybridization, semi-quantitative and quantitative real time PCR were employed to study gene expression pattern. MatInspector was used to identify several potential putative Msx2 binding sites upstream of the murine Sp6 promoter region. Chromatin Immunoprecipitation (chIP) was used to confirm the binding of Msx2 to Sp6 promoter at the putative sites.ResultsUsing the above methods we identified that (i) Msx2 and Sp6 exhibit overlapping expression in secretory ameloblasts, (ii) Sp6 expression is reduced in the Msx2 mouse mutant secretoty ameloblasts, and (iii) that Msx2, like Sp6 inhibits follistatin expression. Specifically, our loss-of function studies by silencing Msx2 and/or Sp6 in mouse dental epithelial (LS8) cells showed significant downregulation of Sp6 but upregulation of Fst expression. Transient transfection of Msx2 overexpression plasmid, up-regulated Sp6 and downregulated Fst expression. Additionally, using MatInspector, we identified several potential putative Msx2 binding sites, 3.5 kb upstream of the murine Sp6 promoter region. By chIP, we confirmed the binding of Msx2 to Sp6 promoter at these sites, thus suggesting that Sp6 is a direct target of Msx2.ConclusionCollectively, these results show that Sp6 and Msx2 work in a concerted manner to form part of a network of transcription factors that operate during later stages of tooth development controlling ameloblast life cycle and amelogenesis.

Msx1 Heterozygosity in Mice Enhances Susceptibility to Phenytoin-Induced Hypoxic Stress Causing Cleft Palate.

Cleft palate is among the most frequent congenital defects in humans. While gene-environment multifactorial threshold models have been proposed to explain this cleft palate formation, only a few experimental models have verified this theory. This study aimed to clarify whether gene-environment interaction can cause cleft palate through a combination of specific genetic and environmental factors. Msx1 heterozygosity in mice (Msx1+/-) was selected as a genetic factor since human MSX1 gene mutations may cause nonsyndromic cleft palate. As an environmental factor, hypoxic stress was induced in pregnant mice by administration of the antiepileptic drug phenytoin, a known arrhythmia inducer, during palatal development from embryonic day (E) 11 to E14. Embryos were dissected at E13 for histological analysis or at E17 for recording of the palatal state. Phenytoin administration downregulated cell proliferation in palatal processes in both wild-type and Msx1+/- embryos. Bone morphogenetic protein 4 (Bmp4) expression was slightly downregulated in the anterior palatal process of Msx1+/- embryos. Although Msx1+/- embryos do not show cleft palate under normal conditions, phenytoin administration induced a significantly higher incidence of cleft palate in Msx1+/- embryos compared to wild-type littermates. Our data suggest that cleft palate may occur because of the additive effects of Bmp4 downregulation as a result of Msx1 heterozygosity and decreased cell proliferation upon hypoxic stress. Human carriers of MSX1 mutations may have to take more precautions during pregnancy to avoid exposure to environmental risks.

Polarized Sonic Hedgehog Protein Localization and a Shift in the Expression of Region-Specific Molecules Is Associated With the Secondary Palate Development in the Veiled Chameleon.

Secondary palate development is characterized by the formation of two palatal shelves on the maxillary prominences, which fuse in the midline in mammalian embryos. However, in reptilian species, such as turtles, crocodilians, and lizards, the palatal shelves of the secondary palate develop to a variable extent and morphology. While in most Squamates, the palate is widely open, crocodilians develop a fully closed secondary palate. Here, we analyzed developmental processes that underlie secondary palate formation in chameleons, where large palatal shelves extend horizontally toward the midline. The growth of the palatal shelves continued during post-hatching stages and closure of the secondary palate can be observed in several adult animals. The massive proliferation of a multilayered oral epithelium and mesenchymal cells in the dorsal part of the palatal shelves underlined the initiation of their horizontal outgrowth, and was decreased later in development. The polarized cellular localization of primary cilia and Sonic hedgehog protein was associated with horizontal growth of the palatal shelves. Moreover, the development of large palatal shelves, supported by the pterygoid and palatine bones, was coupled with the shift in Meox2, Msx1, and Pax9 gene expression along the rostro-caudal axis. In conclusion, our results revealed distinctive developmental processes that contribute to the expansion and closure of the secondary palate in chameleons and highlighted divergences in palate formation across amniote species.

A role for Msx genes in mammalian embryonic diapause.

Mammalian embryonic diapause is a reproductive phenomenon defined by the reversible arrest in blastocyst development and metabolic activity within the uterus which synchronously becomes quiescent to implantation. This natural strategy, evident in over 130 species across eight orders, can temporally uncouple conception from delivery until conditions are favorable for the survival of the mother and newborn. While the maternal endocrine milieu has been shown to be important for this process, the local molecular mechanisms by which the uterus and embryo achieve quiescence, maintain blastocyst survival and then resumes blastocyst activation with subsequent implantation in response to endocrine cues remains unclear. Here we review the first evidence that the proximal molecular control of embryonic diapause is conserved in three unrelated mammalian species which employ different endocrine programs to initiate diapause. In particular, uterine expression of muscle segment homeobox (Msx) genes Msx1 or Msx2 persists during diapause, followed by downregulation with blastocyst reactivation and implantation. Mice (Mus musculus) with conditional inactivation of Msx1 and Msx2 in the uterus fail to achieve diapause and reactivation. Remarkably, the mink (Neovison vison) and tammar wallaby (Macropus eugenii) share this pattern of MSX1 or MSX2 expression as in mice during delay - it persists during diapause and is rapidly downregulated upon implantation. Therefore, these findings were the first to provide evidence that there are common conserved molecular regulators in the uterus of unrelated mammals during embryonic diapause.