Development of the Goose Tongue Filiform Papillae: Could it be Tooth-Like Sense Organs? / Desarrollo de las Papilas Filiformes de la Lengua de Ganso: ¿Podrían ser Órganos Sensoriales Parecidos a los Dientes?

SUMMARY: The geese's tongue filiform papillae are particularly long, and exhibit the same morphology of a tooth, evoking the lingual teeth of several fishes. In adult animals, they contain numerous mechanical Herbst's corpuscles but no taste buds. In the embryo, they appear since stage 38 and acquire their definitive shape between stages 38 and 42. They express several proteins associated with mammalian tooth development (BMP4, β-catenin, SHH, PITX2, PAX9), also known to be linked to parrot's pseudoteeth and goose's denticulations development. Neurofilaments are early present in the papillae primordia, and appear particularly numerous in adult papillae. Our results suggest that these papillae constitute a mechanical organ with a « tooth shape » derived from ancestral odontodes, whose development is controlled by numerous genes involved in classical odontogenesis.

Las papilas filiformes de la lengua de los gansos son particularmente largas y exhiben la morfología de un diente, evocando los dientes linguales presentes en varios peces. En los animales adultos, contienen numerosos corpúsculos de Herbst mecánicos, aunque una ausencia de papilas gustativas. En el embrión, aparecen a partir del estadio 38 y adquieren su forma definitiva entre los estadios 38 y 42. Expresan varias proteínas asociadas al desarrollo dentario de los mamíferos (BMP4, β-catenina, SHH, PITX2, PAX9), también conocidas por estar asociadas al desarrollo de pseudodientes en el loro y denticulaciones en el ganso. Los neurofilamentos están presentes tempranamente en los primordios de las papilas y aparecen particularmente numerosos en las papilas adultas. Nuestros resultados sugieren que estas papilas constituyen un órgano mecánico con «forma de diente» derivado de odontoides ancestrales, cuyo desarrollo está controlado por numerosos genes implicados en la odontogénesis clásica.

[miR-181b-5p promotes cell proliferation and induces apoptosis in human acute myeloid leukemia by targeting PAX9].

Objective To investigate the effects of miR-181b-5p on cells proliferation and apoptosis in acute myeloid leukemia (AML) by targeting paired box 9 (PAX9). Methods The relationship between expression level of PAX9 and prognosis in AML patients was analyzed by gene expression profiling interactive analysis (GEPIA) database and The Cancer Genome Atlas (TCGA) database. Kasumi-1 and AML5 cells were transfected with empty vector (Vector group) or PAX9 (PAX9 group). The proliferation activity was detected by CCK-8 assay, and cells cycle and apoptosis were detected by flow cytometry. Expressions of cyclin-dependent kinase 2 (CDK2), cyclin B1 (CCNB1), B-cell lymphoma 2 (Bcl2) and Bcl2-associated X protein (BAX) were detected by Western blot analysis. The targeted microRNA (miRNA) by PAX9 was predicted by bioinformatics analysis, and the targeted effect was verified by luciferase reporter assay. The level of PAX9 mRNA was detected by real-time quantitative PCR, and expression of PAX9 protein was detected by Western blot analysis. Kasumi-1 and AML5 cells were transfected with miR-NC (miR-NC group) or miR-181b-5p (miR-181b-5p group). The cells were further transfected with PAX9 (miR-181b-5p combined with PAX9 group) in miR-181b-5p group. The proliferation, cycle and apoptosis of cells were detected by the above methods.Results GEPIA and TCGA databases showed that the expression of PAX9 was down-regulated in AML patients, which was correlated with poor prognosis. In Kasumi-1 and AML5 cells, compared with Vector group, proliferation activity of cells, percentage of cells in S phase, and expressions of CDK2, CCNB1 and Bcl2 proteins were decreased, while percentage of cells in G0/G1 phase, apoptosis rate and the expression of BAX protein were increased in PAX9 group. It was confirmed by double luciferase reporter assay that PAX9 was the target gene of miR-181b-5p. Compared with miR-NC group, proliferation activity of cells, percentage of cells in S phase, and expressions of CDK2, CCNB1 and Bcl2 proteins were increased, while percentage of cells in G0/G1 phase, apoptosis rate and the expression of BAX protein were decreased in miR-181b-5p group. Compared with miR-181b-5p group, proliferation activity of cells, percentage of cells in S phase, and expressions of CDK2, CCNB1 and Bcl2 proteins were decreased, while percentage of cells in G0/G1 phase, apoptosis rate and the expression of BAX protein were increased in miR-181b-5p combined with PAX9 group. Conclusion The miR-181b-5p can promote the proliferation of AML cells and delay apoptosis by inhibiting PAX9.

The phenotype and genotype of PAX9 mutations causing tooth agenesis.

OBJECTIVES: The purpose of this study was to identify associations between PAX9 mutations and clinical features of non-syndromic tooth agenesis patients. MATERIALS AND METHODS: Non-syndromic tooth agenesis patients were found to have mutations by whole exome sequencing (WES). Additionally, conservation analysis and three-dimensional structure prediction were also applied to identify mutated proteins. RESULTS: Eight non-syndromic tooth agenesis probands were identified with PAX9 mutations (c.C112T; C.131_134del; c.G151A; c.189delG; c.305delT; c.C365A; c.394delG; c.A679C). All of the probands were missing more than six teeth (oligodontia). The mutations (c.131_134del,p.R44fs; c.189delG,p.T63fs; c.305delT,p.I102fs and c.394delG,p.G123fs) caused premature termination of the PAX9 protein. The c.C112T(p.R38X) mutation created a truncated protein. Bioinformatic prediction demonstrated that the three missense mutations change the PAX9 structure suggesting the corresponding functional impairments. CONCLUSIONS: We reported that eight mutations of PAX9 caused non-syndromic tooth agenesis and analyzed the relationship between PAX9 mutations and non-syndromic tooth agenesis. CLINICAL RELEVANCE: Our study revealed that PAX9 mutations might be the mutations most associated with non-syndromic tooth agenesis in humans, which greatly broadened the mutation spectrum of PAX9-related non-syndromic tooth agenesis.

PAX9 mutations and genetic synergism in familial tooth agenesis.

Familial tooth agenesis (FTA) is one of the most common craniofacial anomalies in humans. Loss-of-function mutations in PAX9 and WNT10A have been known to cause FTA with various expressivity. In this study, we identified five FTA kindreds with novel PAX9 disease-causing mutations: p.(Glu7Lys), p.(Val83Leu), p.(Pro118Ser), p.(Ser197Argfs*23), and c.771+4A>G. Concomitant PAX9 and WNT10A pathogenic variants found in two probands with severe phenotypes suggested an effect of mutational synergism. All overexpressed PAX9s showed proper nuclear localization, excepting the p.(Pro118Ser) mutant. Various missense mutations caused differential loss of PAX9 transcriptional ability. PAX9 overexpression in dental pulp cells upregulated LEF1 and AXIN2 expression, indicating a positive regulatory role for PAX9 in canonical Wnt signaling. Analyzing 176 cases with 63 different mutations, we observed a distinct pattern of tooth agenesis for PAX9-associated FTA: Maxillary teeth are in general more frequently affected than mandibular ones. Along with all second molars, maxillary bicuspids and first molars are mostly involved, while maxillary lateral incisors and mandibular bicuspids are relatively less affected. Genotypically, missense mutations are associated with fewer missing teeth than frameshift and nonsense variants. This study significantly expands the phenotypic and genotypic spectrums of PAX9-associated disorders and reveals a molecular mechanism of genetic synergism underlying FTA variable expressivity.

Novel PAX9 compound heterozygous variants in a Chinese family with non-syndromic oligodontia and genotype-phenotype analysis of PAX9 variants.

OBJECTIVE: Studies have reported that >91.9% of non-syndromic tooth agenesis cases are caused by seven pathogenic genes. To report novel heterozygous PAX9 variants in a Chinese family with non-syndromic oligodontia and summarize the reported genotype-phenotype relationship of PAX9 variants. METHODOLOGY: We recruited 28 patients with non-syndromic oligodontia who were admitted to the Hospital of Stomatology Hebei Medical University (China) from 2018 to 2021. Peripheral blood was collected from the probands and their core family members for whole-exome sequencing (WES) and variants were verified by Sanger sequencing. Bioinformatics tools were used to predict the pathogenicity of the variants. SWISS-MODEL homology modeling was used to analyze the three-dimensional structural changes of variant proteins. We also analyzed the genotype-phenotype relationships of PAX9 variants. RESULTS: We identified novel compound heterozygous PAX9 variants (reference sequence NM_001372076.1) in a Chinese family with non-syndromic oligodontia: a new missense variant c.1010C>A (p.T337K) in exon 4 and a new frameshift variant c.330_331insGT (p.D113Afs*9) in exon 2, which was identified as the pathogenic variant in this family. This discovery expands the known variant spectrum of PAX9; then, we summarized the phenotypes of non-syndromic oligodontia with PAX9 variants. CONCLUSION: We found that PAX9 variants commonly lead to loss of the second molars.

An inclusive study of deleterious missense PAX9 variants using user-friendly tools reveals structural, functional alterations, as well as potential therapeutic targets.

Mutations in the PAX9 are responsible for non-syndromic tooth agenesis in humans, although their structural and functional consequences on protein phenotype, stability, and posttranslational modifications (PTMs) have not yet been adequately investigated. This in silico study focuses on retrieving the six most deleterious mutations (L21P, R26W, R28P, G51S, I87F, and K91E) of PAX9 that has been linked to severe oligodontia. Several computational algorithm methods were used to determine the deleterious effects of PAX9 mutations. Analysis of gene ontology, protein interactions, and PTMs indicated significant functional changes caused by PAX9 mutations. The structural superimposition of the wild-type and mutant PAX9 variants revealed structural changes in locations that were present in the structures of all six variations. The conserved domain analysis revealed that the areas shared by all six variations contained unique sections that lacked DNA binding or protein-protein interaction sites, suggesting prospective drug target sites for functional restoration. The protein-protein interaction network showed KDM5B as PAX9's strongest interacting partner similar to MSX1. The PAX9 protein's structural conformations, compactness, stiffness, and function may all be impacted by changes, according to MD simulations. In addition, research on cell lines and animal models may be valuable in establishing their specific roles in functional annotations.

An in vitro and computational validation of a novel loss-of-functional mutation in PAX9 associated with non-syndromic tooth agenesis.

Congenital tooth agenesis (CTA) is one of the most common craniofacial anomalies. Its frequency varies among different population depending upon the genetic heterogeneity. CTA could be of familial or sporadic and syndromic or non-syndromic. Five major genes are found to be associated with non-syndromic CTA, namely PAX9, MSX1, EDA1, AXIN2, and WNT10A. Very few studies have been carried out so far on CTA on this Indian population making this study unique and important. This study was initiated to identify potential pathogenic variant associated with congenital tooth agenesis in an India family with molar tooth agenesis. CTA was investigated and a novel c.336C > G variation was identified in the exon 3 of PAX9, leading to substitution of evolutionary conserved Cys with Trp at 112th amino acid position located at the functionally significant DNA-binding paired domain region. Functional analysis revealed that p.Cys112Trp mutation did not prevent the nuclear localization although mutant protein had higher cytoplasmic retention. EMSA using e5 probe revealed that mutant protein was unable to bind with the paired-domain-binding site. Subsequently, GST pull-down assay revealed lower binding activity of the mutant protein with its known interactor MSX1. These in vitro results were consistent with the computational results. The in vitro and computational observations altogether suggest that c.336C > G (p.Cys112Trp) variation leads to loss of function of PAX9 leading to CTA in this family.

Detection of novel variant and functional study in a Chinese family with nonsyndromic oligodontia.

OBJECTIVES: To investigate the pathogenic gene of a patient with nonsyndromic oligodontia, and analyze its possible pathogenic mechanism. SUBJECTS AND METHODS: The variant was detected by whole exome sequencing (WES) and Sanger sequencing in a family with oligodontia. Bioinformatic and structural analyses were used to analyze variant. Functional studies including western blotting and immunofluorescent analyses and luciferase reporter assay were conducted to explore the functional effects. RESULTS: We identified a novel frameshift variant of PAX9 (c.491-510delGCCCT-ATCACGGCGGCGGCC, p.P165Qfs*145) outside the DNA-binding domain causing an autosomal-dominant nonsyndromic oligodontia in a Chinese family. Bioinformatic and structural analyses revealed that the variant is pathogenic and conserved evolutionarily, and the changes might affect protein stability or folding. Functional studies demonstrate dramatically reduced ability in activating transcription activity of BMP4 promoter and a marked decrease in protein production, as evaluated by western blotting and immunofluorescent analyses. CONCLUSIONS: We found a novel frameshift variant of PAX9 causing nonsyndromic oligodontia in a Chinese family. Our findings indicate that frameshift variants cause loss of function of PAX9 protein during the patterning of the dentition and the subsequent tooth agenesis, providing new molecular insights into the role of frameshift variant of PAX9 and broaden the pathogenic spectrum of PAX9 variants.

Four Novel PAX9 Variants and the PAX9-Related Non-Syndromic Tooth Agenesis Patterns.

The purpose of this research was to investigate and identify PAX9 gene variants in four Chinese families with non-syndromic tooth agenesis. We identified pathogenic gene variants by whole-exome sequencing (WES) and Sanger sequencing and then studied the effects of these variants on function by bioinformatics analysis and in vitro experiments. Four novel PAX9 heterozygous variants were identified: two missense variants (c.191G > T (p.G64V) and c.350T > G (p.V117G)) and two frameshift variants (c.352delC (p.S119Pfs*2) and c.648_649insC(p.Y217Lfs*100)). The bioinformatics analysis showed that these variants might be pathogenic. The tertiary structure analysis showed that these four variants could cause structural damage to PAX9 proteins. In vitro functional studies demonstrated that (1) the p.Y217Lfs*100 variant greatly affects mRNA stability, thereby affecting endogenous expression; (2) the p. S119Pfs* 2 variant impairs the subcellular localization of the nuclear expression of the wild-type PAX9 protein; and (3) the four variants (p.G64V, p.V117G, p.S119Pfs*2, and p.Y217Lfs*100) all significantly affect the downstream transcriptional activity of the BMP4 gene. In addition, we summarized and analyzed tooth missing positions caused by PAX9 variants and found that the maxillary second molar (84.11%) and mandibular second molar (84.11%) were the most affected tooth positions by summarizing and analyzing the PAX9-related non-syndromic tooth agenesis positions. Our results broaden the variant spectrum of the PAX9 gene related to non-syndromic tooth agenesis and provide useful information for future genetic counseling.

Mutation Detection and Functional Analysis of MSX1, PAX9, AXIN2, and BMP in Nonsyndromic Congenital Missing Teeth Based on Intelligent Image Detection.

Due to the complexity of clinical manifestations and the lack of standardized diagnostic criteria, it is still difficult to distinguish the etiological types of congenital edentulousness corresponding to genetic defects. This paper studies the application of deep learning image processing and digital image processing in medical images in detail and analyzes the functions of congenital edentulous hotspot genes. The cases in the control group and the study group were collected, and the gene mutations of direct sequence MSX1, PAX9, AXIN2, and BMP were analyzed, and new pathogens were found. The experimental results suggest that PAX9 and MSX1 genes may have a synergistic effect in nonsyndromic congenital edentulous patients. In severely missing teeth, the role of PAX9 may be greater than that of MSX1. The experimental results will help us lay the foundation for further understanding of the disease in the future.

PAX9 in Cancer Development.

Paired box 9 (PAX9) is a transcription factor of the PAX family functioning as both a transcriptional activator and repressor. Its functional roles in the embryonic development of various tissues and organs have been well studied. However, its roles and molecular mechanisms in cancer development are largely unknown. Here, we review the current understanding of PAX9 expression, upstream regulation of PAX9, and PAX9 downstream events in cancer development. Promoter hypermethylation, promoter SNP, microRNA, and inhibition of upstream pathways (e.g., NOTCH) result in PAX9 silencing or downregulation, whereas gene amplification and an epigenetic axis upregulate PAX9 expression. PAX9 may contribute to carcinogenesis through dysregulation of its transcriptional targets and related molecular pathways. In summary, extensive studies on PAX9 in its cellular and tissue contexts are warranted in various cancers, in particular, HNSCC, ESCC, lung cancer, and cervical SCC.

PAX9 reactivation by inhibiting DNA methyltransferase triggers antitumor effect in oral squamous cell carcinoma.

Aberrant DNA hypermethylation is associated with oral carcinogenesis. Procaine, a local anesthetic, is a DNA methyltransferase (DNMT) inhibitor that activates anticancer mechanisms. However, its effect on silenced tumor suppressor gene (TSG) activation and its biological role in oral squamous cell carcinoma (OSCC) remain unknown. Here, we report procaine inhibited DNA methylation by suppressing DNMT activity and increased the expression of PAX9, a differentiation gene in OSCC cells. Interestingly, the reactivation of PAX9 by procaine found to inhibit cell growth and trigger apoptosis in OSCC in vitro and in vivo. Likely, the enhanced PAX9 expression after exposure to procaine controls stemness and differentiation through the autophagy-dependent pathway in OSCC cells. PAX9 inhibition abrogated procaine-induced apoptosis, autophagy, and inhibition of stemness. In OSCC cells, procaine improved anticancer drug sensitivity through PAX9, and its deficiency significantly blunted the anticancer drug sensitivity mediated by procaine. Additionally, NRF2 activation by procaine facilitated the antitumor response of PAX9, and pharmacological inhibition of NRF2 by ML385 reduced death and prevented the decrease in the orosphere-forming potential of OSCC cells. Furthermore, procaine promoted antitumor activity in FaDu xenografts in athymic nude mice, and immunohistochemistry data showed that PAX9 expression was significantly enhanced in the procaine group compared to the vehicle control. In conclusion, PAX9 reactivation in response to DNMT inhibition could trigger a potent antitumor mechanism to provide a new therapeutic strategy for OSCC.

Genome-wide screening for deubiquitinase subfamily identifies ubiquitin-specific protease 49 as a novel regulator of odontogenesis.

Proteins expressed by the paired box gene 9 (PAX9) and Msh Homeobox 1 (MSX1) are intimately involved in tooth development (odontogenesis). The regulation of PAX9 and MSX1 protein turnover by deubiquitinating enzymes (DUBs) plausibly maintain the required levels of PAX9 and MSX1 during odontogenesis. Herein, we used a loss-of-function CRISPR-Cas9-mediated DUB KO library kit to screen for DUBs that regulate PAX9 and MSX1 protein levels. We identify and demonstrate that USP49 interacts with and deubiquitinates PAX9 and MSX1, thereby extending their protein half-lives. On the other hand, the loss of USP49 reduces the levels of PAX9 and MSX1 proteins, which causes transient retardation of odontogenic differentiation in human dental pulp stem cells and delays the differentiation of human pluripotent stem cells into the neural crest cell lineage. USP49 depletion produced several morphological defects during tooth development, such as reduced dentin growth with shrunken enamel space, and abnormal enamel formation including irregular mineralization. In sum, our results suggest that deubiquitination of PAX9 and MSX1 by USP49 stabilizes their protein levels to facilitate successful odontogenesis.

Patterning of cartilaginous condensations in the developing facial skeleton.

Adult endochondral bones are prefigured in the embryo as cellular condensations within fields of more loosely distributed skeletal progenitors. How these early condensations are initiated and shaped has remained enigmatic, despite the wealth of research on later stages of cartilage differentiation and endochondral ossification. Using the simple larval zebrafish facial skeleton as a model, we reevaluate the involvement of the master cartilage regulator Sox9 in shaping facial condensations and find it to be largely dispensable. We then use new lineage-tracing tools to definitively show that precartilaginous condensations originate from neighboring clusters of cells termed mesenchymal condensations. These cartilage-generating mesenchymal condensations express a cohort of transcription factors that are also expressed in odontogenic mesenchyme in mammals, including barx1, lhx6a/8a, and pax9. We hypothesized that the position of each mesenchymal condensation determines the axis of growth of its corresponding precartilaginous condensation, thus influencing its final shape. Consistent with this idea, we find that positive Fgf and inhibitory Jagged-Notch signals intersect to precisely position a mesenchymal condensation in the dorsal half of the second pharyngeal arch, with loss of pathway function leading to predictable shape changes in the resulting cartilage element. Deciphering the full array of signals that control the spatial distribution of mesenchymal condensations and regulate their maturation into precartilaginous condensations thus offers a promising approach for understanding the origins of skeletal form.

Patterns of molar agenesis associated with p.P20L and p.R77Q variants in PAX9.

Nonsyndromic tooth agenesis is associated with variants in several genes. There are numerous genotype-phenotype publications involving many patients and kindreds. Here, we identified six Thai individuals in two families with nonsyndromic tooth agenesis, performed exome sequencing, and conducted functional experiments. Family 1 had four affected members carrying the heterozygous PAX9 variant, c.59C>T (p.Pro20Leu). The p.Pro20Leu was previously reported in two families having four and three affected members. These seven cases and Proband-1 had agenesis of at least three third molars. Family 2 comprised two affected members with agenesis of all 12 molars. Both individuals were heterozygous for c.230G>A (p.Arg77Gln) in PAX9, which has not been reported previously. This variant is predicted to be damaging, evolutionarily conserved, and resides in the PAX9 linking peptide. The BMP4 RNA levels in Proband-1's leukocytes were not significantly different from those in the controls, whereas BMP4 levels observed in Proband-2 were significantly increased. Moreover, the p.Arg77Gln variant demonstrated nuclear localization similar to the wild-type but resulted in significantly impaired transactivation of BMP4, a PAX9 downstream gene. In conclusion, we demonstrate that the PAX9 p.Pro20Leu is highly associated with absent third molars, while the novel PAX9 p.Arg77Gln impairs BMP4 transactivation and is associated with total molar agenesis.

The role of the histone methyltransferase SET domain bifurcated 1 during palatal development.

The histone methyltransferase SET domain bifurcated 1 (SETDB1) catalyzes the trimethylation of lysine 9 of histone H3, thereby regulating gene expression. In this study, we used conditional knockout mice, where Setdb1 was deleted only in neural crest cells (Setdb1fl/fl,Wnt1-Cre + mice), to clarify the role of SETDB1 in palatal development. Setdb1fl/fl,Wnt1-Cre + mice died shortly after birth due to a cleft palate with full penetration. Reduced palatal mesenchyme proliferation was seen in Setdb1fl/fl,Wnt1-Cre + mice, which might be a possible mechanism of cleft palate development. Quantitative RT-PCR and in situ hybridization showed that expression of the Pax9, Bmp4, Bmpr1a, Wnt5a, and Fgf10 genes, known to be important for palatal development, were markedly decreased in the palatal mesenchyme of Setdb1fl/fl,Wnt1-Cre + mice. Along with these phenomena, SMAD1/5/9 phosphorylation was decreased by the loss of Setdb1. Our results demonstrated that SETDB1 is indispensable for palatal development partially through its proliferative effect. Taken together with previous reports that PAX9 regulates BMP signaling during palatal development which implies that loss of Setdb1 may be involved in the cleft palate development by decreasing SMAD-dependent BMP signaling through Pax9.

PAX9 functions as a tumor suppressor gene for cervical cancer via modulating cell proliferation and apoptosis.

To investigate the effect of PAX9 on the progression of cervical cancer (CC). PAX9 expression was quantified in CC tissues and adjacent normal tissues, as well as human CC cell lines and human cervical epithelial cells (HCerEpiC). PAX9-overexpression lentiviral vectors were transfected into CC cell lines, followed by the measurement of proliferation and apoptosis and the quantification of apoptosis-related proteins. In vivo, mice were subcutaneously injected with CaSki cells transfected with PAX9-overexpression lentiviral vectors and control vectors. Then, the volume and weight of tumors were measured followed by hematoxylin and eosin (HE) staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and immunohistochemistry. PAX9 expression in the CC tissues was lower than that in the adjacent normal tissues, which was correlated with the FIGO stage, tumor size, infiltration depth, parametrium invasion, lympho-vascular space invasion tumor-positive lymph nodes, and prognosis. Furthermore, PAX9 in CC cell lines was also lower than in HCerEpiC. PAX9 inhibits the CC cell proliferation and promotes the apoptosis, with the up-regulations of caspase-3, poly(ADP-ribose) polymerase (PARP), and Bax and the down-regulation of Bcl-2. In vivo experiments demonstrated that in the PAX9 group, the tumor weight and volume were lower than those in the vector group accompanying the decreased Ki-67, cleaved-caspase-3, and Bax expressions and the increased TUNEL and Bcl-2 expression. PAX9 was lowly expressed in the CC tissues and associated with the clinicopathological characteristics and prognosis. PAX9 could inhibit proliferation of CC cell lines and promote the apoptosis, thus suppressing the tumor growth in vivo, indicating its potential therapeutic role for CC treatment.

HES1 is a novel downstream modifier of the SHH-GLI3 Axis in the development of preaxial polydactyly.

Sonic Hedgehog/GLI3 signaling is critical in regulating digit number, such that Gli3-deficiency results in polydactyly and Shh-deficiency leads to digit number reductions. SHH/GLI3 signaling regulates cell cycle factors controlling mesenchymal cell proliferation, while simultaneously regulating Grem1 to coordinate BMP-induced chondrogenesis. SHH/GLI3 signaling also coordinates the expression of additional genes, however their importance in digit formation remain unknown. Utilizing genetic and molecular approaches, we identified HES1 as a downstream modifier of the SHH/GLI signaling axis capable of inducing preaxial polydactyly (PPD), required for Gli3-deficient PPD, and capable of overcoming digit number constraints of Shh-deficiency. Our data indicate that HES1, a direct SHH/GLI signaling target, induces mesenchymal cell proliferation via suppression of Cdkn1b, while inhibiting chondrogenic genes and the anterior autopod boundary regulator, Pax9. These findings establish HES1 as a critical downstream effector of SHH/GLI3 signaling in the development of PPD.

Msx1 haploinsufficiency modifies the Pax9-deficient cardiovascular phenotype.

BACKGROUND: Successful embryogenesis relies on the coordinated interaction between genes and tissues. The transcription factors Pax9 and Msx1 genetically interact during mouse craniofacial morphogenesis, and mice deficient for either gene display abnormal tooth and palate development. Pax9 is expressed specifically in the pharyngeal endoderm at mid-embryogenesis, and mice deficient for Pax9 on a C57Bl/6 genetic background also have cardiovascular defects affecting the outflow tract and aortic arch arteries giving double-outlet right ventricle, absent common carotid arteries and interruption of the aortic arch. RESULTS: In this study we have investigated both the effect of a different genetic background and Msx1 haploinsufficiency on the presentation of the Pax9-deficient cardiovascular phenotype. Compared to mice on a C57Bl/6 background, congenic CD1-Pax9-/- mice displayed a significantly reduced incidence of outflow tract defects but aortic arch defects were unchanged. Pax9-/- mice with Msx1 haploinsufficiency, however, have a reduced incidence of interrupted aortic arch, but more cases with cervical origins of the right subclavian artery and aortic arch, than seen in Pax9-/- mice. This alteration in arch artery defects was accompanied by a rescue in third pharyngeal arch neural crest cell migration and smooth muscle cell coverage of the third pharyngeal arch arteries. Although this change in phenotype could theoretically be compatible with post-natal survival, using tissue-specific inactivation of Pax9 to maintain correct palate development whilst inducing the cardiovascular defects was unable to prevent postnatal death in the mutant mice. Hyoid bone and thyroid cartilage formation were abnormal in Pax9-/- mice. CONCLUSIONS: Msx1 haploinsufficiency mitigates the arch artery defects in Pax9-/- mice, potentially by maintaining the survival of the 3rd arch artery through unimpaired migration of neural crest cells to the third pharyngeal arches. With the neural crest cell derived hyoid bone and thyroid cartilage also being defective in Pax9-/- mice, we speculate that the pharyngeal endoderm is a key signalling centre that impacts on neural crest cell behaviour highlighting the ability of cells in different tissues to act synergistically or antagonistically during embryo development.

PAX7, PAX9 and RYK Expression in Cleft Affected Tissue.

Background and Objectives: Cleft lip with or without cleft palate is one of the most common types of congenital malformations. Transcription factors paired box 7 and 9 (PAX7, PAX9) and receptor-like tyrosine kinase (RYK) have been previously associated with the formation of orofacial clefts but their exact possible involvement and interactions in the tissue of specific cleft types remains uncertain. There is a limited number of morphological studies analyzing these specific factors in cleft affected tissue due to ethical aspects and the limited amount of available tissue material. This study analyses the presence of PAX7, PAX9, and RYK immunopositive structures within different cleft affected tissue to assess their possible involvement in cleft morphopathogenesis. Materials and Methods: Cleft affected tissue was collected from non-syndromic orofacial cleft patients during cleft correcting surgery (36 patients with unilateral cleft lip, 13 patients with bilateral cleft lip, 26 patients with isolated cleft palate). Control group oral cavity tissue was obtained from 7 patients without cleft lip and palate. To evaluate the number of immunopositive structures in the cleft affected tissue and the control group, a semiquantitative counting method was used. Non-parametric statistical methods (Kruskal-Wallis H test, Mann-Whitney U test, and Spearman's rank correlation) were used. Results: Statistically significant differences for the number of PAX7, PAX9, and RYK-positive cells were notified between the controls and the patient groups. Multiple statistically significant correlations between the factors were found in each cleft affected tissue group. Conclusions: PAX7, PAX9, and RYK have a variable involvement and interaction in postnatal morphopathogenesis of orofacial clefts. PAX7 is more associated with the formation of unilateral cleft lip, while PAX9 relates more towards the isolated cleft palate. The stable presence of RYK in all cleft types indicates its possible participation in different facial cleft formations.