Msx1 deficiency interacts with hypoxia and induces a morphogenetic regulation during mouse lip development.

Nonsyndromic clefts of the lip and palate are common birth defects resulting from gene-gene and gene-environment interactions. Mutations in human MSX1 have been linked to orofacial clefting and we show here that Msx1 deficiency causes a growth defect of the medial nasal process (Mnp) in mouse embryos. Although this defect alone does not disrupt lip formation, Msx1-deficient embryos develop a cleft lip when the mother is transiently exposed to reduced oxygen levels or to phenytoin, a drug known to cause embryonic hypoxia. In the absence of interacting environmental factors, the Mnp growth defect caused by Msx1 deficiency is modified by a Pax9-dependent 'morphogenetic regulation', which modulates Mnp shape, rescues lip formation and involves a localized abrogation of Bmp4-mediated repression of Pax9 Analyses of GWAS data revealed a genome-wide significant association of a Gene Ontology morphogenesis term (including assigned roles for MSX1, MSX2, PAX9, BMP4 and GREM1) specifically for nonsyndromic cleft lip with cleft palate. Our data indicate that MSX1 mutations could increase the risk for cleft lip formation by interacting with an impaired morphogenetic regulation that adjusts Mnp shape, or through interactions that inhibit Mnp growth.

Pax9 is required for cardiovascular development and interacts with Tbx1 in the pharyngeal endoderm to control 4th pharyngeal arch artery morphogenesis.

Developmental defects affecting the heart and aortic arch arteries are a significant phenotype observed in individuals with 22q11 deletion syndrome and are caused by a microdeletion on chromosome 22q11. TBX1, one of the deleted genes, is expressed throughout the pharyngeal arches and is considered a key gene, when mutated, for the arch artery defects. Pax9 is expressed in the pharyngeal endoderm and is downregulated in Tbx1 mutant mice. We show here that Pax9-deficient mice are born with complex cardiovascular malformations that affect the outflow tract and aortic arch arteries with failure of the 3rd and 4th pharyngeal arch arteries to form correctly. Transcriptome analysis indicated that Pax9 and Tbx1 may function together, and mice double heterozygous for Tbx1/Pax9 presented with a significantly increased incidence of interrupted aortic arch when compared with Tbx1 heterozygous mice. Using a novel Pax9Cre allele, we demonstrated that the site of this Tbx1-Pax9 genetic interaction is the pharyngeal endoderm, therefore revealing that a Tbx1-Pax9-controlled signalling mechanism emanating from the pharyngeal endoderm is required for crucial tissue interactions during normal morphogenesis of the pharyngeal arch artery system.

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.

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.

Predicting the clinical outcome of oral potentially malignant disorders using transcriptomic-based molecular pathology.

BACKGROUND: This study was undertaken to develop and validate a gene expression signature that characterises oral potentially malignant disorders (OPMD) with a high risk of undergoing malignant transformation. METHODS: Patients with oral epithelial dysplasia at one hospital were selected as the 'training set' (n = 56) whilst those at another hospital were selected for the 'test set' (n = 66). RNA was extracted from formalin-fixed paraffin-embedded (FFPE) diagnostic biopsies and analysed using the NanoString nCounter platform. A targeted panel of 42 genes selected on their association with oral carcinogenesis was used to develop a prognostic gene signature. Following data normalisation, uni- and multivariable analysis, as well as prognostic modelling, were employed to develop and validate the gene signature. RESULTS: A prognostic classifier composed of 11 genes was developed using the training set. The multivariable prognostic model was used to predict patient risk scores in the test set. The prognostic gene signature was an independent predictor of malignant transformation when assessed in the test set, with the high-risk group showing worse prognosis [Hazard ratio = 12.65, p = 0.0003]. CONCLUSIONS: This study demonstrates proof of principle that RNA extracted from FFPE diagnostic biopsies of OPMD, when analysed on the NanoString nCounter platform, can be used to generate a molecular classifier that stratifies the risk of malignant transformation with promising clinical utility.

Gene expression changes associated with malignant transformation of oral potentially malignant disorders.

BACKGROUND: A large number of oral squamous cell carcinomas (OSCCs) are believed to be preceded by oral potentially malignant disorders (OPMD) that have an increased likelihood of malignant transformation compared to clinically normal mucosa. This study was performed to identify differentially expressed genes between OPMDs that underwent malignant transformation (MT) and those that did not, termed "non-transforming" (NT) cases. METHODS: Total RNA was extracted from formalin-fixed paraffin-embedded tissue biopsies of 20 OPMD cases with known clinical outcomes (10 MT vs. 10 NT). Samples were assessed for quantity, quality and integrity of RNA prior to sequencing. Analysis for differential gene expression between MT and NT was performed using statistical packages in R. Genes were considered to be significantly differentially expressed if the False Discovery Rate corrected P-value was < 0.05. RESULTS: RNA yield was variable but RNA purity was good (A260/A280 > 1.90). Analysis of RNA-Sequencing outputs revealed 41 genes (34 protein-coding; 7 non-coding) that were significantly differentially expressed between MT and NT cases. The log2 fold change for the statistically significant differentially expressed genes ranged from -2.63 to 2.48, with 23 protein-coding genes being downregulated and 11 protein-coding genes being upregulated in MT cases compared to NT cases. CONCLUSION: Several candidate genes that may play a role in malignant transformation of OPMD have been identified. Experiments to validate these candidates are underway. It is anticipated that this work will contribute to better understanding of the etiopathogenesis of OPMD and development of novel biomarkers.

PAX9 regulates squamous cell differentiation and carcinogenesis in the oro-oesophageal epithelium.

PAX9 is a transcription factor of the PAX family characterized by a DNA-binding paired domain. Previous studies have suggested a potential role of PAX9 in squamous cell differentiation and carcinogenesis of the oro-oesophageal epithelium. However, its functional roles in differentiation and carcinogenesis remain unclear. In this study, Pax9 deficiency in mouse oesophagus promoted cell proliferation, delayed cell differentiation, and altered the global gene expression profile. Ethanol exposure downregulated PAX9 expression in human oesophageal epithelial cells in vitro and mouse forestomach and tongue in vivo. We further showed that PAX9 was downregulated in human oro-oesophageal squamous cell carcinoma (OESCC), and its downregulation was associated with alcohol drinking and promoter hypermethylation. Moreover, ad libitum feeding with a liquid diet containing ethanol for 40 weeks or Pax9 deficiency promoted N-nitrosomethylbenzylamine-induced squamous cell carcinogenesis in mouse tongue, oesophagus, and forestomach. In conclusion, PAX9 regulates squamous cell differentiation in the oro-oesophageal epithelium. Alcohol drinking and promoter hypermethylation are associated with PAX9 silencing in human OESCC. PAX9 downregulation may contribute to alcohol-associated oro-oesophageal squamous cell carcinogenesis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The formation of endoderm-derived taste sensory organs requires a Pax9-dependent expansion of embryonic taste bud progenitor cells.

In mammals, taste buds develop in different regions of the oral cavity. Small epithelial protrusions form fungiform papillae on the ectoderm-derived dorsum of the tongue and contain one or few taste buds, while taste buds in the soft palate develop without distinct papilla structures. In contrast, the endoderm-derived circumvallate and foliate papillae located at the back of the tongue contain a large number of taste buds. These taste buds cluster in deep epithelial trenches, which are generated by intercalating a period of epithelial growth between initial placode formation and conversion of epithelial cells into sensory cells. How epithelial trench formation is genetically regulated during development is largely unknown. Here we show that Pax9 acts upstream of Pax1 and Sox9 in the expanding taste progenitor field of the mouse circumvallate papilla. While a reduced number of taste buds develop in a growth-retarded circumvallate papilla of Pax1 mutant mice, its development arrests completely in Pax9-deficient mice. In addition, the Pax9 mutant circumvallate papilla trenches lack expression of K8 and Prox1 in the taste bud progenitor cells, and gradually differentiate into an epidermal-like epithelium. We also demonstrate that taste placodes of the soft palate develop through a Pax9-dependent induction. Unexpectedly, Pax9 is dispensable for patterning, morphogenesis and maintenance of taste buds that develop in ectoderm-derived fungiform papillae. Collectively, our data reveal an endoderm-specific developmental program for the formation of taste buds and their associated papilla structures. In this pathway, Pax9 is essential to generate a pool of taste bud progenitors and to maintain their competence towards prosensory cell fate induction.

Scx+/Sox9+ progenitors contribute to the establishment of the junction between cartilage and tendon/ligament.

SRY-box containing gene 9 (Sox9) and scleraxis (Scx) regulate cartilage and tendon formation, respectively. Here we report that murine Scx(+)/Sox9(+) progenitors differentiate into chondrocytes and tenocytes/ligamentocytes to form the junction between cartilage and tendon/ligament. Sox9 lineage tracing in the Scx(+) domain revealed that Scx(+) progenitors can be subdivided into two distinct populations with regard to their Sox9 expression history: Scx(+)/Sox9(+) and Scx(+)/Sox9(-) progenitors. Tenocytes are derived from Scx(+)/Sox9(+) and Scx(+)/Sox9(-) progenitors. The closer the tendon is to the cartilaginous primordium, the more tenocytes arise from Scx(+)/Sox9(+) progenitors. Ligamentocytes as well as the annulus fibrosus cells of the intervertebral discs are descendants of Scx(+)/Sox9(+) progenitors. Conditional inactivation of Sox9 in Scx(+)/Sox9(+) cells causes defective formation in the attachment sites of tendons/ligaments into the cartilage, and in the annulus fibrosus of the intervertebral discs. Thus, the Scx(+)/Sox9(+) progenitor pool is a unique multipotent cell population that gives rise to tenocytes, ligamentocytes and chondrocytes for the establishment of the chondro-tendinous/ligamentous junction.

E-cigarette vapour is not inert and exposure can lead to cell damage.

MATERIALS AND METHODS: In vitro experiments were performed on normal epithelial cells as well as head and neck squamous cell carcinoma (HNSCC) cell lines. The widely available cell line HaCat, a spontaneously transformed immortal keratinocyte and the HNSCC cell lines HN30 and UMSCC10B were used. Cells were exposed to nicotine-containing and nicotine-free vapour extract from two popular e-cigarette brands for periods ranging from 48 hours to eight weeks. Cytotoxicity was assessed using Annexin V flow cytometric analysis, trypan blue exclusion and clonogenic assays. Genotoxicity in the form of DNA strand breaks was quantified using the neutral comet assay and γ-H2AX immunostaining. RESULTS: E-cigarette-exposed cells showed significantly reduced cell viability and clonogenic survival, along with increased rates of apoptosis and necrosis, regardless of e-cigarette vapour nicotine content. They also exhibited significantly increased comet tail length and accumulation of γ-H2AX foci, demonstrating increased DNA strand breaks. CONCLUSIONS: In conclusion, our study strongly suggests that electronic cigarettes are not as safe as their marketing makes them appear to the public. Our in vitro experiments employing two brands of e-cigs show that at biologically relevant doses, vapourised e-cig liquids induce increased DNA strand breaks and cell death, and decreased clono- genic survival in both normal epithelial and HNSCC cell lines independently of nicotine content. Further research is needed to definitively determine the long-term effects of e-cig usage, as well as whether the DNA damage shown in our study as a result of e-cig exposure will lead to mutations that ultimately result in cancer.

Transcription factor SOX9 plays a key role in the regulation of visual cycle gene expression in the retinal pigment epithelium.

The retinal pigment epithelium (RPE) performs specialized functions to support retinal photoreceptors, including regeneration of the visual chromophore. Enzymes and carrier proteins in the visual cycle function sequentially to regenerate and continuously supply 11-cis-retinal to retinal photoreceptor cells. However, it is unknown how the expression of the visual cycle genes is coordinated at the transcriptional level. Here, we show that the proximal upstream regions of six visual cycle genes contain chromatin-accessible sex-determining region Y box (SOX) binding sites, that SOX9 and LIM homeobox 2 (LHX2) are coexpressed in the nuclei of mature RPE cells, and that SOX9 acts synergistically with orthodenticle homeobox 2 (OTX2) to activate the RPE65 and retinaldehyde binding protein 1 (RLBP1) promoters and acts synergistically with LHX2 to activate the retinal G protein-coupled receptor (RGR) promoter. ChIP reveals that SOX9 and OTX2 bind to the promoter regions of RPE65, RLBP1, and RGR and that LHX2 binds to those of RPE65 and RGR in bovine RPE. ChIP with human fetal RPE cells shows that SOX9 and OTX2 also bind to the human RPE65, RLBP1, and RGR promoters. Conditional inactivation of Sox9 in mouse RPE results in reduced expression of several visual cycle genes, most dramatically Rpe65 and Rgr. Furthermore, bioinformatic analysis predicts that multiple common microRNAs (miRNAs) regulate visual cycle genes, and cotransfection of miRNA mimics with luciferase reporter constructs validated some of the predicted miRNAs. These results implicate SOX9 as a key regulator of visual cycle genes, reveal for the first time the functional role of LHX2 in the RPE, and suggest the possible regulation of visual cycle genes by common miRNAs.

Endothelial cells drive organ fibrosis in mice by inducing expression of the transcription factor SOX9.

Fibrosis is a hallmark of chronic disease. Although fibroblasts are involved, it is unclear to what extent endothelial cells also might contribute. We detected increased expression of the transcription factor Sox9 in endothelial cells in several different mouse fibrosis models. These models included systolic heart failure induced by pressure overload, diastolic heart failure induced by high-fat diet and nitric oxide synthase inhibition, pulmonary fibrosis induced by bleomycin treatment, and liver fibrosis due to a choline-deficient diet. We also observed up-regulation of endothelial SOX9 in cardiac tissue from patients with heart failure. To test whether SOX9 induction was sufficient to cause disease, we generated mice with endothelial cell-specific overexpression of Sox9, which promoted fibrosis in multiple organs and resulted in signs of heart failure. Endothelial Sox9 deletion prevented fibrosis and organ dysfunction in the two mouse models of heart failure as well as in the lung and liver fibrosis mouse models. Bulk and single-cell RNA sequencing of mouse endothelial cells across multiple vascular beds revealed that SOX9 induced extracellular matrix, growth factor, and inflammatory gene expression, leading to matrix deposition by endothelial cells. Moreover, mouse endothelial cells activated neighboring fibroblasts that then migrated and deposited matrix in response to SOX9, a process partly mediated by the secreted growth factor CCN2, a direct SOX9 target; endothelial cell-specific Sox9 deletion reversed these changes. These findings suggest a role for endothelial SOX9 as a fibrosis-promoting factor in different mouse organs during disease and imply that endothelial cells are an important regulator of fibrosis.

Mice lacking Ly49E show normal NK cell development and provide evidence for probabilistic expression of Ly49E in NK cells and T cells.

Ly49E is an unusual member of the Ly49 family that is expressed on fetal NK cells, epithelial T cells, and NKT cells, but not on resting adult NK cells. Ly49E(bgeo/bgeo) mice in which the Ly49E gene was disrupted by inserting a ß-geo transgene were healthy, fertile, and had normal numbers of NK and T cells in all organs examined. Their NK cells displayed normal expression of Ly49 and other NK cell receptors, killed tumor and MHC class I-deficient cells efficiently, and produced normal levels of IFN-γ. In heterozygous Ly49E(+/bgeo) mice, the proportion of epidermal T cells, NKT cells, and IL-2-activated NK cells that expressed Ly49E was about half that found in wild-type mice. Surprisingly, although splenic T cells rarely expressed Ly49E, IL-2-activated splenic T cells from Ly49E(bgeo/bgeo) mice were as resistant to growth in G418 as NK cells and expressed similar levels of ß-geo transcripts, suggesting that disruption of the Ly49E locus had increased its expression in these cells to the same level as that in NK cells. Importantly, however, the proportion of G418-resistant heterozygous Ly49E(+/bgeo) cells that expressed Ly49E from the wild-type allele was similar to that observed in control cells. Collectively, these findings demonstrate that Ly49E is not required for the development or homeostasis of NK and T cell populations or for the acquisition of functional competence in NK cells and provide compelling evidence that Ly49E is expressed in a probabilistic manner in adult NK cells and T cells.

Hydroureternephrosis due to loss of Sox9-regulated smooth muscle cell differentiation of the ureteric mesenchyme.

Congenital ureter anomalies, including hydroureter, affect up to 1% of the newborn children. Despite the prevalence of these developmental abnormalities in young children, the underlying molecular causes are only poorly understood. Here, we show that the high mobility group domain transcription factor Sox9 plays an important role in ureter development in the mouse. Transient Sox9 expression was detected in the undifferentiated ureteric mesenchyme and inactivation of Sox9 in this domain resulted in strong proximal hydroureter formation due to functional obstruction. Loss of Sox9 did not affect condensation, proliferation and apoptosis of the undifferentiated mesenchyme, but perturbed cyto-differentiation into smooth muscle cells (SMCs). Expression of genes encoding extracellular matrix (ECM) components was strongly reduced, suggesting that deficiency in ECM composition and/or signaling may underlie the observed defects. Prolonged expression of Sox9 in the ureteric mesenchyme led to increased deposition of ECM components and SMC dispersal. Furthermore, Sox9 genetically interacts with the T-box transcription factor 18 gene (Tbx18) during ureter development at two levels--as a downstream mediator of Tbx18 function and in a converging pathway. Together, our results argue that obstructive uropathies in campomelic dysplasia patients that are heterozygous for mutations in and around SOX9 arise from a primary requirement of Sox9 in the development of the ureteric mesenchyme.

Sox9 regulates cell proliferation and is required for Paneth cell differentiation in the intestinal epithelium.

The HMG-box transcription factor Sox9 is expressed in the intestinal epithelium, specifically, in stem/progenitor cells and in Paneth cells. Sox9 expression requires an active beta-catenin-Tcf complex, the transcriptional effector of the Wnt pathway. This pathway is critical for numerous aspects of the intestinal epithelium physiopathology, but processes that specify the cell response to such multipotential signals still remain to be identified. We inactivated the Sox9 gene in the intestinal epithelium to analyze its physiological function. Sox9 inactivation affected differentiation throughout the intestinal epithelium, with a disappearance of Paneth cells and a decrease of the goblet cell lineage. Additionally, the morphology of the colon epithelium was severely altered. We detected general hyperplasia and local crypt dysplasia in the intestine, and Wnt pathway target genes were up-regulated. These results highlight the central position of Sox9 as both a transcriptional target and a regulator of the Wnt pathway in the regulation of intestinal epithelium homeostasis.

Loss of Sox9 in the periotic mesenchyme affects mesenchymal expansion and differentiation, and epithelial morphogenesis during cochlea development in the mouse.

Sox9 encodes an HMG-domain transcription factor that is critically required in numerous developmental processes such as chondrogenesis and otic placode formation. Here, we show that Sox9 is expressed in the mesenchyme surrounding the developing cochlea in the mouse suggesting that Sox9 may also control development of the otic fibrocyte compartment and the surrounding otic capsule. Tissue-specific inactivation of Sox9 in the periotic mesenchyme using a Tbx18(Cre) mouse line results in arrest of early chondrogenesis and consequently, in a lack of cochlear otic capsule formation. Furthermore, loss of Sox9 severely compromises expansion, differentiation and remodeling of the otic fibrocyte compartment. Early cell proliferation defects in the entire periotic mesenchyme of Sox9-deficient inner ears suggest a cell-autonomous function of Sox9 for the development of the inner mesenchymal compartment. Abnormal cochlear duct morphogenesis in Sox9 mutants including disruption of the coiling process is tightly associated with the onset of mesenchymal defects whereas the absence of major differentiation defects in the otic epithelium suggests that Sox9-dependent mesenchymal signals primarily control epithelial morphogenesis.

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.

Haploinsufficiency of the germ cell-specific nuclear RNA binding protein hnRNP G-T prevents functional spermatogenesis in the mouse.

Human HNRNPGT, encoding the protein hnRNP G-T, is one of several autosomal retrogenes derived from RBMX. It has been suggested that HNRNPGT functionally replaces the sex-linked RBMX and RBMY genes during male meiosis. We show here that during normal mouse germ cell development, hnRNP G-T protein is strongly expressed during and after meiosis when proteins expressed from Rbmx or Rbmx-like genes are absent. Amongst these Rbmx-like genes, DNA sequence analyses indicate that two other mouse autosomal Rbmx-derived retrogenes have evolved recently in rodents and one already shows signs of degenerating into a non-expressed pseudogene. In contrast, orthologues of Hnrnpgt are present in all four major groups of placental mammals. The sequence of Hnrnpgt is under considerable positive selection suggesting it performs an important germ cell function in eutherians. To test this, we inactivated Hnrnpgt in ES cells and studied its function during spermatogenesis in chimaeric mice. Although germ cells heterozygous for this targeted allele could produce sperm, they did not contribute to the next generation. Chimaeric mice with a high level of mutant germ cells were infertile with low sperm counts and a high frequency of degenerate seminiferous tubules and abnormal sperm. Chimaeras made from a 1:1 mix of targeted and wild-type ES cell clones transmitted wild-type germ cells only. Our data show that haploinsufficiency of Hnrnpgt results in abnormal sperm production in the mouse. Genetic defects resulting in reduced levels of HNRNPGT could, therefore, be a cause of male infertility in humans.

Loss of Sox9 function results in defective chondrocyte differentiation of mouse embryonic stem cells in vitro.

The transcription factor Sox9 plays an important role during chondrogenesis. After early conditional inactivation of Sox9 in mesenchymal limb bud cells of mice, mesenchymal condensations as well as cartilage and bone are completely absent in the developing limbs. We analyzed chondrogenic differentiation of Sox9-/- mouse embryonic stem cells in vitro, using two clones with different targeted mutations. We found that the development of mature and hypertrophic chondrocytes is completely inhibited in the absence of Sox9 confirming that Sox9 is required for the formation of cartilage. In contrast, Sox9+/- mouse embryonic stem cells showed continuous but reduced differentiation into mature chondrocytes. Interestingly, the formation of early chondrogenic condensations expressing characteristic marker genes such as scleraxis, Sox5 and Sox6 was not inhibited in the absence of Sox9 in vitro. Thus, we propose that the earliest step of chondrogenesis could be regulated by a non cell-autonomous function of Sox9.

Metalloprotease-Dependent Attenuation of BMP Signaling Restricts Cardiac Neural Crest Cell Fate.

In higher vertebrates, cephalic neural crest cells (NCCs) form craniofacial skeleton by differentiating into chondrocytes and osteoblasts. A subpopulation of cephalic NCCs, cardiac NCCs (CNCCs), migrates to the heart. However, CNCCs mostly do not yield skeletogenic derivatives, and the molecular mechanisms of this fate restriction remain elusive. We identify a disintegrin and metalloprotease 19 (Adam19) as a position-specific fate regulator of NCCs. Adam19-depleted mice abnormally form NCC-derived cartilage in their hearts through the upregulation of Sox9 levels in CNCCs. Moreover, NCC-lineage-specific Sox9-overexpressing mice recapitulate CNCC chondrogenesis. In vitro experiments show that Adam19 mediates the cleavage of bone morphogenic protein (BMP) type I receptor Alk2 (Acvr1), whereas pharmacogenetic approaches reveal that Adam19 inhibits CNCC chondrogenesis by suppressing the BMP-Sox9 cascade, presumably through processing Alk2. These findings suggest a metalloprotease-dependent mechanism attenuating cellular responsiveness to BMP ligands, which is essential for both the positional restriction of NCC skeletogenesis and normal heart development.