RESUMO
Using dynamic liquid-state NMR spectroscopy a degenerate double proton tautomerism was detected in tetramethyl reductic acid (TMRA) dissolved in toluene-d8 and in CD2Cl2. Similar to vitamin C, TMRA belongs to the class of reductones of biologically important compounds. The tautomerism involves an intramolecular HH transfer that interconverts the peripheric and the central positions of the two OH groups. It is slow in the NMR time scale around 200 K and fast at room temperature. Pseudo-first-order rate constants of the HH transfer and of the HD transfer after suitable deuteration were obtained by line shape analyses. Interestingly, the chemical shifts were found to be temperature dependent carrying information about an equilibrium between a hydrogen bonded dimer and a monomer forming two weak intramolecular hydrogen bonds. The structures of the monomer and the dimer are discussed. The latter may consist of several rapidly interconverting hydrogen-bonded associates. A way was found to obtain the enthalpies and entropies of dissociation, which allowed us to convert the pseudo-first-order rate constants of the reaction mixture into first-order rate constants of the tautomerization of the monomer. Surprisingly, these intrinsic rate constants were the same for toluene-d8 and CD2Cl2, but in the latter solvent more monomer is formed. This finding is attributed to the dipole moment of the TMRA monomer, compensated in the dimer, and to the larger dielectric constant of CD2Cl2. Within the margin of error, the kinetic HH/HD isotope effects were found to be of the order of 3 but independent of temperature. That finding indicates a stepwise HH transfer involving a tunnel mechanism along a double barrier pathway. The Arrhenius curves were described in terms of the Bell-Limbach tunneling model.
RESUMO
Patients with 46,XY gonadal dysgenesis (GD) exhibit genital anomalies, which range from hypospadias to complete male-to-female sex reversal. However, a molecular diagnosis is made in only 30% of cases. Heterozygous mutations in the human FGFR2 gene cause various craniosynostosis syndromes including Crouzon and Pfeiffer, but testicular defects were not reported. Here, we describe a patient whose features we would suggest represent a new FGFR2-related syndrome, craniosynostosis with XY male-to-female sex reversal or CSR. The craniosynostosis patient was chromosomally XY, but presented as a phenotypic female due to complete GD. DNA sequencing identified the FGFR2c heterozygous missense mutation, c.1025G>C (p.Cys342Ser). Substitution of Cys342 by Ser or other amino acids (Arg/Phe/Try/Tyr) has been previously reported in Crouzon and Pfeiffer syndrome. We show that the 'knock-in' Crouzon mouse model Fgfr2c(C342Y/C342Y) carrying a Cys342Tyr substitution displays XY gonadal sex reversal with variable expressivity. We also show that despite FGFR2c-Cys342Tyr being widely considered a gain-of-function mutation, Cys342Tyr substitution in the gonad leads to loss of function, as demonstrated by sex reversal in Fgfr2c(C342Y/-) mice carrying the knock-in allele on a null background. The rarity of our patient suggests the influence of modifier genes which exacerbated the testicular phenotype. Indeed, patient whole exome analysis revealed several potential modifiers expressed in Sertoli cells at the time of testis determination in mice. In summary, this study identifies the first FGFR2 mutation in a 46,XY GD patient. We conclude that, in certain rare genetic contexts, maintaining normal levels of FGFR2 signaling is important for human testis determination.
Assuntos
Craniossinostoses/genética , Disgenesia Gonadal 46 XY/genética , Mutação de Sentido Incorreto , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Adolescente , Animais , Craniossinostoses/metabolismo , Análise Mutacional de DNA , Modelos Animais de Doenças , Feminino , Técnicas de Introdução de Genes , Humanos , Masculino , Camundongos , Camundongos Mutantes , SíndromeRESUMO
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.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Cartilagem/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Ligamentos/crescimento & desenvolvimento , Fatores de Transcrição SOX9/genética , Células-Tronco/citologia , Tendões/crescimento & desenvolvimento , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Osso e Ossos/fisiologia , Mesoderma/crescimento & desenvolvimento , Camundongos , Camundongos Transgênicos , Fatores de Transcrição SOX9/metabolismoRESUMO
BACKGROUND: SOX9 mutations cause the skeletal malformation syndrome campomelic dysplasia in combination with XY sex reversal. Studies in mice indicate that SOX9 acts as a testis-inducing transcription factor downstream of SRY, triggering Sertoli cell and testis differentiation. An SRY-dependent testis-specific enhancer for Sox9 has been identified only in mice. A previous study has implicated copy number variations (CNVs) of a 78 kb region 517-595 kb upstream of SOX9 in the aetiology of both 46,XY and 46,XX disorders of sex development (DSD). We wanted to better define this region for both disorders. RESULTS: By CNV analysis, we identified SOX9 upstream duplications in three cases of SRY-negative 46,XX DSD, which together with previously reported duplications define a 68 kb region, 516-584 kb upstream of SOX9, designated XXSR (XX sex reversal region). More importantly, we identified heterozygous deletions in four families with SRY-positive 46,XY DSD without skeletal phenotype, which define a 32.5 kb interval 607.1-639.6 kb upstream of SOX9, designated XY sex reversal region (XYSR). To localise the suspected testis-specific enhancer, XYSR subfragments were tested in cell transfection and transgenic experiments. While transgenic experiments remained inconclusive, a 1.9 kb SRY-responsive subfragment drove expression specifically in Sertoli-like cells. CONCLUSIONS: Our results indicate that isolated 46,XY and 46,XX DSD can be assigned to two separate regulatory regions, XYSR and XXSR, far upstream of SOX9. The 1.9 kb SRY-responsive subfragment from the XYSR might constitute the core of the Sertoli-cell enhancer of human SOX9, representing the so far missing link in the genetic cascade of male sex determination.
Assuntos
Variações do Número de Cópias de DNA , Transtornos do Desenvolvimento Sexual/genética , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição SOX9/genética , Animais , Linhagem Celular , Estudos de Coortes , Feminino , Humanos , Masculino , Camundongos , LinhagemRESUMO
Sox9 plays a critical role in early chondrocyte initiation and promotion as well as repression of later maturation. Fellow Sox family members L-Sox5 and Sox6 also function as regulators of cartilage development by boosting Sox9 activation of chondrocyte-specific genes such as Col2a1 and Agc1; however, the regulatory mechanism and other target genes are largely unknown. MicroRNAs are a class of short, non-coding RNAs that act as negative regulators of gene expression by promoting target mRNA degradation and/or repressing translation. Analysis of genetically modified mice identified miR-140 as a cartilage-specific microRNA that could be a critical regulator of cartilage development and homeostasis. Recent findings suggest Sox9 promotes miR-140 expression, although the detailed mechanisms are not fully understood. In this study we demonstrate that the proximal upstream region of pri-miR-140 has chondrogenic promoter activity in vivo. We found an L-Sox5/Sox6/Sox9 (Sox trio) response element and detailed binding site in the promoter region. Furthermore, detailed analysis suggests the DNA binding and/or transactivation ability of Sox9 as a homodimer is boosted by L-Sox5 and Sox6. These findings provide new insight into cartilage-specific gene regulation by the Sox trio.
Assuntos
Cartilagem/metabolismo , MicroRNAs/metabolismo , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOXD/metabolismo , Animais , Condrócitos/citologia , Imunoprecipitação da Cromatina , Dimerização , Regulação da Expressão Gênica , Células HEK293 , Proteínas de Grupo de Alta Mobilidade/genética , Humanos , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase/métodos , Isoformas de Proteínas , Ativação Transcricional , TransgenesRESUMO
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.
Assuntos
Diferenciação Celular , Hidronefrose/genética , Hidronefrose/patologia , Mesoderma/patologia , Miócitos de Músculo Liso/patologia , Fatores de Transcrição SOX9/genética , Ureter/patologia , Animais , Diferenciação Celular/genética , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Matriz Extracelular/genética , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Rim/metabolismo , Rim/patologia , Mesoderma/metabolismo , Camundongos , Mutação/genética , Miócitos de Músculo Liso/metabolismo , Fatores de Transcrição SOX9/metabolismo , Ureter/crescimento & desenvolvimento , Ureter/metabolismoRESUMO
The sex-determining gene Sry and its target gene Sox9 initiate the early steps of testis development in mammals. Of the related Sox genes Sox8, Sox9, and Sox10, all expressed during Sertoli cell differentiation, only inactivation of Sox9 before the sex determination stage at Embryonic Day 11.5 (E11.5) causes XY sex reversal, while Sox9 inactivation after this stage has no effect on testis cord differentiation. We have previously shown that both Sox9 and Sox8 are essential for maintaining testicular function in post-E14.0 Sertoli cells. To gain insight into the molecular and cellular processes underlying the abnormal development of Sox9 and Sox8 mutant testes, we performed a detailed developmental study of embryonic and neonatal stages. We observe a progressive disruption of the basal lamina surrounding the testis cords that starts at E17.5 and already at E15.5 reduced expression levels of collagen IV, collagen IXa3 and testatin, structural components of the basal lamina, and the extracellular matrix transcriptional regulator Scleraxis. Lineage tracing reveals that mutant Sertoli cells delaminate from testis cords and are present as isolated cells between remaining cords. Also, Sox10 expression is strongly reduced in the absence of Sox9 and/or Sox8. Finally, we document increasing expression of the ovarian marker FOXL2 in mutant cords starting at E15.5, indicating progressive transdifferentiation of mutant Sertoli cells. This study shows that Sox9 and Sox8 maintain integrity of the basal lamina to prevent testis cord disintegration and that both factors actively suppress the ovarian program during early testis development.
Assuntos
Membrana Basal/fisiologia , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição SOX9/fisiologia , Fatores de Transcrição SOXE/fisiologia , Testículo/embriologia , Testículo/ultraestrutura , Animais , Membrana Basal/metabolismo , Permeabilidade da Membrana Celular/genética , Transdiferenciação Celular/genética , Feminino , Proteína Forkhead Box L2 , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Gravidez , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOXE/genética , Fatores de Transcrição SOXE/metabolismo , Células de Sertoli/metabolismo , Células de Sertoli/fisiologia , Testículo/citologia , Testículo/metabolismoRESUMO
Tyrosinemia Type III is caused by the deficiency of 4-hydroxyphenylpyruvate dioxygenase (4-HPPD), an enzyme involved in the catabolic pathway of tyrosine. To our knowledge, only a few patients presenting with this disease have been described in the literature, and the clinical phenotype remains variable and unclear. We report the case of a boy with tyrosinemia Type III detected using neonatal screening, who is homozygous for the splice donor mutation IVS11+1G>A in intron 11 of the HPD gene. At the age of 30 months, the boy's outcome under mild protein restriction was characterized by normal growth and psychomotor development.
Assuntos
4-Hidroxifenilpiruvato Dioxigenase/genética , Dieta com Restrição de Proteínas , Tirosina/metabolismo , Tirosinemias/dietoterapia , 4-Hidroxifenilpiruvato Dioxigenase/deficiência , Pré-Escolar , Gerenciamento Clínico , Homozigoto , Humanos , Recém-Nascido , Íntrons , Masculino , Mutação , Triagem Neonatal , Desempenho Psicomotor , Resultado do Tratamento , Tirosinemias/genética , Tirosinemias/metabolismoRESUMO
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.
Assuntos
Colo/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Celulas de Paneth/metabolismo , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Colo/citologia , Proteínas de Grupo de Alta Mobilidade/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Celulas de Paneth/citologia , Fatores de Transcrição SOX9 , Fatores de Transcrição/genéticaRESUMO
Members of the evolutionarily conserved T-box family of transcription factors are important players in developmental processes that include mesoderm formation and patterning and organogenesis both in vertebrates and invertebrates. The importance of T-box genes for human development is illustrated by the association between mutations in several of the 17 human family members and congenital errors of morphogenesis that include cardiac, craniofacial, and limb malformations. We identified two unrelated individuals with a complex cranial, cervical, auricular, and skeletal malformation syndrome with scapular and pelvic hypoplasia (Cousin syndrome) that recapitulates the dysmorphic phenotype seen in the Tbx15-deficient mice, droopy ear. Both affected individuals were homozygous for genomic TBX15 mutations that resulted in truncation of the protein and addition of a stretch of missense amino acids. Although the mutant proteins had an intact T-box and were able to bind to their target DNA sequence in vitro, the missense amino acid sequence directed them to early degradation, and cellular levels were markedly reduced. We conclude that Cousin syndrome is caused by TBX15 insufficiency and is thus the human counterpart of the droopy ear mouse.
Assuntos
Estatura/genética , Anormalidades Craniofaciais/genética , Mutação , Pelve/patologia , Escápula/patologia , Proteínas com Domínio T/genética , Criança , Anormalidades Craniofaciais/diagnóstico por imagem , Feminino , Humanos , Mutação de Sentido Incorreto , Pelve/diagnóstico por imagem , Radiografia , Escápula/diagnóstico por imagem , Síndrome , Proteínas com Domínio T/metabolismo , Adulto JovemRESUMO
A girl, born to unrelated Spanish parents, presented at 6 months of age with photophobia, keratitis, palmar hyperkeratosis and high plasma tyrosine levels, indicative of tyrosinaemia type II. Analysis of the tyrosine aminotransferase (TAT) gene revealed a paternally inherited frameshift mutation c.1213delCinsAG at codon 405 causing a premature stop codon, and a maternally inherited deletion of 193kb encompassing the complete TAT gene and three neighbouring genes. This is the first complete TAT deletion in tyrosinaemia type II described so far.
Assuntos
Deleção de Genes , Tirosina Transaminase/genética , Tirosinemias/genética , Sequência de Bases , Códon sem Sentido , Primers do DNA/genética , Feminino , Mutação da Fase de Leitura/genética , Humanos , Lactente , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , EspanhaRESUMO
Sox9 and Sox8 are transcription factors expressed in embryonic and postnatal Sertoli cells of the mouse testis. Sox9 inactivation prior to the sex determination stage leads to complete XY sex reversal. In contrast, there is normal embryonic testis development in Sox8 mutants which are initially fertile, but later develop progressive seminiferous tubule failure and infertility. To determine whether Sox9 is required for testis development after the initial steps of sex determination, we crossed Sox9(flox) mice with an AMH-Cre transgenic line thereby completely deleting Sox9 in Sertoli cells by E14.0. Conditional Sox9 null mutants show normal embryonic testis development and are initially fertile, but, like Sox8(-/-) mutants, become sterile from dysfunctional spermatogenesis at about 5 months. To see whether Sox8 may compensate for the absence of Sox9 during embryonic testis differentiation, we generated a Sox9 conditional knockout on a Sox8 mutant background. In the double mutants, differentiation of testis cords into seminiferous testis tubules ceases after P6 in the absence of one Sox8 allele, and after P0 in the absence of both Sox8 alleles, leading to complete primary infertility. Sox9,Sox8 double nullizygous testes show upregulation of early ovary-specific markers and downregulation of Sertoli intercellular junctions at E15.5. Their very low Amh levels still cause complete regression of the Müllerian duct but with reduced penetrance. This study shows that testis cord differentiation is independent of Sox9, and that concerted Sox9 and Sox8 function in post E14.0 Sertoli cells is essential for the maintenance of testicular function.
Assuntos
Morfogênese/fisiologia , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOXE/metabolismo , Processos de Determinação Sexual , Testículo , Animais , Biomarcadores/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Feminino , Fertilidade/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOXE/genética , Espermatogênese/fisiologia , Testículo/anatomia & histologia , Testículo/embriologiaRESUMO
BACKGROUND: Sox9 (Sry box containing gene 9) is a DNA-binding transcription factor involved in chondrocyte development and sex determination. The protein's absence in testicular Sertoli nurse cells has been shown to disrupt testicular function in adults but little is known at the genome-wide level about molecular events concomitant with testicular break-down. METHODS: To determine the genome-wide effect on mRNA concentrations triggered by the absence of Sox9 in Sertoli cells we analysed adult testicular tissue from wild-type versus mutant mice with high-density oligonucleotide microarrays and integrated the output of this experiment with regulatory motif predictions and protein-protein network data. RESULTS: We report the genome-wide mRNA signature of adult testes lacking Sox9 in Sertoli cells before and after the onset of late spermatogenic failure as compared to fertile controls. The GeneChip data integrated with evolutionarily conserved Sox9 DNA binding motifs and regulatory network data identified genes involved in feminization, stress response and inflammation. CONCLUSIONS: Our results extend previous observations that genes required for female gonadogenesis are up-regulated in the absence of Sox9 in fetal Sertoli cells to the adult stage. Importantly, we identify gene networks involved in immunological processes and stress response which is reminiscent of a phenomenon occurring in a sub-group of infertile men. This suggests mice lacking Sox9 in their Sertoli cells to be a potentially useful model for adult human testicular failure.
Assuntos
Feminização/genética , Inflamação/genética , Fatores de Transcrição SOX9/genética , Células de Sertoli/metabolismo , Espermatogênese/fisiologia , Estresse Fisiológico/genética , Animais , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Masculino , Camundongos , Modelos Animais , RNA Mensageiro/metabolismo , Testículo/embriologia , Testículo/metabolismoRESUMO
Von Hippel-Lindau disease (VHL) is an autosomal dominant cancer syndrome. Affected individuals are predisposed to multiple tumors, primarily of the central nervous system (CNS), eyes, adrenals, and kidneys. The VHL tumor suppressor gene on chromosome 3p26-25 is partially or completely deleted in 20 to 30% of families with VHL. We identified deletions ranging from 0.5 kb to 250 kb affecting part of or the entire VHL and flanking genes in 54 families. In 33 of the index patients, the breakpoints were precisely characterized by DNA sequencing. Of the 66 breakpoints, 90% were located in Alu elements, revealing Alu-mediated recombination as the major mechanism for large germline deletions of the VHL gene, which lies in a region of high Alu density. Interestingly, an AluYa5 element in VHL intron 2, the evolutionarily youngest Alu element and the only such element in the entire region, was found to be the most recombinogenic, involved in 7 out of the 33 deletions. In comparison to VHL patients in general, the 54 index cases and their affected relatives showed a higher occurrence of renal cell carcinomas (RCC) and of CNS hemangioblastomas. We not only noted the association of RCC with retention of the HSPC300 gene, but also observed a significant correlation between retention of HSPC300 and the development of retinal angiomas (AR). This study reveals that germline VHL deletions provide a particularly rich source for the study of Alu-mediated unequal crossover events, and provides evidence for a protective role of the loss of the actin-regulator gene HSPC300 for the development of both RCC and AR.
Assuntos
Elementos Alu/genética , Mutação em Linhagem Germinativa/genética , Recombinação Genética/genética , Deleção de Sequência/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Doença de von Hippel-Lindau/genética , Sequência de Bases , Quebra Cromossômica , Sequência Consenso , Genótipo , Humanos , Fenótipo , Mapeamento Físico do Cromossomo , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
In mammals, sex is determined in the bipotential embryonic gonad by a balanced network of gene actions which when altered causes disorders of sexual development (DSD, formerly known as intersex). In the XY gonad, presumptive Sertoli cells begin to differentiate when SRY up-regulates SOX9, which in turn activates FGF9 and PGDS to maintain its own expression. This study identifies a new and essential component of FGF signaling in sex determination. Fgfr2 mutant XY mice on a mixed 129/C57BL6 genetic background had either normal testes, or developed ovotestes, with predominantly testicular tissue. However, backcrossing to C57BL6 mice resulted in a wide range of gonadal phenotypes, from hypoplastic testes to ovotestes with predominantly ovarian tissue, similar to Fgf9 knockout mice. Since typical male-specific FGF9-binding to the coelomic epithelium was abolished in Fgfr2 mutant XY gonads, these results suggest that FGFR2 acts as the receptor for FGF9. Pgds and SOX9 remained expressed within the testicular portions of Fgfr2 mutant ovotestes, suggesting that the Prostaglandin pathway acts independently of FGFR2 to maintain SOX9 expression. We could further demonstrate that double-heterozygous Fgfr2/Sox9 knockout mice developed ovotestes, demonstrating that both Fgfr2 and Sox9 can act as modifier intersex genes in the heterozygous state. In summary, we provide evidence that FGFR2 is important for male sex determination in mice, thereby rendering human FGFR2 a candidate gene for unsolved DSD cases such as 10q26 deletions.
Assuntos
Transtornos do Desenvolvimento Sexual , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/fisiologia , Processos de Determinação Sexual , Animais , Feminino , Fator 9 de Crescimento de Fibroblastos/genética , Fator 9 de Crescimento de Fibroblastos/fisiologia , Gônadas/citologia , Gônadas/embriologia , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/fisiologia , Masculino , Camundongos , Camundongos Knockout , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Fatores de Transcrição SOX9 , Células de Sertoli/citologia , Células de Sertoli/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologiaRESUMO
The cis-regulatory regions of many developmental regulators and transcription factors are believed to be highly conserved in the genomes of vertebrate species, suggesting specific regulatory mechanisms for these gene classes. We functionally characterized five notochord enhancers, whose sequence is highly conserved, and systematically mutated two of them. Two subregions were identified to be essential for expression in the notochord of the zebrafish embryo. Synthetic enhancers containing the two essential regions in front of a TATA-box drive expression in the notochord while concatemerization of the subregions alone is not sufficient, indicating that the combination of the two sequence elements is required for notochord expression. Both regions are present in the five functionally characterized notochord enhancers. However, the position, the distance and relative orientation of the two sequence motifs can vary substantially within the enhancer sequences. This suggests that the regulatory grammar itself does not dictate the high evolutionary conservation between these orthologous cis-regulatory sequences. Rather, it represents a less well-conserved layer of sequence organization within these sequences.
Assuntos
Notocorda/metabolismo , Elementos Reguladores de Transcrição , Peixe-Zebra/metabolismo , Animais , Sequência de Bases , Sequência Conservada , Análise Mutacional de DNA , Elementos Facilitadores Genéticos , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas HMGB/genética , Humanos , Regiões Promotoras Genéticas , Fatores de Transcrição SOX9 , Alinhamento de Sequência , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The HMG-domain-containing transcription factor Sox9 is an important regulator of chondrogenesis, testis formation and development of several other organs. Sox9 is expressed in the otic placodes, the primordia of the inner ear, and studies in Xenopus have provided evidence that Sox9 is required for otic specification. Here we report novel and different functions of Sox9 during mouse inner ear development. We show that in mice with a Foxg1(Cre)-mediated conditional inactivation of Sox9 in the otic ectoderm, otic placodes form and express markers of otic specification. However, mutant placodes do not attach to the neural tube, fail to invaginate, and subsequently degenerate by apoptosis, resulting in a complete loss of otic structures. Transmission-electron microscopic analysis suggests that cell-cell contacts in the Sox9 mutant placodes are abnormal, although E-cadherin, N-cadherin, and beta-catenin protein expression are unchanged. In contrast, expression of Epha4 was downregulated in mutant placodes. In embryos with a Keratin-19(Cre)-mediated mosaic inactivation of Sox9, Sox9-negative and Sox9-positive cells in the otic ectoderm sort out from one another. In these embryos only Sox9-positive cells invaginate and form one or several micro-vesicles, whereas Sox9-negative cells stay behind and die. Our findings demonstrate that, in contrast to Xenopus, Sox9 is not required for the initial specification of the otic placode in the mouse, but instead controls adhesive properties and invagination of placodal cells in a cell-autonomous manner.
Assuntos
Orelha Interna/embriologia , Proteínas de Grupo de Alta Mobilidade/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Orelha Interna/citologia , Ectoderma/metabolismo , Embrião de Mamíferos/metabolismo , Proteínas de Grupo de Alta Mobilidade/genética , Camundongos , Receptor EphA4/metabolismo , Fatores de Transcrição SOX9 , Fatores de Transcrição SOXE , Gânglio Espiral da Cóclea/citologia , Gânglio Espiral da Cóclea/embriologia , Fatores de Transcrição/genéticaRESUMO
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.
Assuntos
Condrócitos/química , Condrócitos/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Proteínas de Grupo de Alta Mobilidade/deficiência , Fatores de Transcrição/deficiência , Sequência de Aminoácidos , Animais , Sequência de Bases , Cartilagem/citologia , Cartilagem/crescimento & desenvolvimento , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Condrogênese/genética , Condrogênese/fisiologia , Células Clonais , Colágeno Tipo II/genética , DNA/genética , Éxons , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/fisiologia , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOX9 , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologiaRESUMO
46,XY gonadal dysgenesis (46,XY GD) is a disorder of sexual development caused by mutations in genes involved in early gonadal development (bipotential gonads) and testis differentiation. In 46,XY GD individuals, mutations of the SRY gene are detected most frequently, followed by mutations in the NR5A1 (SF-1) gene, but in a lot of cases, the underlying molecular mechanism remains elusive. In this study, we retrospectively performed sequence analyses of the NR5A1 (SF-1) gene in 84 patients with complete, partial, and syndromic forms of 46,XY GD. In total, 7 heterozygous mutations were found in 6 of 84 patients (7.1%). Among these, we identified 4 mutations that, to the best of our knowledge, have not been reported before (c.268G>T, c.369del, c.871-1G>C, and c.893T>C). Transfection of different mutations revealed altered subcellular localization of the mutant SF-1 protein in the case of the frameshift mutations, indicating an impaired protein function. In conclusion, we present 4 novel mutations of the NR5A1 gene associated with 46,XY GD together with in vitro data pointing towards a possible functional impairment of the mutant SF-1 proteins.
Assuntos
Mutação da Fase de Leitura/genética , Disgenesia Gonadal 46 XY/genética , Fator Esteroidogênico 1/metabolismo , Feminino , Células HEK293 , Humanos , Masculino , Mutação/genética , Estudos Retrospectivos , Fator Esteroidogênico 1/genéticaRESUMO
BACKGROUND: Campomelic dysplasia (CD) is a semilethal developmental disorder caused by mutations in and around SOX9. CD is characterized by multiple skeletal malformations including bending (campomelia) of long bones. Surviving patients frequently have the acampomelic form of CD (ACD). METHODS: This is a single case report on a patient with clinical and radiological features of ACD who has no mutation in the SOX9 protein-coding sequence nor a translocation with breakpoint in the SOX9 regulatory domain. We include functional studies of the novel mutant protein in vitro and in cultured cells. RESULTS: The patient was found to have a de novo heterozygous mutation c.-185G>A in the SOX9 5'UTR. The mutation creates an upstream translation start codon, uAUG, with a much better fit of its flanking sequence to the Kozak consensus than the wild-type AUG. By in vitro transcription-translation and transient transfection into COS-7 cells, we show that the uAUG leads to translation of a short peptide from a reading frame that terminates just after the wild-type AUG start codon. This results in reduced translation of the wild-type protein, compatible with the milder phenotype of the patient. CONCLUSION: Findings support the notion that more mildly affected, surviving CD/ACD patients carry mutant SOX9 alleles with residual expression of SOX9 wild-type protein. Although rarely described in human genetic disease and for the first time here for CD, mutations creating upstream AUG codons may be more common than generally assumed.