Key Spec Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|R, H, M||WB, IHC||Rb||Affinity Purified||Polyclonal Antibody|
|Presentation||Purified rabbit polyclonal in buffer containing 0.1M Tris -Glycine, 0.15M NaCl, 0.05% sodium azide, pH7.4.|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Stable for 6 months at 2-8°C in undiluted aliquots from date of receipt.|
|Material Size||100 µg|
|Anti-Sox9 - 2383973||2383973|
|Anti-Sox9 - 2424751||2424751|
|Anti-Sox9 - 2462860||2462860|
|Anti-Sox9 - 1969702||1969702|
|Anti-Sox9 - 1997395||1997395|
|Anti-Sox9 - 2031330||2031330|
|Anti-Sox9 - 2065999||2065999|
|Anti-Sox9 - 2167153||2167153|
|Anti-Sox9 - 2262679||2262679|
|Anti-Sox9 - 2361818||2361818|
|Anti-Sox9 - 2494029||2494029|
|Anti-Sox9 - LV1478872||LV1478872|
|Anti-Sox9 - LV1580835||LV1580835|
|Anti-Sox9 - LV1679322||LV1679322|
|Anti-Sox9 - LV1738269||LV1738269|
|Anti-Sox9 - LV1762669||LV1762669|
|Anti-Sox9 - NG1839530||NG1839530|
|Anti-Sox9 - NG1884650||NG1884650|
|Anti-Sox9 - NG1901550||NG1901550|
|Anti-Sox9 - NG1922963||NG1922963|
|RABBIT ANTI-POTASSIUM CHANNEL MAXI K (BKCa) - 2494610||2494610|
References | 54 Available | See All References
|Reference overview||Pub Med ID|
|Mutant IDH inhibits HNF-4α to block hepatocyte differentiation and promote biliary cancer. |
Saha, SK; Parachoniak, CA; Ghanta, KS; Fitamant, J; Ross, KN; Najem, MS; Gurumurthy, S; Akbay, EA; Sia, D; Cornella, H; Miltiadous, O; Walesky, C; Deshpande, V; Zhu, AX; Hezel, AF; Yen, KE; Straley, KS; Travins, J; Popovici-Muller, J; Gliser, C; Ferrone, CR; Apte, U; Llovet, JM; Wong, KK; Ramaswamy, S; Bardeesy, N
Nature 513 110-4 2014
Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 are among the most common genetic alterations in intrahepatic cholangiocarcinoma (IHCC), a deadly liver cancer. Mutant IDH proteins in IHCC and other malignancies acquire an abnormal enzymatic activity allowing them to convert α-ketoglutarate (αKG) to 2-hydroxyglutarate (2HG), which inhibits the activity of multiple αKG-dependent dioxygenases, and results in alterations in cell differentiation, survival, and extracellular matrix maturation. However, the molecular pathways by which IDH mutations lead to tumour formation remain unclear. Here we show that mutant IDH blocks liver progenitor cells from undergoing hepatocyte differentiation through the production of 2HG and suppression of HNF-4α, a master regulator of hepatocyte identity and quiescence. Correspondingly, genetically engineered mouse models expressing mutant IDH in the adult liver show an aberrant response to hepatic injury, characterized by HNF-4α silencing, impaired hepatocyte differentiation, and markedly elevated levels of cell proliferation. Moreover, IDH and Kras mutations, genetic alterations that co-exist in a subset of human IHCCs, cooperate to drive the expansion of liver progenitor cells, development of premalignant biliary lesions, and progression to metastatic IHCC. These studies provide a functional link between IDH mutations, hepatic cell fate, and IHCC pathogenesis, and present a novel genetically engineered mouse model of IDH-driven malignancy.
|Testicular development in mice lacking receptors for follicle stimulating hormone and androgen. |
Peter J O'Shaughnessy,Ana Monteiro,Margaret Abel
PloS one 7 2012
Post-natal testicular development is dependent on gonadotrophin and androgen stimulation. Follicle stimulating hormone (FSH) acts through receptors (FSHR) on the Sertoli cell to stimulate spermatogenesis while androgens promote testis growth through receptors (AR) on the Sertoli cells, Leydig cells and peritubular myoid cells. In this study we have examined the effects on testis development of ablating FSHRs (FSHRKO mice) and/or ARs ubiquitously (ARKO mice) or specifically on the Sertoli cells (SCARKO mice). Cell numbers were measured using stereological methods. In ARKO mice Sertoli cell numbers were reduced at all ages from birth until adulthood. FSHR ablation also caused small reductions in Sertoli cell numbers up to day 20 with more marked effects seen in the adult. Germ cell numbers were unaffected by FSHR and/or AR ablation at birth. By day 20 ubiquitous AR or FSHR ablation caused a marked reduction in germ cell numbers with a synergistic effect of losing both receptors (germ cell numbers in FSHRKO.ARKO mice were 3% of control). Germ cell numbers in SCARKO mice were less affected. By adulthood, in contrast, clear synergistic control of germ cell numbers had become established between the actions of FSH and androgen through the Sertoli cells. Leydig cell numbers were normal on day 1 and day 5 in all groups. By day 20 and in adult animals total AR or FSHR ablation significantly reduced Leydig cell numbers but Sertoli cell specific AR ablation had no effect. Results show that, prior to puberty, development of most testicular parameters is more dependent on FSH action than androgen action mediated through the Sertoli cells although androgen action through other cells types is crucial. Post-pubertally, germ cell numbers and spermatogenesis are dependent on FSH and androgen action through the Sertoli cells.
|Regulation of human lung alveolar multipotent cells by a novel p38α MAPK/miR-17-92 axis. |
Feride Oeztuerk-Winder,Anna Guinot,Anna Ochalek,Juan-Jose Ventura
The EMBO journal 31 2012
The cellular and molecular mechanisms that control lung homeostasis and regeneration are still poorly understood. It has been proposed that a population of cells exists in the mouse lung with the potential to differentiate into all major lung bronchioalveolar epithelium cell types in homeostasis or in response to virus infection. A new population of E-Cad/Lgr6(+) putative stem cells has been isolated, and indefinitely expanded from human lungs, harbouring both, self-renewal capacity and the potency to differentiate in vitro and in vivo. Recently, a putative population of human lung stem cells has been proposed as being c-Kit(+). Unlike Integrin-α6(+) or c-Kit(+) cells, E-Cad/Lgr6(+) single-cell injections in the kidney capsule produce differentiated bronchioalveolar tissue, while retaining self-renewal, as they can undergo serial transplantations under the kidney capsule or in the lung. In addition, a signalling network involving the p38α pathway, the activation of p53 and the regulation of the miR-17-92 cluster has been identified. Disruption of the proper cross-regulation of this signalling axis might be involved in the promotion of human lung diseases.
|BAC-Dkk3-EGFP transgenic mouse: an in vivo analytical tool for Dkk3 expression. |
Yuki Muranishi,Takahisa Furukawa
Journal of biomedicine & biotechnology 2012 2012
Dickkopf (DKK) family proteins are secreted modulators of the Wnt signaling pathway and are capable of regulating the development of many organs and tissues. We previously identified Dkk3 to be a molecule predominantly expressed in the mouse embryonic retina. However, which cell expresses Dkk3 in the developing and mature mouse retina remains to be elucidated. To examine the precise expression of the Dkk3 protein, we generated BAC-Dkk3-EGFP transgenic mice that express EGFP integrated into the Dkk3 gene in a BAC plasmid. Expression analysis using the BAC-Dkk3-EGFP transgenic mice revealed that Dkk3 is expressed in retinal progenitor cells (RPCs) at embryonic stages and in Müller glial cells in the adult retina. Since Müller glial cells may play a potential role in retinal regeneration, BAC-Dkk3-EGFP mice could be useful for retinal regeneration studies.
|GATA4 and GATA6 control mouse pancreas organogenesis. |
Manuel Carrasco,Irene Delgado,Bernat Soria,Francisco Mart,Anabel Rojas
The Journal of clinical investigation 122 2012
Recently, heterozygous mutations in GATA6 have been found in neonatal diabetic patients with failed pancreatic organogenesis. To investigate the roles of GATA4 and GATA6 in mouse pancreas organogenesis, we conditionally inactivated these genes within the pancreas. Single inactivation of either gene did not have a major impact on pancreas formation, indicating functional redundancy. However, double Gata4/Gata6 mutant mice failed to develop pancreata, died shortly after birth, and displayed hyperglycemia. Morphological defects in Gata4/Gata6 mutant pancreata were apparent during embryonic development, and the epithelium failed to expand as a result of defects in cell proliferation and differentiation. The number of multipotent pancreatic progenitors, including PDX1+ cells, was reduced in the Gata4/Gata6 mutant pancreatic epithelium. Remarkably, deletion of only 1 Gata6 allele on a Gata4 conditional knockout background severely reduced pancreatic mass. In contrast, a single WT allele of Gata4 in Gata6 conditional knockout mice was sufficient for normal pancreatic development, indicating differential contributions of GATA factors to pancreas formation. Our results place GATA factors at the top of the transcriptional network hierarchy controlling pancreas organogenesis.
|Isolation, differentiation, and characterisation of skeletal stem cells from human bone marrow in vitro and in vivo. |
Rahul S Tare,Peter D Mitchell,Janos Kanczler,Richard O C Oreffo
Methods in molecular biology (Clifton, N.J.) 816 2012
In this chapter, we describe techniques for the isolation and characterization of skeletal stem cells from human bone marrow. The methods for enrichment of STRO-1 positive cells using magnetic activated cell sorting are described and we also cover techniques for establishing and characterising osteogenic, adipogenic, and chondrogenic cultures from these cells. Finally, we present methods for studying the ability of these cells to produce bone in vivo using diffusion chambers which have been implanted subcutaneously in mice.
|Oncogenicity of the developmental transcription factor Sox9. |
Ander Matheu,Manuel Collado,Clare Wise,Lorea Manterola,Lina Cekaite,Angela J Tye,Marta Canamero,Luis Bujanda,Andreas Schedl,Kathryn S E Cheah,Rolf I Skotheim,Ragnhild A Lothe,Adolfo López de Munain,James Briscoe,Manuel Serrano,Robin Lovell-Badge,Adolfo L
Cancer research 72 2012
SOX9 [sex-determining region Y (SRY)-box 9 protein], a high mobility group box transcription factor, plays critical roles during embryogenesis and its activity is required for development, differentiation, and lineage commitment in various tissues including the intestinal epithelium. Here, we present functional and clinical data of a broadly important role for SOX9 in tumorigenesis. SOX9 was overexpressed in a wide range of human cancers, where its expression correlated with malignant character and progression. Gain of SOX9 copy number is detected in some primary colorectal cancers. SOX9 exhibited several pro-oncogenic properties, including the ability to promote proliferation, inhibit senescence, and collaborate with other oncogenes in neoplastic transformation. In primary mouse embryo fibroblasts and colorectal cancer cells, SOX9 expression facilitated tumor growth and progression whereas its inactivation reduced tumorigenicity. Mechanistically, we have found that Sox9 directly binds and activates the promoter of the polycomb Bmi1, whose upregulation represses the tumor suppressor Ink4a/Arf locus. In agreement with this, human colorectal cancers showed a positive correlation between expression levels of SOX9 and BMI1 and a negative correlation between SOX9 and ARF in clinical samples. Taken together, our findings provide direct mechanistic evidence of the involvement of SOX9 in neoplastic pathobiology, particularly, in colorectal cancer.
|Sox9 directs hypertrophic maturation and blocks osteoblast differentiation of growth plate chondrocytes. |
Peter Dy,Weihuan Wang,Pallavi Bhattaram,Qiuqing Wang,Lai Wang,R Tracy Ballock,Véronique Lefebvre
Developmental cell 22 2012
The transcription factor Sox9 is necessary for early chondrogenesis, but its subsequent roles in the cartilage growth plate, a highly specialized structure that drives skeletal growth and endochondral ossification, remain unclear. Using a doxycycline-inducible Cre transgene and Sox9 conditional null alleles in the mouse, we show that Sox9 is required to maintain chondrocyte columnar proliferation and generate cell hypertrophy, two key features of functional growth plates. Sox9 keeps Runx2 expression and ?-catenin signaling in check and thereby inhibits not only progression from proliferation to prehypertrophy, but also subsequent acquisition of an osteoblastic phenotype. Sox9 protein outlives Sox9 RNA in upper hypertrophic chondrocytes, where it contributes with Mef2c to directly activate the major marker of these cells, Col10a1. These findings thus reveal that Sox9 remains a central determinant of the lineage fate and multistep differentiation program of growth plate chondrocytes and thereby illuminate our understanding of key molecular mechanisms underlying skeletogenesis.
|Slug and Sox9 cooperatively determine the mammary stem cell state. |
Guo, Wenjun, et al.
Cell, 148: 1015-28 (2012) 2012
Regulatory networks orchestrated by key transcription factors (TFs) have been proposed to play a central role in the determination of stem cell states. However, the master transcriptional regulators of adult stem cells are poorly understood. We have identified two TFs, Slug and Sox9, that act cooperatively to determine the mammary stem cell (MaSC) state. Inhibition of either Slug or Sox9 blocks MaSC activity in primary mammary epithelial cells. Conversely, transient coexpression of exogenous Slug and Sox9 suffices to convert differentiated luminal cells into MaSCs with long-term mammary gland-reconstituting ability. Slug and Sox9 induce MaSCs by activating distinct autoregulatory gene expression programs. We also show that coexpression of Slug and Sox9 promotes the tumorigenic and metastasis-seeding abilities of human breast cancer cells and is associated with poor patient survival, providing direct evidence that human breast cancer stem cells are controlled by key regulators similar to those operating in normal murine MaSCs.
|The BMP Antagonist Follistatin-Like 1 Is Required for Skeletal and Lung Organogenesis. |
Sylva M, Li VS, Buffing AA, van Es JH, van den Born M, van der Velden S, Gunst Q, Koolstra JH, Moorman AF, Clevers H, van den Hoff MJ.
PloS one 6 e22616 2011
Follistatin-like 1 (Fstl1) is a secreted protein of the BMP inhibitor class. During development, expression of Fstl1 is already found in cleavage stage embryos and becomes gradually restricted to mesenchymal elements of most organs during subsequent development. Knock down experiments in chicken and zebrafish demonstrated a role as a BMP antagonist in early development. To investigate the role of Fstl1 during mouse development, a conditional Fstl1 KO allele as well as a Fstl1-GFP reporter mouse were created. KO mice die at birth from respiratory distress and show multiple defects in lung development. Also, skeletal development is affected. Endochondral bone development, limb patterning as well as patterning of the axial skeleton are perturbed in the absence of Fstl1. Taken together, these observations show that Fstl1 is a crucial regulator in BMP signalling during mouse development.
|SOX9 and SF1 are involved in cyclic AMP-mediated upregulation of anti-Mullerian gene expression in the testicular prepubertal Sertoli cell line |
Lasala C, Schteingart HF, Arouche N, Bedecarrás P, Grinspon RP, Picard JY, Josso N, di Clemente N, Rey RA
American journal of physiology Endocrinology and metabolism 301 E539-47. Epub 2011 Jun 21. 2011
In Sertoli cells, anti-Müllerian hormone (AMH) expression is upregulated by FSH via cyclic AMP (cAMP), although no classical cAMP response elements exist in the AMH promoter. The response to cAMP involves NF-κB and AP2; however, targeted mutagenesis of their binding sites in the AMH promoter do not completely abolish the response. In this work we assessed whether SOX9, SF1, GATA4, and AP1 might represent alternative pathways involved in cAMP-mediated AMH upregulation, using real-time RT-PCR (qPCR), targeted mutagenesis, luciferase assays, and immunocytochemistry in the Sertoli cell line SMAT1. We also explored the signaling cascades potentially involved. In qPCR experiments, Amh, Sox9, Sf1, and Gata4 mRNA levels increased after SMAT1 cells were incubated with cAMP. Blocking PKA abolished the effect of cAMP on Sox9, Sf1, and Gata4 expression, inhibiting PI3K/PKB impaired the effect on Sf1 and Gata4, and reducing MEK1/2 and p38 MAPK activities curtailed Gata4 increase. SOX9 and SF1 translocated to the nucleus after incubation with cAMP. Mutations of the SOX9 or SF1 sites, but not of GAT4 or AP1 sites, precluded the response of a 3,063-bp AMH promoter to cAMP. In conclusion, in the Sertoli cell line SMAT1 cAMP upregulates SOX9, SF1, and GATA4 expression and induces SOX9 and SF1 nuclear translocation mainly through PKA, although other kinases may also participate. SOX9 and SF1 binding to the AMH promoter is essential to increase the activity of the AMH promoter in response to cAMP.
|Extensive Pancreas Regeneration Following Acinar-Specific Disruption of Xbp1 in Mice. |
Hess DA, Humphrey SE, Ishibashi J, Damsz B, Lee AH, Glimcher LH, Konieczny SF
|Ascl1 expression defines a subpopulation of lineage-restricted progenitors in the mammalian retina. |
Brzezinski JA 4th, Kim EJ, Johnson JE, Reh TA
Development (Cambridge, England) 138 3519-31. Epub 2011 Jul 19. 2011
The mechanisms of cell fate diversification in the retina are not fully understood. The seven principal cell types of the neural retina derive from a population of multipotent progenitors during development. These progenitors give rise to multiple cell types concurrently, suggesting that progenitors are a heterogeneous population. It is thought that differences in progenitor gene expression are responsible for differences in progenitor competence (i.e. potential) and, subsequently, fate diversification. To elucidate further the mechanisms of fate diversification, we assayed the expression of three transcription factors made by retinal progenitors: Ascl1 (Mash1), Ngn2 (Neurog2) and Olig2. We observed that progenitors were heterogeneous, expressing every possible combination of these transcription factors. To determine whether this progenitor heterogeneity correlated with different cell fate outcomes, we conducted Ascl1- and Ngn2-inducible expression fate mapping using the CreER™/LoxP system. We found that these two factors gave rise to markedly different distributions of cells. The Ngn2 lineage comprised all cell types, but retinal ganglion cells (RGCs) were exceedingly rare in the Ascl1 lineage. We next determined whether Ascl1 prevented RGC development. Ascl1-null mice had normal numbers of RGCs and, interestingly, we observed that a subset of Ascl1+ cells could give rise to cells expressing Math5 (Atoh7), a transcription factor required for RGC competence. Our results link progenitor heterogeneity to different fate outcomes. We show that Ascl1 expression defines a competence-restricted progenitor lineage in the retina, providing a new mechanism to explain fate diversification.
|Blimp1 regulates the transition of neonatal to adult intestinal epithelium. |
Vanesa Muncan,Jarom Heijmans,Stephen D Krasinski,Nikè V Büller,Manon E Wildenberg,Sander Meisner,Marijana Radonjic,Kelly A Stapleton,Wout H Lamers,Izak Biemond,Marius A van den Bergh Weerman,Dónal O'Carroll,James C Hardwick,Daniel W Hommes,Gijs R van den Brink
Nature communications 2 2011
In many mammalian species, the intestinal epithelium undergoes major changes that allow a dietary transition from mother's milk to the adult diet at the end of the suckling period. These complex developmental changes are the result of a genetic programme intrinsic to the gut tube, but its regulators have not been identified. Here we show that transcriptional repressor B lymphocyte-induced maturation protein 1 (Blimp1) is highly expressed in the developing and postnatal intestinal epithelium until the suckling to weaning transition. Intestine-specific deletion of Blimp1 results in growth retardation and excessive neonatal mortality. Mutant mice lack all of the typical epithelial features of the suckling period and are born with features of an adult-like intestine. We conclude that the suckling to weaning transition is regulated by a single transcriptional repressor that delays epithelial maturation.Full Text Article
|Distinct Levels of Sox9 Expression Mark Colon Epithelial Stem Cells that Form Colonoids in Culture. |
Ramalingam S, Daughtridge GW, Johnston MJ, Gracz AD, Magness ST
American journal of physiology Gastrointestinal and liver physiology 2011
Sox9 is an HMG-box transcription factor that is expressed in the stem cell zone of the small intestine and colon. We have previously used a Sox9EGFP mouse model to demonstrate that discrete levels of Sox9 expression mark small intestine epithelial stem cells that form crypt/villus-like structures in a three-dimensional culture system. In the present study we hypothesized that discrete levels of Sox9 expression would also mark colonic epithelial stem cells (CESCs). Using the Sox9EGFP mouse model, we show that lower levels of Sox9 mark cells in the transit-amplifying progenitor cell zone while higher levels of Sox9 mark cells in the colonic crypt base. Further, we demonstrate that variable SOX9 levels persist in cells of colonic adenomas from mice and humans. Cells expressing lower Sox9 levels demonstrate gene expression profiles consistent with more differentiated populations, and cells expressing higher Sox9EGFP levels are consistent with less differentiated populations. Introducing FACS-isolated colonic epithelial cells into culturing conditions demonstrated that cells expressing the highest levels of Sox9EGFP formed \'colonoids\', which are defined as bodies of cultured colonic epithelial cells that possess multiple crypt-like structures and a pseudolumen. Cells expressing the highest levels of Sox9 also demonstrate multipotency and self-renewal in vitro, indicating functional stemness. These data suggest a dose-dependent role for Sox9 in normal CESCs and cells comprising colon tumors. Furthermore, distinct Sox9 levels represent a new biomarker to study CESC and progenitor biology in physiologic and disease states.
|The maturation of photoreceptors in the avian retina is stimulated by thyroid hormone. |
Fischer AJ, Bongini R, Bastaki N, Sherwood P
Neuroscience 178 250-60. Epub 2011 Jan 19. 2011
During retinal development, the cell-fate of photoreceptors is committed long before maturation, which entails the expression of opsins and functional transduction of light. The mechanisms that delay the maturation of photoreceptors remain unknown. We have recently reported that immature photoreceptors express the LIM domain transcription factors Islet2 and Lim3, as well as the cell-surface glycoprotein axonin1 [Fischer et al., (2008a) J Comp Neurol 506:584-603]. As the photoreceptors mature to form outer segments and express photopigments, the expression of the Islet2, Lim3 and axonin1 is diminished. The purpose of this study was to investigate whether thyroid hormone (TH) influences the maturation of photoreceptors. We studied the maturation of photoreceptors across the gradient of maturity that exists in far peripheral regions of the post-natal chicken retina [Ghai et al., (2008) Brain Res 1192:76-89]. We found that intraocular injections of TH down-regulated Islet2, Lim3 and axonin1 in photoreceptors in far peripheral regions of the retina. By contrast, TH stimulated the up-regulation of red-green opsin, violet opsin, rhodopsin and calbindin in photoreceptors. We found a correlation between the onset of RLIM (RING finger LIM-domain binding protein) and down-regulation of Islet2 and Lim3 in maturing photoreceptors; RLIM is known to interfere with the transcriptional activity of LIM-domain transcription factors. We conclude that TH stimulates the maturation of photoreceptors in the avian retina. We propose that TH inhibits the expression of Islet2 and Lim3, which thereby permits photoreceptor maturation and the onset of photopigment-expression.Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.Full Text Article
|FSH and Its Second Messenger cAMP Stimulate the Transcription of Human Anti-Mullerian Hormone in Cultured Granulosa Cells. |
Taieb J, Grynberg M, Pierre A, Arouche N, Massart P, Belville C, Hesters L, Frydman R, Catteau-Jonard S, Fanchin R, Picard JY, Josso N, Rey RA, di Clemente N
Mol Endocrinol 2011
Anti-Müllerian hormone (AMH), also called Müllerian-inhibiting substance, a member of the TGF-ß family, is responsible for the regression of Müllerian ducts in the male fetus. In females, AMH is synthesized by granulosa cells of preantral and small antral follicles, and production wanes at later stages of follicle maturation. Using RT-PCR in luteal granulosa cells in primary culture and reporter gene techniques in the KK1 granulosa cell line, we show that FSH and cAMP enhance AMH transcription, and LH has an additive effect. Gonadotropins and cAMP act through protein kinase A and p38 MAPK signaling pathways and involve the GATA binding factor-4 and steroidogenic factor-1 transcription factors, among others. The expression profile of AMH and the dynamics of serum AMH after gonadotropin stimulation have been interpreted as a down-regulating effect of FSH upon AMH production by granulosa cells. The specific effect of gonadotropins upon granulosa cells may be obscured in vivo by the effect of FSH upon follicular maturation and by the presence of other hormones and growth factors, acting individually or in concert.
|Ras mutation cooperates with ?-catenin activation to drive bladder tumourigenesis. |
I Ahmad,R Patel,Y Liu,L B Singh,M M Taketo,X-R Wu,H Y Leung,O J Sansom
Cell death & disease 2 2011
Mutations in the Ras family of proteins (predominantly in H-Ras) occur in approximately 40% of urothelial cell carcinoma (UCC). However, relatively little is known about subsequent mutations/pathway alterations that allow tumour progression. Indeed, expressing mutant H-Ras within the mouse bladder does not lead to tumour formation, unless this is expressed at high levels. The Wnt signalling pathway is deregulated in approximately 25% of UCC, so we examined if this correlated with the activation of MAPK signalling in human UCC and found a significant correlation. To test the functional significance of this association we examined the impact of combining Ras mutation (H-Ras(Q61L) or K-Ras(G12D)) with an activating ?-catenin mutation within the mouse bladder using Cre-LoxP technology. Although alone, neither Ras mutation nor ?-catenin activation led to UCC (within 12 months), mice carrying both mutations rapidly developed UCC. Mechanistically this was associated with reduced levels of p21 with dependence on the MAPK signalling pathway. Moreover, tumours from these mice were sensitive to MEK inhibition. Importantly, in human UCC there was a negative correlation between levels of p-ERK and p21 suggesting that p21 accumulation may block tumour progression following Ras mutation. Taken together these data definitively show Ras pathway activation strongly cooperates with Wnt signalling to drive UCC in vivo.Full Text Article
|Distinct ATOH1 and Neurog3 requirements define tuft cells as a new secretory cell type in the intestinal epithelium. |
Gerbe F, van Es JH, Makrini L, Brulin B, Mellitzer G, Robine S, Romagnolo B, Shroyer NF, Bourgaux JF, Pignodel C, Clevers H, Jay P
J Cell Biol 192 767-80. 2011
The unique morphology of tuft cells was first revealed by electron microscopy analyses in several endoderm-derived epithelia. Here, we explore the relationship of these cells with the other cell types of the intestinal epithelium and describe the first marker signature allowing their unambiguous identification. We demonstrate that although mature tuft cells express DCLK1, a putative marker of quiescent stem cells, they are post-mitotic, short lived, derive from Lgr5-expressing epithelial stem cells, and are found in mouse and human tumors. We show that whereas the ATOH1/MATH1 transcription factor is essential for their differentiation, Neurog3, SOX9, GFI1, and SPDEF are dispensable, which distinguishes these cells from enteroendocrine, Paneth, and goblet cells, and raises from three to four the number of secretory cell types in the intestinal epithelium. Moreover, we show that tuft cells are the main source of endogenous intestinal opioids and are the only epithelial cells that express cyclooxygenase enzymes, suggesting important roles for these cells in the intestinal epithelium physiopathology.Full Text Article
|Pax6-positive müller glia cells express cell cycle markers but do not proliferate after photoreceptor injury in the mouse retina. |
Joly S, Pernet V, Samardzija M, Grimm C
|An EGFR-ERK-SOX9 signaling cascade links urothelial development and regeneration to cancer. |
Ling S, Chang X, Schultz L, Lee TK, Chaux A, Marchionni L, Netto GJ, Sidransky D, Berman DM
Cancer research 71 3812-21. Epub 2011 Apr 21. 2011
Like many carcinomas, urothelial carcinoma (UroCa) is associated with chronic injury. A better understanding of this association could inform improved strategies for preventing and treating this disease. We investigated the expression, regulation, and function of the transcriptional regulator SRY-related high-mobility group box 9 (Sox9) in urothelial development, injury repair, and cancer. In mouse bladders, Sox9 levels were high during periods of prenatal urothelial development and diminished with maturation after birth. In adult urothelial cells, Sox9 was quiescent but was rapidly induced by a variety of injuries, including exposure to the carcinogen cyclophosphamide, culture with hydrogen peroxide, and osmotic stress. Activation of extracellular signal-regulated kinases 1/2 (ERK1/2) was required for Sox9 induction in urothelial injury and resulted from activation of the epidermal growth factor receptor (Egfr) by several Egfr ligands that were dramatically induced by injury. In UroCa cell lines, SOX9 expression was constitutively upregulated and could be suppressed by EGFR or ERK1/2 blockade. Gene knockdown showed a role for SOX9 in cell migration and invasion. Accordingly, SOX9 protein levels were preferentially induced in invasive human UroCa tissue samples (n = 84) compared with noninvasive cancers (n = 56) or benign adjacent urothelium (n = 49). These results identify a novel, potentially oncogenic signaling axis linking urothelial injury to UroCa. Inhibiting this axis is feasible through a variety of pharmacologic approaches and may have clinical utility.
|Pleiotropic role for MYCN in medulloblastoma. |
Swartling FJ, Grimmer MR, Hackett CS, Northcott PA, Fan QW, Goldenberg DD, Lau J, Masic S, Nguyen K, Yakovenko S, Zhe XN, Flynn Gilmer HC, Collins R, Nagaoka M, Phillips JJ, Jenkins RB, Tihan T, Vandenberg SR, James CD, Tanaka K, Taylor MD, Weiss WA, Chesler L
Genes Dev 24 1059-72. 2010
Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Sonic Hedgehog (SHH) signaling drives a minority of MB, correlating with desmoplastic pathology and favorable outcome. The majority, however, arises independently of SHH and displays classic or large cell anaplastic (LCA) pathology and poor prognosis. To identify common signaling abnormalities, we profiled mRNA, demonstrating misexpression of MYCN in the majority of human MB and negligible expression in normal cerebella. We clarified a role in pathogenesis by targeting MYCN (and luciferase) to cerebella of transgenic mice. MYCN-driven MB showed either classic or LCA pathologies, with Shh signaling activated in approximately 5% of tumors, demonstrating that MYCN can drive MB independently of Shh. MB arose at high penetrance, consistent with a role for MYCN in initiation. Tumor burden correlated with bioluminescence, with rare metastatic spread to the leptomeninges, suggesting roles for MYCN in both progression and metastasis. Transient pharmacological down-regulation of MYCN led to both clearance and senescence of tumor cells, and improved survival. Targeted expression of MYCN thus contributes to initiation, progression, and maintenance of MB, suggesting a central role for MYCN in pathogenesis.
|Sox9-Expression Marks a Subset of CD24-expressing Small Intestine Epithelial Stem Cells that Form Organoids in vitro. |
Gracz AD, Ramalingam S, Magness ST
American journal of physiology. Gastrointestinal and liver 298 G590-600 Epub 2010 Feb 25 2010
The inability to identify, isolate, and culture intestinal epithelial stem cells (IESCs) has been prohibitive to the study and therapeutic utilization of these cells. Using a Sox9(EGFP) mouse model, we demonstrate that Sox9(EGFP) fluorescence signatures can be used to differentiate between and enrich for progenitors (Sox9(EGFPsubLo)) and multipotent IESCs (Sox9(EGFPlo)). Sox9(EGFPlo) cells generate "organoids" in a recently defined culture system that mimics the native IESC niche. These organoids possess all four differentiated cell types of the small intestine epithelium, demonstrating the multipotent capacity of Sox9(EGFPlo) cells. Our results are consistent with the previously reported observation that single IESCs generate cryptlike units without a detectable mesenchymal cell component. A prospective search revealed that CD24 is expressed in the Sox9(EGFPlo) population and marks IESCs that form organoids in culture. CD24 represents the first cell surface marker that facilitates fluorescence-activated cell sorting enrichment of IESCs with widely available antibodies without requiring a specialized fluorescent reporter gene mouse model.
|Inhibition of hypoxia-inducible factor-targeting prolyl hydroxylase domain-containing protein 2 (PHD2) enhances matrix synthesis by human chondrocytes. |
Brendan L Thoms,Christopher L Murphy
The Journal of biological chemistry 285 2010
Human articular cartilage is an avascular tissue, and therefore it functions in a hypoxic environment. Cartilage cells, the chondrocytes, have adapted to this and actually use hypoxia to drive tissue-specific functions. We have previously shown that human chondrocytes enhance cartilage matrix synthesis in response to hypoxia specifically through hypoxia-inducible factor 2alpha (HIF-2alpha)-mediated up-regulation of master regulator transcription factor SOX9, which in turn drives expression of the main cartilage-specific extracellular matrix genes. HIF-alpha isoforms are themselves regulated by specific prolyl hydroxylase domain-containing proteins, which target them for proteosomal degradation. In fact, prolyl hydroxylase domains are the direct oxygen sensors because they require molecular oxygen as a co-substrate. Here, we have identified PHD2 as the dominant isoenzyme regulating HIF-2alpha stability in human chondrocytes. Moreover, specific inhibition of PHD2 using RNA interference-mediated depletion caused an up-regulation of SOX9 and enhanced extracellular matrix protein production. Depletion of PHD2 resulted in greater HIF-2alpha levels and therefore enhanced SOX9-induced cartilage matrix production compared with the levels normally found in hypoxia (1% oxygen) implying that PHD2 inhibition offers a novel means to enhance cartilage repair in vivo. The need for HIF-specific hydroxylase inhibition was highlighted because treatment with the 2-oxoglutarate analogue dimethyloxalylglycine (which also inhibits the collagen prolyl 4-hydroxylases) prevented secretion of type II collagen, a critical cartilage matrix component.Full Text Article
|Deficiency of splicing factor 1 suppresses the occurrence of testicular germ cell tumors. |
Zhu R, Heaney J, Nadeau JH, Ali S, Matin A
Cancer Res 70 7264-72. Epub 2010 Aug 24. 2010
Testicular germ cell tumors (TGCT) originate from germ cells. The 129-Ter and M19 (129.MOLF-Chr19 consomic) mouse strains have extremely high incidences of TGCTs. We found that the expression levels of Sf1-encoded splicing factor 1 (SF1) can modulate the incidence of TGCTs. We generated mice with inactivated Sf1. Sf1 null mice (Sf1-/-) died before birth. Mice with one intact allele of Sf1 (Sf1+/-) were viable but expressed reduced levels of Sf1. When Sf1-deficient mice (Sf1+/-) were crossed to the 129-Ter and M19 strains, we observed decreased incidence of TGCTs in Sf1+/-;Ter and Sf1+/-;M19/+ mice compared with that in control cohorts. Therefore, Sf1 deficiency protects against TGCT development in both strains. Sf1 is expressed in the testes. We found that Sf1 levels vary significantly in the testes of inbred strains such as 129 and MOLF, and as such Sf1 is an oncogenic tumor-susceptibility factor from 129. Our results also highlight the complications involved in evaluating Sf1 levels and TGCT incidences. When a large number of tumor-promoting factors are present in a strain, the protective effect of lower Sf1 levels is masked. However, when the dosage of tumor-promoting factors is reduced, the protective effect of lower Sf1 levels becomes apparent. SF1 is involved in splicing of specific pre-mRNAs in cells. Alternate splicing generates the complex proteosome in eukaryotic cells. Our data indicate that Sf1 levels in mouse strains correlate with their incidences of TGCTs and implicate the importance of splicing mechanisms in germ cell tumorigenesis.Full Text Article
|Oestrogen blocks the nuclear entry of SOX9 in the developing gonad of a marsupial mammal. |
Andrew J Pask,Natalie E Calatayud,Geoff Shaw,William M Wood,Marilyn B Renfree
BMC biology 8 2010
Hormones are critical for early gonadal development in nonmammalian vertebrates, and oestrogen is required for normal ovarian development. In contrast, mammals determine sex by the presence or absence of the SRY gene, and hormones are not thought to play a role in early gonadal development. Despite an XY sex-determining system in marsupial mammals, exposure to oestrogen can override SRY and induce ovarian development of XY gonads if administered early enough. Here we assess the effect of exogenous oestrogen on the molecular pathways of mammalian gonadal development.Full Text Article
|Increased expression of FoxM1 transcription factor in respiratory epithelium inhibits lung sacculation and causes Clara cell hyperplasia. |
I-Ching Wang,Yufang Zhang,Jonathan Snyder,Mardi J Sutherland,Michael S Burhans,John M Shannon,Hyun Jung Park,Jeffrey A Whitsett,Vladimir V Kalinichenko
Developmental biology 347 2010
Foxm1 is a member of the Forkhead Box (Fox) family of transcription factors. Foxm1 (previously called Foxm1b, HFH-11B, Trident, Win, or MPP2) is expressed in multiple cell types and plays important roles in cellular proliferation, differentiation and tumorigenesis. Genetic deletion of Foxm1 from mouse respiratory epithelium during initial stages of lung development inhibits lung maturation and causes respiratory failure after birth. However, the role of Foxm1 during postnatal lung morphogenesis remains unknown. In the present study, Foxm1 expression was detected in epithelial cells of conducting and peripheral airways and changing dynamically with lung maturation. To discern the biological role of Foxm1 in the prenatal and postnatal lung, a novel transgenic mouse line that expresses a constitutively active form of FoxM1 (FoxM1 N-terminal deletion mutant or FoxM1-?N) under the control of lung epithelial-specific SPC promoter was produced. Expression of the FoxM1-?N transgene during embryogenesis caused epithelial hyperplasia, inhibited lung sacculation and expression of the type II epithelial marker, pro-SPC. Expression of FoxM1-?N mutant during the postnatal period did not influence alveologenesis but caused focal airway hyperplasia and increased proliferation of Clara cells. Likewise, expression of FoxM1-?N mutant in conducting airways with Scgb1a1 promoter was sufficient to induce Clara cell hyperplasia. Furthermore, FoxM1-?N cooperated with activated K-Ras to induce lung tumor growth in vivo. Increased activity of Foxm1 altered lung sacculation, induced proliferation in the respiratory epithelium and accelerated lung tumor growth, indicating that precise regulation of Foxm1 is critical for normal lung morphogenesis and development of lung cancer.Full Text Article
|Expression of master regulatory genes controlling skeletal development in benign cartilage and bone forming tumors. |
Jane Y Dancer,Stephen P Henry,Jolanta Bondaruk,Sangkyou Lee,Alberto G Ayala,Benoit de Crombrugghe,Bogdan Czerniak
Human pathology 41 2010
Recent progress in skeletal molecular biology has led to the clarification of the transcriptional mechanisms of chondroblastic and osteoblastic lineage differentiation. Three master transcription factors-Sox9, Runx2, and Osterix-were shown to play an essential role in determining the skeletal progenitor cells' fate. The present study evaluates the expression of these factors in 4 types of benign bone tumors-chondromyxoid fibroma, chondroblastoma, osteoid osteoma, and osteoblastoma-using immunohistochemistry and tissue microarrays. Osteoid osteoma and osteoblastoma showed strong nuclear expression of Osterix and Runx2. In contrast, only a few chondroblastomas showed positive nuclear expression of Osterix. Strong nuclear expression of Sox9 was detected in all chondroblastomas, whereas nearly half of the osteoblastomas showed focal weak cytoplasmic expression of Sox9.
|SOX9, through interaction with microphthalmia-associated transcription factor (MITF) and OTX2, regulates BEST1 expression in the retinal pigment epithelium. |
Tomohiro Masuda,Noriko Esumi
The Journal of biological chemistry 285 2010
BEST1 is highly and preferentially expressed in the retinal pigment epithelium (RPE) and causes Best macular dystrophy when mutated. We previously demonstrated that the human BEST1 upstream region -154 to +38 bp is sufficient to direct expression in the RPE of transgenic mice, and microphthalmia-associated transcription factor (MITF) and OTX2 regulate this BEST1 promoter. However, a number of questions remained. Here, we show that yeast one-hybrid screen with bait corresponding to BEST1 -120 to -88 bp identified the SOX-E factors, SOX8, SOX9, and SOX10. A paired SOX site was found in this bait, and mutation of either of the paired sites significantly decreased BEST1 promoter activity in RPE primary cultures. Among the SOX-E genes, SOX9 is highly and preferentially expressed in the RPE, and chromatin immunoprecipitation with fresh RPE cells revealed binding of SOX9, but not SOX10, to the BEST1 region where the paired SOX site is located. BEST1 promoter activity was increased by SOX9 overexpression and decreased by siRNA-mediated SOX9 knockdown. Importantly, SOX9 physically interacted with MITF and OTX2 and orchestrated synergistic activation of the BEST1 promoter with the paired SOX site playing essential roles. A combination of the expression patterns of SOX9, MITF, and OTX2 yielded tissue distribution remarkably similar to that of BEST1. Lastly, the BEST1 promoter was also active in Sertoli cells of the testis in transgenic mice where SOX9 is highly expressed. These results define SOX9 as a key regulator of BEST1 expression and demonstrate for the first time its functional role in the RPE.Full Text Article
|Upregulation of SOX9 in lung adenocarcinoma and its involvement in the regulation of cell growth and tumorigenicity. |
Jiang SS, Fang WT, Hou YH, Huang SF, Yen BL, Chang JL, Li SM, Liu HP, Liu YL, Huang CT, Li YW, Jang TH, Chan SH, Yang SJ, Hsiung CA, Wu CW, Wang LH, Chang IS
Clin Cancer Res 16 4363-73. Epub 2010 Jul 22. 2010
PURPOSE: SOX9 is an important transcription factor required for development and has been implicated in several types of cancer. However, SOX9 has never been linked to lung cancer to date. Here, we show that SOX9 expression is upregulated in lung adenocarcinoma and show how it is associated with cancer cell growth.
|Antitumoural immunity by virus-mediated immunogenic apoptosis inhibits metastatic growth of hepatocellular carcinoma. |
Boozari B, Mundt B, Woller N, Strüver N, Gürlevik E, Schache P, Kloos A, Knocke S, Manns MP, Wirth TC, Kubicka S, Kühnel F
Background and aims Viral infection of a dying cell dictates the immune response against intracellular antigens, suggesting that virotherapy may be an effective tool to induce immunogenic cell death during systemic cancer treatment. Since viruses and proteasome inhibitors both induce accumulation of misfolded proteins, endoplasmic reticulum (ER) stress and immune responses during treatment of hepatocellular carcinoma (HCC) with bortezomib and the tumour-specifically replicating virus hTert-Ad (human telomerase reverse transcriptase promoter-regulated adenovirus) were investigated. Methods Unfolded protein response (UPR) pathways and ER stress-mediated apoptosis were investigated by western blots, caspase-3 assays, 4',6-diamidino-2-phenylindole (DAPI) and Annexin V staining in HCC cells following hTert-Ad/bortezomib treatment. Oncolysis was assessed in subcutaneous HCC mouse models. Antiviral/antitumoural immune responses were characterised in immunocompetent HCC mouse models by ELISA, ELISpot assays and pentamer staining. Systemic efficacy of antitumoural immunity was investigated by determination of lung metastases burden. Results Bortezomib and hTert-Ad trigger complementary UPR pathways but negatively interfere with important recovery checkpoints, resulting in enhanced apoptosis of HCC cells in vitro and improved oncolysis in vivo. In immunocompetent mice, bortezomib inhibited antiviral immune responses, whereas ER stress-induced apoptosis of infected HCC resulted in caspase-dependent triggering of antitumoural immunity. In therapeutic settings in immunocompetent, but not in immunodeficient or CD8-depleted mice, virotherapy-induced antitumoural immunity efficiently inhibited outgrowth of non-infected lung metastases. Immunotherapeutic efficacy could be significantly improved by bortezomib in experiments with low viral doses. Conclusion Proteasome inhibition during virotherapy disrupts the UPR, leading to enhanced ER stress-induced apoptosis, improved local oncolysis and antitumoural immunity. The results suggest that combining intratumoural virotherapy with adjuvant systemic therapies, which specifically support the function of the virotherapy as an antitumoural vaccine, is a promising immunotherapeutic strategy against HCC.
|Upregulation of SOX9 inhibits the growth of human and mouse melanomas and restores their sensitivity to retinoic acid. |
Thierry Passeron,Julio C Valencia,Takeshi Namiki,Wilfred D Vieira,Hélène Passeron,Yoshinori Miyamura,Vincent J Hearing
The Journal of clinical investigation 119 2009
Treatments for primary and metastatic melanomas are rarely effective. Even therapeutics such as retinoic acid (RA) that are successfully used to treat several other forms of cancer are ineffective. Recent evidence indicates that the antiproliferative effects of RA are mediated by the transcription factor SOX9 in human cancer cell lines. As we have previously shown that SOX9 is expressed in normal melanocytes, here we investigated SOX9 expression and function in human melanomas. Although SOX9 was expressed in normal human skin, it was increasingly downregulated as melanocytes progressed to the premalignant and then the malignant and metastatic states. Overexpression of SOX9 in both human and mouse melanoma cell lines induced cell cycle arrest by increasing p21 transcription and restored sensitivity to RA by downregulating expression of PRAME, a melanoma antigen. Furthermore, SOX9 overexpression in melanoma cell lines inhibited tumorigenicity both in mice and in a human ex vivo model of melanoma. Treatment of melanoma cell lines with PGD2 increased SOX9 expression and restored sensitivity to RA. Thus, combined treatment with PGD2 and RA substantially decreased tumor growth in human ex vivo and mouse in vivo models of melanoma. The results of our experiments targeting SOX9 provide insight into the pathophysiology of melanoma. Further, the effects of SOX9 on melanoma cell proliferation and RA sensitivity suggest the encouraging possibility of a noncytotoxic approach to the treatment of melanoma.Full Text Article
|Intrahepatic bile ducts develop according to a new mode of tubulogenesis regulated by the transcription factor SOX9. |
Antoniou, Aline, et al.
Gastroenterology, 136: 2325-33 (2009) 2009
BACKGROUND & AIMS: A number of diseases are characterized by defective formation of the intrahepatic bile ducts. In the embryo, hepatoblasts differentiate to cholangiocytes, which give rise to the bile ducts. Here, we investigated duct development in mouse liver and characterized the role of the SRY-related HMG box transcription factor 9 (SOX9). METHODS: We identified SOX9 as a new biliary marker and used it in immunostaining experiments to characterize bile duct morphogenesis. The expression of growth factors was determined by in situ hybridization and immunostaining, and their role was studied on cultured hepatoblasts. SOX9 function was investigated by phenotyping mice with a liver-specific inactivation of Sox9. RESULTS: Biliary tubulogenesis started with formation of asymmetrical ductal structures, lined on the portal side by cholangiocytes and on the parenchymal side by hepatoblasts. When the ducts grew from the hilum to the periphery, the hepatoblasts lining the asymmetrical structures differentiated to cholangiocytes, thereby allowing formation of symmetrical ducts lined only by cholangiocytes. We also provide evidence that transforming growth factor-beta promotes differentiation of the hepatoblasts lining the asymmetrical structures. In the absence of SOX9, the maturation of asymmetrical structures into symmetrical ducts was delayed. This was associated with abnormal expression of CCAAT/Enhancer Binding Protein alpha and Homolog of Hairy/Enhancer of Split-1, as well as of the transforming growth factor-beta receptor type II, which are regulators of biliary development. CONCLUSIONS: Our results suggest that biliary development proceeds according to a new mode of tubulogenesis characterized by transient asymmetry and whose timing is controlled by SOX9.
|Derivation of a novel undifferentiated human foetal phenotype in serum-free cultures with BMP-2. |
Mirmalek-Sani SH, Stokes PJ, Tare RS, Ralph EJ, Inglis S, Hanley NA, Houghton FD, Oreffo RO
J Cell Mol Med 13 3541-55. Epub 2009 Mar 6. 2009
Skeletal stem and progenitor populations provide a platform for cell-based tissue regeneration strategies. Optimized conditions for ex vivo expansion will be critical and use of serum-free culture may allow enhanced modelling of differentiation potential. Maintenance of human foetal femur-derived cells in a chemically defined medium (CDM) with activin A and fibroblast growth factor-2 generated a unique undifferentiated cell population in comparison to basal cultures, with significantly reduced amino acid depletion, appearance and turnover, reduced alkaline phosphatase (ALP) activity and loss of type I and II collagen expression demonstrated by fluorescence immunocytochemistry. Microarray analysis demonstrated up-regulation of CLU, OSR2, POSTN and RABGAP1 and down-regulation of differentiation-associated genes CRYAB, CSRP1, EPAS1, GREM1, MT1X and SRGN as validated by quantitative real-time polymerase chain reaction. Application of osteogenic conditions to CDM cultures demonstrated partial rescue of ALP activity. In contrast, the addition of bone morphogenetic protein-2 (BMP-2) resulted in reduced ALP levels, increased amino acid metabolism and, strikingly, a marked shift to a cobblestone-like cellular morphology, with expression of SOX-2 and SOX-9 but not STRO-1 as shown by immunocytochemistry, and significantly altered expression of metabolic genes (GFPT2, SC4MOL and SQLE), genes involved in morphogenesis (SOX15 and WIF1) and differentiation potential (C1orf19, CHSY-2,DUSP6, HMGCS1 and PPL). These studies demonstrate the use of an intermediary foetal cellular model for differentiation studies in chemically defined conditions and indicate the in vitro reconstruction of the mesenchymal condensation phenotype in the presence of BMP-2, with implications therein for rescue studies, screening assays and skeletal regeneration research.
|Cyclic GMP-dependent protein kinase II inhibits cell proliferation, Sox9 expression and Akt phosphorylation in human glioma cell lines. |
F J Swartling, M Ferletta, M Kastemar, W A Weiss, B Westermark, F J Swartling, M Ferletta, M Kastemar, W A Weiss, B Westermark
Oncogene 28 3121-31 2009
Earlier we used a glioma model to identify loci in the mouse genome, which were repeatedly targeted by platelet-derived growth factor (PDGF)-containing Moloney murine leukemia viruses. The gene Prkg2, encoding cyclic guanosine monophosphate (cGMP)-dependent protein kinase II, cGKII, was tagged by retroviral insertions in two brain tumors. The insertions were both situated upstream of the kinase domain and suggested creating a truncated form of the cGKII protein. We transfected different human glioma cell lines with Prkg2 and found an overall reduction in colony formation and cell proliferation compared with controls transfected with truncated Prkg2 (lacking the kinase domain) or empty vector. All glioma cells transfected with the cGKII phosphorylate vasodilator-stimulated phosphoprotein, VASP, after cGMP analog treatment. Glioma cell lines positive for the Sox9 transcription factor showed reduced Sox9 expression when Prkg2 was stably transfected. When cGKII was activated by cGMP analog treatment, Sox9 was phosphorylated, Sox9 protein expression was suppressed and the glioma cell lines displayed loss of cell adhesion, inhibition of Akt phosphorylation and G1 arrest. Sox9 repression by siRNA was similarly shown to reduce glioma cell proliferation. Expression analysis of stem and glial lineage cell markers also suggests that cGKII induces differentiation of glioma cell lines. These findings describe an anti-proliferative role of cGKII in human glioma biology and would further explain the retroviral tagging of the cGKII gene during brain tumor formation in PDGF-induced tumors.
|BDNF/MAPK/ERK-Induced BMP7 Expression in the Developing Cerebral Cortex Induces Premature Radial Glia Differentiation and Impairs Neuronal Migration. |
Ortega JA, AlcÃ¡ntara S
Cereb Cortex 2009
During development of the mammalian nervous system, a combination of genetic and environmental factors governs the sequential generation of neurons and glia and the initial establishment of the neural circuitry. Here, we demonstrate that brain-derived neurotrophic factor (BDNF), one of those local acting factors, induces Bone Morphogenetic Protein 7 (BMP7) expression in embryonic neurons by activating Mitogen-Activated Protein Kinase/Extracellular signal-Regulated Kinase signaling and by the negative regulation of p53/p73 function. We also show that intraventricular injection of BMP7 at midgestation induces the early differentiation of radial glia into glial precursors and astrocytes and the expression of mature glial markers such as the antiadhesive protein SC1. As a result of this precocious radial glia maturation, the laminar distribution of late-born pyramidal neurons is altered, most likely by the termination of radial glia ability to support neuronal migration and the early neuronal detachment from the glial rail. We propose a mechanism for BDNF regulation of BMP7 in which local activity-driven BDNF-induced BMP7 expression at the end of neurogenesis instructs competent precursors to generate astrocytes. Such a mechanism might ensure synchronic neuronal and glial maturation at the beginning of cortical activity.
|Epistasis between RET and BBS mutations modulates enteric innervation and causes syndromic Hirschsprung disease. |
de Pontual L, Zaghloul NA, Thomas S, Davis EE, McGaughey DM, Dollfus H, Baumann C, Bessling SL, Babarit C, Pelet A, Gascue C, Beales P, Munnich A, Lyonnet S, Etchevers H, Attie-Bitach T, Badano JL, McCallion AS, Katsanis N, Amiel J
Proceedings of the National Academy of Sciences of the United States of America 106 13921-6 2009
Hirschsprung disease (HSCR) is a common, multigenic neurocristopathy characterized by incomplete innervation along a variable length of the gut. The pivotal gene in isolated HSCR cases, either sporadic or familial, is RET. HSCR also presents in various syndromes, including Shah-Waardenburg syndrome (WS), Down (DS), and Bardet-Biedl (BBS). Here, we report 3 families with BBS and HSCR with concomitant mutations in BBS genes and regulatory RET elements, whose functionality is tested in physiologically relevant assays. Our data suggest that BBS mutations can potentiate HSCR predisposing RET alleles, which by themselves are insufficient to cause disease. We also demonstrate that these genes interact genetically in vivo to modulate gut innervation, and that this interaction likely occurs through complementary, yet independent, pathways that converge on the same biological process.Full Text Article
|Hypoxia promotes the differentiated human articular chondrocyte phenotype through SOX9-dependent and -independent pathways. |
Jérôme E Lafont, Sonia Talma, Christine Hopfgarten, Christopher L Murphy
The Journal of biological chemistry 283 4778-86 2008
The chondrocyte is solely responsible for synthesis and maintenance of the resilient articular cartilage matrix that gives this load-bearing tissue its mechanical integrity. When the differentiated cell phenotype is lost, the matrix becomes compromised and cartilage function begins to fail. We have recently shown that hypoxia promotes the differentiated phenotype through hypoxia-inducible factor 2alpha (HIF-2alpha)-mediated SOX9 induction of the main matrix genes. However, to date, only a few genes have been shown to be SOX9 targets, while little is known about SOX9-independent regulators. We therefore performed a detailed microarray study to address these issues. Analysis involved 35 arrays on chondrocytes obtained from seven healthy, non-elderly human cartilage samples. Genes were selected that were down-regulated with serial passage in culture (as this causes loss of the differentiated phenotype) and subsequently up-regulated in hypoxia. The importance of key findings was further probed using the technique of RNA interference on these human articular chondrocytes. Our results show that hypoxia has a broader beneficial effect on the chondrocyte phenotype than has been previously described. Of special note, we report new hypoxia-inducible and SOX9-regulated genes, Gdf10 and Chm-I. In addition, Mig6 and InhbA were induced by hypoxia, predominantly via HIF-2alpha, but were not regulated by SOX9. Therefore, hypoxia, and more specifically HIF-2alpha, promotes both SOX9-dependent and -independent factors important for cartilage homeostasis. HIF-2alpha may therefore represent a new and promising therapeutic target for cartilage repair.
|Components of the CCR4-NOT complex function as nuclear hormone receptor coactivators via association with the NRC-interacting Factor NIF-1. |
Shivani Garapaty, Muktar A Mahajan, Herbert H Samuels
The Journal of biological chemistry 283 6806-16 2008
CCR4-NOT is an evolutionarily conserved, multicomponent complex known to be involved in transcription as well as mRNA degradation. Various subunits (e.g. CNOT1 and CNOT7/CAF1) have been reported to be involved in influencing nuclear hormone receptor activities. Here, we show that CCR4/CNOT6 and RCD1/CNOT9, members of the CCR4-NOT complex, potentiate nuclear receptor activity. RCD1 interacts in vivo and in vitro with NIF-1 (NRC-interacting factor), a previously characterized nuclear receptor cotransducer that activates nuclear receptors via its interaction with NRC. As with NIF-1, RCD1 and CCR4 do not directly associate with nuclear receptors; however, they enhance ligand-dependent transcriptional activation by nuclear hormone receptors. CCR4 mediates its effect through the ligand binding domain of nuclear receptors and small interference RNA-mediated silencing of endogenous CCR4 results in a marked decrease in nuclear receptor activation. Furthermore, knockdown of CCR4 results in an attenuated stimulation of RARalpha target genes (e.g. Sox9 and HoxA1) as shown by quantitative PCR assays. The silencing of endogenous NIF-1 also resulted in a comparable decrease in the RAR-mediated induction of both Sox9 and HoxA1. Furthermore, CCR4 associates in vivo with NIF-1. In addition, the CCR4-enhanced transcriptional activation by nuclear receptors is dependent on NIF-1. The small interference RNA-mediated knockdown of NIF-1 blocks the ligand-dependent potentiating effect of CCR4. Our results suggest that CCR4 plays a role in the regulation of certain endogenous RARalpha target genes and that RCD1 and CCR4 might mediate their function through their interaction with NIF-1.
|Expression patterns of epiplakin1 in pancreas, pancreatic cancer and regenerating pancreas. |
Tetsu Yoshida,Nobuaki Shiraki,Hideo Baba,Mizuki Goto,Sakuhei Fujiwara,Kazuhiko Kume,Shoen Kume
Genes to cells : devoted to molecular & cellular mechanisms 13 2008
Epiplakin1 (Eppk1) is a plakin family gene with its function remains largely unknown, although the plakin genes are known to function in interconnecting cytoskeletal filaments and anchoring them at plasma membrane-associated adhesive junction. Here we analyzed the expression patterns of Eppk1 in the developing and adult pancreas in the mice. In the embryonic pancreas, Eppk1+/Pdx1+ and Eppk1+/Sox9+ pancreatic progenitor cells were observed in early pancreatic epithelium. Since Pdx1 expression overlapped with that of Sox9 at this stage, these multipotent progenitor cells are Eppk1+/Pdx1+/Sox9+ cells. Then Eppk1 expression becomes confined to Ngn3+ or Sox9+ endocrine progenitor cells, and p48+ exocrine progenitor cells, and then restricted to the duct cells and a cells at birth. In the adult pancreas, Eppk1 is expressed in centroacinar cells (CACs) and in duct cells. Eppk1 is observed in pancreatic intraepithelial neoplasia (PanIN), previously identified as pancreatic ductal adenocarcinoma (PDAC) precursor lesions. In addition, the expansion of Eppk1-positive cells occurs in a caerulein-induced acute pancreatitis, an acinar cell regeneration model. Furthermore, in the partial pancreatectomy (Px) regeneration model using mice, Eppk1 is expressed in ducts in foci, a tubular structure transiently induced. These results suggest that Eppk1 serves as a useful marker for detecting pancreatic progenitor cells in developing and regenerating pancreas.
|L-Sox5 and Sox6 drive expression of the aggrecan gene in cartilage by securing binding of Sox9 to a far-upstream enhancer. |
Yu Han, Véronique Lefebvre, Yu Han, Véronique Lefebvre, Yu Han, Véronique Lefebvre
Molecular and cellular biology 28 4999-5013 2008
The Sry-related high-mobility-group box transcription factor Sox9 recruits the redundant L-Sox5 and Sox6 proteins to effect chondrogenesis, but the mode of action of the trio remains unclear. We identify here a highly conserved 359-bp sequence 10 kb upstream of the Agc1 gene for aggrecan, a most essential cartilage proteoglycan and key marker of chondrocyte differentiation. This sequence directs expression of a minimal promoter in both embryonic and adult cartilage in transgenic mice, in a manner that matches Agc1 expression. The chondrogenic trio is required and sufficient to mediate the activity of this enhancer. It acts directly, Sox9 binding to a critical cis-acting element and L-Sox5/Sox6 binding to three additional elements, which are cooperatively needed. Upon binding to their specific sites, L-Sox5/Sox6 increases the efficiency of Sox9 binding to its own recognition site and thereby robustly potentiates the ability of Sox9 to activate the enhancer. L-Sox5/Sox6 similarly secures Sox9 binding to Col2a1 (encoding collagen-2) and other cartilage-specific enhancers. This study thus uncovers critical cis-acting elements and transcription factors driving Agc1 expression in cartilage and increases understanding of the mode of action of the chondrogenic Sox trio.Full Text Article
|A cellular study of human testis development. |
H Ostrer, H Y Huang, R J Masch, E Shapiro
Sexual development : genetics, molecular biology, evolution, endocrinology, embryology, and pathology of sex determination and differentiation 1 286-92 2007
This study catalogs the cellular events underlying the formation of a human testis. These events were identified by immunocytochemistry using antibodies that served as markers for specific cell types, then contrasted with the events occurring in the developing mouse testis. The presence of germ cells in the embryonic gonadal ridge and of coelomic epithelial cells that give rise to Sertoli cells was observed at 7 weeks. This was followed by the appearance of Sertoli cells in testicular tubules and of Leydig cells at 9 weeks and by the appearance of vascular endothelial cells and peritubular myoid cells at 12 weeks. Overall the temporal sequence of events in humans was similar, albeit longer, than what occurs in mice. Notably, Leydig cell differentiation occurs earlier in the sequence of events and germ cell maturation occurs during fetal life. The candidate testis-determining genes, FGF9, GATA4, FOG2, EMX2, and CBX2 were expressed at 7 weeks suggesting a role in early gonadal development, such as that observed in mice. In addition, expression of FGF9 in germ cells following testis determination suggests a role in germ cell maturation.
|The Wilms tumor gene, Wt1, is required for Sox9 expression and maintenance of tubular architecture in the developing testis. |
Fei Gao, Sourindra Maiti, Nargis Alam, Zhen Zhang, Jian Min Deng, Richard R Behringer, Charlotte Lécureuil, Florian Guillou, Vicki Huff
Proceedings of the National Academy of Sciences of the United States of America 103 11987-92 2006
Mutation of the transcription factor and tumor suppressor gene WT1 results in a range of genitourinary anomalies in humans, including 46,XY gonadal dysgenesis, indicating that WT1 plays a critical role in sex determination. However, because knockout of Wt1 in mice results in apoptosis of the genital ridge, it is unknown whether WT1 is required for testis development after the initial steps of sex determination. To address this question, we generated a mouse strain carrying a Wt1 conditional knockout allele and ablated Wt1 function specifically in Sertoli cells by embryonic day 14.5, several days after testis determination. Wt1 knockout resulted in disruption of developing seminiferous tubules and subsequent progressive loss of Sertoli cells and germ cells such that postnatal mutant testes were almost completely devoid of these cell types and were severely hypoplastic. Thus, Wt1 is essential for the maintenance of Sertoli cells and seminiferous tubules in the developing testes. Of particular note, expression of the testis-determining gene Sox9 in mutant Sertoli cells was turned off at embryonic day 14.5 after Wt1 ablation, suggesting that WT1 regulates Sox9, either directly or indirectly, after Sry expression ceases. Our data, along with previous work demonstrating the role of Wt1 at early stages of gonadal development, thus indicate that Wt1 is essential at multiple steps in testicular development.Full Text Article
|Characterization and multipotentiality of human fetal femur-derived cells: implications for skeletal tissue regeneration. |
Sayed-Hadi Mirmalek-Sani, Rahul S Tare, Suzanne M Morgan, Helmtrud I Roach, David I Wilson, Neil A Hanley, Richard O C Oreffo
Stem cells (Dayton, Ohio) 24 1042-53 2006
To date, the plasticity, multipotentiality, and characteristics of progenitor cells from fetal skeletal tissue remain poorly defined. This study has examined cell populations from human fetal femurs in comparison with adult-derived mesenchymal cell populations. Real-time quantitative polymerase chain reaction demonstrated expression of mesenchymal progenitor cell markers by fetal-derived cells in comparison with unselected adult-derived and immunoselected STRO-1-enriched adult populations. Multipotentiality was examined using cells derived from femurs and single-cell clones, culture-expanded from explants, and maintained in basal medium prior to exposure to adipogenic, osteogenic, and chondrogenic conditions. Adipocyte formation was confirmed by Oil Red O lipid staining and aP2 immunocytochemistry, with expression of peroxisome proliferation-activated receptor-gamma detected only in adipogenic conditions. In chondrogenic pellets, chondrocytes lodged within lacunae and embedded within dense proteoglycan matrix were observed using Alcian blue/Sirius red staining and type II collagen immunocytochemistry. Osteogenic differentiation was confirmed by alkaline phosphatase staining and type I collagen immunocytochemistry as well as by gene expression of osteopontin and osteocalcin. Single-cell clonal analysis was used to demonstrate multipotentiality of the fetal-derived populations with the formation of adipogenic, chondrogenic, and osteogenic populations. Mineralization and osteoid formation were observed after culture on biomimetic scaffolds with extensive matrix accumulation both in vitro and in vivo after subcutaneous implantation in severely compromised immunodeficient mice. These studies demonstrate the proliferative and multipotential properties of fetal femur-derived cells in comparison with adult-derived cells. Selective differentiation and immunophenotyping will determine the potential of these fetal cells as a unique alternative model and cell source in the restoration of damaged tissue.
|Concordant regulation of gene expression by hypoxia and 2-oxoglutarate-dependent dioxygenase inhibition: the role of HIF-1alpha, HIF-2alpha, and other pathways. |
Gareth P Elvidge, Louisa Glenny, Rebecca J Appelhoff, Peter J Ratcliffe, Jiannis Ragoussis, Jonathan M Gleadle
The Journal of biological chemistry 281 15215-26 2006
Studies of gene regulation by oxygen have revealed novel signal pathways that regulate the hypoxia-inducible factor (HIF) transcriptional system through post-translational hydroxylation of specific prolyl and asparaginyl residues in HIF-alpha subunits. These oxygen-sensitive modifications are catalyzed by members of the 2-oxoglutarate (2-OG) dioxygenase family (PHD1, PHD2, PHD3, and FIH-1), raising an important question regarding the extent of involvement of these and other enzymes of the same family in directing the global changes in gene expression that are induced by hypoxia. To address this, we compared patterns of gene expression induced by hypoxia and by a nonspecific 2-OG-dependent dioxygenase inhibitor, dimethyloxalylglycine (DMOG), among a set of 22,000 transcripts, by microarray analysis of MCF7 cells. By using short interfering RNA-based suppression of HIF-alpha subunits, we also compared responses that were dependent on, or independent of, the HIF system. Results revealed striking concordance between patterns of gene expression induced by hypoxia and by DMOG, indicating the central involvement of 2-OG-dependent dioxygenases in oxygen-regulated gene expression. Many of these responses were suppressed by short interfering RNAs directed against HIF-1alpha and HIF-2alpha, with HIF-1alpha suppression manifesting substantially greater effects than HIF-2alpha suppression, supporting the importance of HIF pathways. Nevertheless, the definition of genes regulated by both hypoxia and DMOG, but not HIF, distinguished other pathways most likely involving the action of 2-OG-dependent dioxygenases on non-HIF substrates.
|Temperature regulates SOX9 expression in cultured gonads of Lepidochelys olivacea, a species with temperature sex determination. |
Moreno-Mendoza, N, et al.
Dev. Biol., 229: 319-26 (2001) 2001
Although sex determination starts in the gonads, this may not be the case for species with temperature sex determination (TSD). Since temperature affects the whole embryo, extragonadal thermosensitive cells may produce factors that induce gonadal sex determination as a secondary event. To establish if gonads of a species with TSD respond directly to temperature, pairs of gonads were cultured, one at female-promoting temperature (FPT) and the contralateral at male-promoting temperature (MPT). Histological and immunohistochemical detection of SOX9 revealed that the response to temperature of isolated gonads was similar to that of the gonads of whole embryos. While gonads cultured at MPT maintained SOX9 expression, it was downregulated in gonads at FPT. Downregulation of SOX9 took longer in gonads cultured at stage 23 than in gonads cultured at stage 24, suggesting that a developmental clock was already established at the onset of culture. To find out if sex commitment occurs in vitro, gonads were switched from FPT to MPT at different days. Results showed that the ovarian pathway was established after 4 days of culture. The present demonstration that gonads have an autonomous temperature detector that regulates SOX9 expression provides a useful starting point from which the molecular pathways underlying TSD can be elucidated.
|Dexamethasone enhances SOX9 expression in chondrocytes. |
Sekiya, I, et al.
J. Endocrinol., 169: 573-9 (2001) 2001
SOX9 is a transcription factor that activates type II procollagen (Col2a1) gene expression during chondrocyte differentiation. Glucocorticoids are also known to promote chondrocyte differentiation via unknown molecular mechanisms. We therefore investigated the effects of a synthetic glucocorticoid, dexamethasone (DEX), on Sox9 gene expression in chondrocytes prepared from rib cartilage of newborn mice. Sox9 mRNA was expressed at high levels in these chondrocytes. Treatment with DEX enhanced Sox9 mRNA expression within 24 h and this effect was observed at least up to 48 h. The effect of DEX was dose dependent, starting at 0.1 nM and maximal at 10 nM. The half life of Sox9 mRNA was approximately 45 min in the presence or absence of DEX. Western blot analysis revealed that DEX also enhanced the levels of SOX9 protein expression. Treatment with DEX enhanced Col2a1 mRNA expression in these chondrocytes and furthermore, DEX enhanced the activity of Col2-CAT (chloramphenicol acetyltransferase) construct containing a 1.6 kb intron fragment where chondrocyte-specific Sry/Sox- consensus sequence is located. The enhancing effect of DEX was specific to SOX9, as DEX did not alter the levels of Sox6 mRNA expression. These data suggest that DEX promotes chondrocyte differentiation through enhancement of SOX9.
|Identification of an interaction between SOX9 and HSP70. |
Marshall, O J and Harley, V R
FEBS Lett., 496: 75-80 (2001) 2001
The campomelic dysplasia/autosomal sex reversal protein SOX9 is an important developmental transcription factor, required for correct bone and testis formation. Through in vitro and in vivo studies we have identified the heat shock protein HSP70 as an interacting partner for SOX9 in chondrocyte and testicular cell lines. HSP70 forms a ternary complex with DNA-bound SOX9. The interaction between HSP70 and SOX9 is ATP-independent and involves a highly conserved region of SOX9 hitherto of unknown function and the C-terminal region of HSP70. Our results implicate HSP70-SOX9 interactions in the assembly of multi-protein complexes during SOX9-mediated transcription.
|Localisation of the SRY-related HMG box protein, SOX9, in rodent brain. |
Pompolo, S and Harley, V R
Brain Res., 906: 143-8 (2001) 2001
Human mutations in the transcription factor gene, SOX9, cause campomelic dysplasia (CD), a severe dwarfism associated with brain abnormalities including dilation of lateral ventricles, hypoplasia of the corpus callosum and cerebellum defects. To improve our understanding of how SOX9 contributes to the molecular genetic pathway of brain development we sought to investigate the distribution of SOX9 protein in rat and mouse brain. The regions of SOX9 expression identified in this study correlated with the sites of reported brain abnormalities in CD patients. SOX9 immunoreactivity was observed in nuclei of scattered cells throughout the brain, in the ependymal layer and cells of the choroid plexus. In the forebrain most SOX9-immunoreactive nuclei co-localised with the glial astrocyte marker S-100. In the cerebellum, SOX9 was observed mostly in cells surrounding Purkinje cells, which were identified, by electron microscopy, as Golgi epithelial cells, also known as Bergmann glia. Using SOX9 antibody as a marker for the precursors of Bergmann glia, we traced their origin during mouse development. At embryonic day (E)14.5 and E16.5, SOX9 immunoreactivity was present mainly in the primordial choroid plexus, and ventricular zone. By E18.5, SOX9 was observed in the granular cell and Purkinje cell layers but no labelling was detectable in the external granular layer. These results suggest that SOX9 immunoreactivity is a marker for Bergmann cells during development and favour the proposed origin of the secondary glial scaffold arising from Bergmann cells derived exclusively from the ventricular zone.
|Accelerated up-regulation of L-Sox5, Sox6, and Sox9 by BMP-2 gene transfer during murine fracture healing. |
Uusitalo, H, et al.
J. Bone Miner. Res., 16: 1837-45 (2001) 2001
|Sox9 protein in rat sertoli cells is age and stage dependent. |
Fröjdman, K, et al.
Histochem. Cell Biol., 113: 31-6 (2000) 2000
We studied the location of Sox9 protein in the embryonic, juvenile, and adult rat testis by immunohistochemistry and immunoblotting. Sox9 belongs to a family of Sox proteins that are transcription factors and important in several developmental processes. In the incipient embryonic testis, Sox9 was prominently present in the gonadal blastema. With further embryonic differentiation, Sox9-positive cells arranged in the periphery of the testicular cords, showing the location of the Sertoli cells. Thereafter the immunoreaction for Sox9 gradually declined and was only weakly detectable in the 2-day-old postnatal rat testis. This situation remained for some period of time. In the 15-day-old rat testis, Sox9 protein strongly reappeared in the testicular cords. In the adult, the Sertoli cells of most regions of the seminiferous tubules were positive for Sox9. The strongest reaction for Sox9 was found in the dark zone. However, clearly negative or only weakly positive spermatogenic stages for the protein were also found, as seen for example in the pale zone. In fertile 1-year-old rats this basic situation was still detectable. Analyzed rat ovaries were all negative for Sox9, showing the sex-specific nature of Sox9. The results showed that Sox9 protein is distinctly present in the developing and mature Sertoli cells, but that its presence and amount is dependent on the age and the spermatogenetic stage within the seminiferous tubuli. The prominent presence of Sox9 in the incipient testis and at puberty suggests that this protein is needed at important phases of aggregation and reorganization of the Sertoli cells. The age and stage-specific presence of Sox9 in the testicular cords and in the seminiferous tubules of the adult suggests that Sox9 also may have a pivotal role in germ cell differentiation.
|Characterization of the model for experimental testicular teratoma in 129/SvJ-mice. |
Sundström, J, et al.
Br. J. Cancer, 80: 149-60 (1999) 1999
An animal model of experimental testicular teratoma has been established to study how a teratoma affects the host testis and how the host testis reacts against the teratoma. 129/SvJ-mice were used as experimental animals. To induce the experimental testicular teratoma, male gonadal ridges from 12-day-old 129/SvJ-mouse fetuses were grafted into the testes of adult mice for 1-12 weeks. The developing tumour was analysed by light and electron microscopy and by immunocytochemical localization of transcription factors SOX9 and c-kit, glial fibrillary acidic protein (GFAP) and type IV collagen. Testicular teratoma was observed in 36 out of 124 testes with implanted fetal gonadal ridges (frequency 29%). One spontaneous testicular teratoma was observed in this material from 70 male mice (1.5%). One week after implantation intracordal clusters of cells were seen in embryonic testicular cords of the graft as the first sign of testicular teratomas. Four weeks after implantation the embryonic testicular cords had totally disappeared from grafts with teratomas, and the tumour tissue had enlarged the testis and invaded the interstitium of the host testis. It consisted of solitary pieces of immature cartilage as well as of glial cells and of primitive neuroepithelium. Six to eight weeks after implantation the tumour tissue had expanded so that the enlarged testis could be detected by macroscopic enlargement of the scrotum. The testicular tissue of the host had practically disappeared, and only solitary disrupted seminiferous tubules of the host were seen surrounding the teratoma. Neuroepithelial structures of some teratomas cultured for 8 weeks had cells with a granular nucleus as a sign of obvious apoptosis. Eleven to 12 weeks after implantation the growth of the teratoma had stopped, and the histology corresponded to that of a mature cystic teratoma. GFAP, SOX9 and type IV collagen were strongly positive in some parts of the tumours cultured for 4 and 8 weeks, while only occasional c-kit-positive areas were observed in tumours cultured for 8 weeks. As conclusions: (1) the metastasizing capacity of the experimental testicular teratoma is very low during 12 weeks, but the behaviour of the tumour in the testicular tissue of the graft is invasive; (2) the growth of experimental testicular teratomas cease 6-8 weeks after implantation of the fetal gonadal ridges with the obvious apoptosis of the immature tissue components; (3) the model of experimental testicular teratoma in the mouse is suitable for studying how the teratoma affects the host testis and how the host testis reacts to teratoma.
|SOX9 is a potent activator of the chondrocyte-specific enhancer of the pro alpha1(II) collagen gene. |
Lefebvre, V, et al.
Mol. Cell. Biol., 17: 2336-46 (1997) 1997
The identification of mutations in the SRY-related SOX9 gene in patients with campomelic dysplasia, a severe skeletal malformation syndrome, and the abundant expression of Sox9 in mouse chondroprogenitor cells and fully differentiated chondrocytes during embryonic development have suggested the hypothesis that SOX9 might play a role in chondrogenesis. Our previous experiments with the gene (Col2a1) for collagen II, an early and abundant marker of chondrocyte differentiation, identified a minimal DNA element in intron 1 which directs chondrocyte-specific expression in transgenic mice. This element is also a strong chondrocyte-specific enhancer in transient transfection experiments. We show here that Col2a1 expression is closely correlated with high levels of SOX9 RNA and protein in chondrocytes. Our experiments indicate that the minimal Col2a1 enhancer is a direct target for Sox9. Indeed, SOX9 binds to a sequence of the minimal Col2a1 enhancer that is essential for activity in chondrocytes, and SOX9 acts as a potent activator of this enhancer in cotransfection experiments in nonchondrocytic cells. Mutations in the enhancer that prevent binding of SOX9 abolish enhancer activity in chondrocytes and suppress enhancer activation by SOX9 in nonchondrocytic cells. Other SOX family members are ineffective. Expression of a truncated SOX9 protein lacking the transactivation domain but retaining DNA-binding activity interferes with enhancer activation by full-length SOX9 in fibroblasts and inhibits enhancer activity in chondrocytes. Our results strongly suggest a model whereby SOX9 is involved in the control of the cell-specific activation of COL2A1 in chondrocytes, an essential component of the differentiation program of these cells. We speculate that in campomelic dysplasia a decrease in SOX9 activity would inhibit production of collagen II, and eventually other cartilage matrix proteins, leading to major skeletal anomalies.
|Sox9 expression during gonadal development implies a conserved role for the gene in testis differentiation in mammals and birds. |
Morais da Silva, S, et al.
Nat. Genet., 14: 62-8 (1996) 1996
Heterozygous mutations in SOX9 lead to a human dwarfism syndrome, Campomelic dysplasia. Consistent with a role in sex determination, we find that Sox9 expression closely follows differentiation of Sertoli cells in the mouse testis, in experimental sex reversal when fetal ovaries are grafted to adult kidneys and in the chick where there is no evidence for a Sry gene. Our results imply that Sox9 plays an essential role in sex determination, possibly immediately downstream of Sry in mammals, and that it functions as a critical Sertoli cell differentiation factor, perhaps in all vertebrates.
|SNAP i.d. 2.0 System Brochure|
|Anti-Sox9 - Data Sheet|