Expression Pattern Of Sox9 Research Articles

The SoxE factor Sox9 is selectively expressed in indirect pathway striatal projection neurons and regulates synaptogenesis

Striatum, as the largest structure of the basal ganglia, serves as a center for information transmission and is critical for motor function and reward perception. However, the genetic mechanisms underlying its development require further exploration. Here, we found that Sox9, traditionally recognized as a glial marker, is uniquely expressed in striatal medium spiny neurons (MSNs), especially in Drd2-expressing indirect pathway MSNs (D2-MSNs). Intriguingly, Sox9 expression in the striatum, which is conserved in humans, is a dynamic process. It maintains a high level during the perinatal stage, and exhibits low expression levels or vanishes at the embryonic and postnatal stages, respectively. The peak period of Sox9 expression coincides with the transition from neurogenesis to synaptogenesis. Importantly, gene regulatory network analysis and gain-of-function experiments confirmed Sox9 is strongly correlated with synaptogenesis. Moreover, we identified that Sox9 regulates synaptogenesis by repressing Foxp2, a well-known synapse regulator. Furthermore, we demonstrated that the biased expression pattern of Sox9 in D2-MSNs is, at least in part, regulated by another SoxE family member Sox8, which is specifically expressed in Drd1-expressing direct pathway MSNs (D1-MSNs). Taken together, our findings reveal a new marker of D2-MSNs and identify its distinctive function in striatal development.

SRY-box Transcription Factor 6 Is Expressed Not Only in the Dorsal but Also in the Ventral Zone of the Neural Tube and Is Highly Expressed in the Notochord and Chordoma

In the course of SRY-box transcription factor 6 (SOX6) expression profiling in human embryonic tissue, SOX 6 was found to be highly expressed in the notochord, based on the findings of immunohistochemistry (IHC). Sox6 is also expressed in the neural tube and the distribution of SOX6 is located in the ventral and dorsal zones of the neural tube. In contrast to the findings that SOX6-positive cells were located on the floor plate of the neural tube, OLIG2- and NKX2.2-expressing cells were lacking on the floor plate of the neural tube, and their expression was restricted only to the ventral zone of the neural tube. The expression patterns of SOX9 were similar to those of OLIG2 and NKX2.2 in the neural tube. NKX2.2 and OLIG2 are not expressed in the notochord, but SOX9 and SOX6 are. Because Sox6 is highly expressed in the notochord, the present study investigated whether or not SOX6 is an immunohistochemical marker for the pathologic diagnosis of chordoma, a neoplasm derived from the notochord. IHC revealed that chordoma was strongly positive for SOX6 in two cases of chordoma, one of which occurred in the sacrococcygeal region and another that developed at the base of the skull, suggesting that SOX6 is a useful marker for the histopathologic diagnosis of chordoma.

Distinct gene expression patterns of SOX2 and SOX2OT variants in different types of brain tumours

Numerous investigations have been recently published on the dysregulated expression of long-noncoding RNAs (lncRNAs) in various cancer types, emphasizing that abnormal lncRNA expression is a major contributor to tumourigenesis. A broad spectrum of lncRNAs is expressed in the central nervous system, where these RNAs seem to play key roles in brain development and function. In addition to expressing SOX2, a master regulator of pluripotency that lies within its third intron, lncRNA SOX2OT has a proposed role in regulating neural development. Based on our previous studies, alternative splicing of SOX2OT generates two alternatively spliced variants (SOX2OT-S1 and SOX2OT-S2). The present study investigated the expression patterns of SOX2OT variants and SOX2 in three principal types of brain tumours (gliomas, meningiomas and pituitary adenomas) and in four brain tumour cell lines (U87-MG, 1321N1, A172 and DAOY). Total RNAwas extracted from 34 human brain tumour specimens, and the expression profile of target genes was measured using a real-time reverse transcription PCR approach. Our data revealed distinct expression patterns for SOX2OT variants and SOX2 in the brain tumour samples, indicating their potential involvement in brain tumourigenesis. Moreover, our results highlighted the potential usefulness of SOX2OT-S1, SOX2OT-S2, and SOX2 in molecular diagnosis and brain tumour classification.

Integrative Analysis Reveals the Expression Pattern of SOX9 in Satellite Glial Cells after Sciatic Nerve Injury

Several complex cellular and gene regulatory processes are involved in peripheral nerve repair. This study uses bioinformatics to analyze the differentially expressed genes (DEGs) in the satellite glial cells of mice following sciatic nerve injury. R software screens differentially expressed genes, and the WebGestalt functional enrichment analysis tool conducts Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomics (KEGG) pathway analysis. The Search Tool for the Retrieval of Interacting Genes/Proteins constructs protein interaction networks, and the cytoHubba plug-in in the Cytoscape software predicts core genes. Subsequently, the sciatic nerve injury model of mice was established and the dorsal root ganglion satellite glial cells were isolated and cultured. Satellite glial cells-related markers were verified by immunofluorescence staining. Real-time polymerase chain reaction assay and Western blotting assay were used to detect the mRNA and protein expression of Sox9 in satellite glial cells. A total of 991 DEGs were screened, of which 383 were upregulated, and 508 were downregulated. The GO analysis revealed the processes of biosynthesis, negative regulation of cell development, PDZ domain binding, and other biological processes were enriched in DEGs. According to the KEGG pathway analysis, DEGs are primarily involved in steroid biosynthesis, hedgehog signaling pathway, terpenoid backbone biosynthesis, American lateral skeleton, and melanoma pathways. According to various cytoHubba algorithms, the common core genes in the protein-protein interaction network are Atf3, Mmp2, and Sox9. Among these, Sox9 was reported to be involved in the central nervous system and the generation and development of astrocytes and could mediate the transformation between neurogenic and glial cells. The experimental results showed that satellite glial cell marker GS were co-labeled with Sox9; stem cell characteristic markers Nestin and p75NTR were labeled satellite glial cells. The mRNA and protein expression of Sox9 in satellite glial cells were increased after sciatic nerve injury. In this study, bioinformatics was used to analyze the DEGs of satellite glial cells after sciatic nerve injury, and transcription factors related to satellite glial cells were screened, among which Sox9 may be associated with the fate of satellite glial cells.

In vitro production of zebra cloned embryos

Several equids have gone extinct, and many extant equids are currently considered vulnerable to critically endangered. In vitro embryo production has been promoted for genetic rescue and preservation potential over the last few years. This work aimed to evaluate whether domestic horse oocytes support preimplantation development of SCNT reconstructed embryos using zebra fibroblast and compare blastocyst quality through the expression of SOX2 and YAP1. Oocyte collection, SCNT, culture, blastocyst fixation, IF, and statistical analysis were performed as described by Gambini et al. 2020, PLoSONE,15(9):e0238948. Interestingly, the blastocyst rate of zebra SCNT was higher than the control group (Table 1). No significant differences were found in total cell numbers (cell number ± SEM) between horse (386,4±70,89) and zebra (285,8±41,13) SCNT blastocyst. All blastocysts showed a similar expression pattern of SOX2 and nuclear YAP1. Most nuclei were positive for YAP1 (trophectoderm lineage), and most SOX2+ nuclei were clustered and negative for YAP1 (inner cell mass lineage). A few blastocysts of each group showed a scattered pattern expression of the SOX2 protein, probably due to alterations during cellreprogramming of SCNT embryos. We demonstrated that domestic horse oocytes support reprogramming zebra cells and the subsequent preimplantation development without compromising blastocyst cell number neither the differentiation of inner cell mass nor trophectoderm lineages. Our results encourage the use of domestic horse oocytes as a model to study in vitro zebra embryos to preserve valuable genetics in conservation programs for endangered or extinct equid species.

Unique expression patterns of the embryonal stem cell marker SOX2 and hormone receptors suggest the existence of a subpopulation of epithelial stem/progenitor cells in porcine and bovine endometrium.

BackgroundThere are currently insufficient data on the population of endometrial epithelial stem/progenitor cells in farm animals.ObjectivesWith the aim of identifying a potential population of epithelial stem/progenitor cells in the porcine and bovine endometrium, this study immunohistochemically examined the expression patterns of the oestrogen and progesterone receptors, as well as that of the embryonal stem cell marker SOX2.MethodsA total of 24 endometrial tissue samples obtained from cycling pigs (n = 12) and cows (n = 12) were included in our study. Each endometrium was divided into basal, middle and luminal portions. The percentage of marker‐positive cells and the intensity of the immunoreaction in each portion of the endometrium were determined.ResultsInverse expression patterns of SOX2 and progesterone receptors were found in both animal species throughout the oestrous cycle. Strong diffuse SOX2 expression was detected in the basal portions of the glands, while a significant decrease in positivity and a weak immunoreaction were found in the luminal two thirds of the glandular epithelium. Strong progesterone receptor expression was observed in at least 90% of glandular cells in the middle and luminal portions, whereas weak staining and significant decrease in positivity were detected in the basal portions of the glands. One oestrogen receptor expression pattern resembled that of progesterone receptors.ConclusionThe inverse expression patterns of SOX2 and hormone (especially progesterone) receptors suggest that endometrial epithelial stem/progenitor cells represent a subset of cells that reside in the basal portions of the endometrial glands in both the bovine and porcine endometrium.

Tracheal Ring Formation.

The trachea is a long tube that enables air passage between the larynx and the bronchi. C-shaped cartilage rings on the ventral side stabilise the structure. On its esophagus-facing dorsal side, deformable smooth muscle facilitates the passage of food in the esophagus. While the symmetry break along the dorsal-ventral axis is well understood, the molecular mechanism that results in the periodic Sox9 expression pattern that translates into the cartilage rings has remained elusive. Here, we review the molecular regulatory interactions that have been elucidated, and discuss possible patterning mechanisms. Understanding the principles of self-organisation is important, both to define biomedical interventions and to enable tissue engineering.

Expression of Protein SOX9 in Biliary Atresia.

Biliary atresia (BA) is still an enigmatic disease. Deeper knowledge of its pathophysiology could help develop better treatments. SOX9 regulates bile duct development, liver regeneration and fibrosis; therefore, it could be determinant in characterizing BA liver damage. Aim: To study if there is a SOX9 expression pattern in liver biopsies from BA patients. Liver biopsies from BA patients (group BA), from age-matched infants without primary hepatic disease (group Control), and from patients with other liver conditions (group OLC) were compared. Expression of SOX9 was checked for: amount, intensity of immunoreaction, localization within ductular structures, perifibrotic epithelial cells, and lobular cells. The scores were added to create a scale from 0 to 11 that allowed group comparison. SOX9 Scale and liver survival were also looked for a correlation. All BA cases had a score >4, while all controls scored <4. OLC livers scored 1 to 8 (3.5 ± 2.0) (P < 0.001 between all groups). A cut-off at 4 had 100% sensitivity and 88.24% specificity to differentiate BA from Controls and from OLC (area under receiver operating characteristic curve: 0.9989 (95% confidence interval: 0.9964-1.000). Strong expression of SOX9 was observed mainly in the nuclei of proliferated ductules of portal spaces and fibrotic bridges. SOX9 Scale score could not be related to liver survival in this study. In BA livers, SOX9 is mainly expressed in reactive ductular epithelium, following a pattern significantly different from that seen in non-BA patients; thus, SOX9 Scale may have a role in the diagnosis of BA.

Horse ooplasm supports in vitro preimplantation development of zebra ICSI and SCNT embryos without compromising YAP1 and SOX2 expression pattern.

Several equids have gone extinct and many extant equids are currently considered vulnerable to critically endangered. This work aimed to evaluate whether domestic horse oocytes support preimplantation development of zebra embryos obtained by intracytoplasmic sperm injection (ICSI, zebroid) and cloning, and to study the Hippo signaling pathway during the lineage specification of trophectoderm cells and inner cell mass cells. We first showed that zebra and horse sperm cells induce porcine oocyte activation and recruit maternal SMARCA4 during pronuclear formation. SMARCA4 recruitment showed to be independent of the genetic background of the injected sperm. No differences were found in blastocyst rate of ICSI hybrid (zebra spermatozoon into horse egg) embryos relative to the homospecific horse control group. Interestingly, zebra cloned blastocyst rate was significantly higher at day 8. Moreover, most ICSI and cloned horse and zebra blastocysts showed a similar expression pattern of SOX2 and nuclear YAP1 with the majority of the nuclei positive for YAP1, and most SOX2+ nuclei negative for YAP1. Here we demonstrated that horse oocytes support zebra preimplantation development of both, ICSI and cloned embryos, without compromising development to blastocyst, blastocyst cell number neither the expression of SOX2 and YAP1. Our results support the use of domestic horse oocytes as a model to study in vitro zebra embryos on behalf of preservation of valuable genetic.

Prognostic impact of SOX9 in stage II colon cancer: Results from a large nationwide cohort.

183 Background: Up-regulation of the transcription factor SOX9 has been described in colon cancer, and it has been argued that differences in the expression levels dictates cell proliferation. The aim of the present study was to analyze the prognostic impact of SOX9 in patients with stage II colon cancer. Methods: Individual patient data and formalin fixed paraffin embedded tumor tissue were collected from a large unbiased, population-based cohort, representing all patients operated for stage II colon cancer in Denmark in 2002 and 2003. The SOX9 expression was evaluated by immunohistochemistry on whole tumor sections. Patients were classified into three groups dependent on the SOX9 expression gradient in the tumor: luminal, peripheral, and uniform for comparison with the clinical data. The endpoint was disease free survival (DFS). Results: A total of 1,153 patients were included. We detected an expression-dependent relationship between SOX9 and prognosis. Patients with tumors exhibiting a luminal expression pattern (N = 267, increasing SOX9 expression towards the luminal compartment of the tumor) were characterized by a significantly better DFS compared to the uniform (N = 846) and peripheral (N = 40) patterns, p = 0.0070. The five-year DFS rates were 74%, 67%, and 56%, respectively. Multiple Cox regression analysis, confirmed an independent prognostic advantage of the luminal SOX9 expression pattern, as compared to the uniform pattern, hazard ratio (HR) 0.7211 (95% confidence interval (CI) 0.5561-0.9351), p = 0.0137, whereas the possible disadvantage of the periphery pattern could not be verified, p = 0.1405. Conclusions: The present results support a prognostic impact of SOX9 in stage II colon cancer. The consequence of an altered SOX9 expression seems to differ between intra-tumoral compartments, and we propose that a gradient-dependent evaluation should be applied to provide the most clinically relevant information about this transcription factor.

Sox2 expression in the visual system of two teleost species

The visual system of teleost fish shows growth and regeneration capacities during the entire animal’s life. Thus, the visual system of adult fish serves as a model for studying neurogenesis in the vertebrate central nervous system (CNS). Our study focused on the expression pattern of Sox2 in the fish visual system. Sox2 is a transcription factor known for its function in keeping stem cell properties, and as a regulator of cell fate during development, especially in the visual system. We used two different fish species: Astatotilapia burtoni and Danio rerio. In the visual system of fish, we identified Sox2 positive cells in the stem cell niche in the peripheral retina, in Müller cells and amacrine cells in the differentiated retina, and glial cells in the optic nerve (ON). We did not observe hardly any Sox2 expression in the optic nerve head (ONH). In the ON, Sox2 positive glial cells were lining the fascicles of new axons. Taking together, the broad spectrum of Sox2 expression indicates that this protein has different functions in the CNS of adult vertebrates. The results suggest that Sox2 has functions associated with the pathway of new axons from the retina. To understand the variety of cell types and subtypes and their plasticity potential in the visual system of fish will be essential to comprehend the growing and regenerating CNS in adult vertebrates.

SOX9 dependent FOXA1 expression promotes tumorigenesis in lung carcinoma

The malignant proliferation is one of the major characteristic for tumor cells, however the mechanism of lung cancer cells uncontrollable proliferation is still confusing. This study investigated the mechanism of up-regulated FOXA1 in lung cancer and its tumorigenic function in lung cancer. FOXA1 showed an increasing expression pattern with the pathological progression in lung cancer, and SOX9 expression pattern is positively correlated with FOXA1. Furthermore, combined up-regulated FOXA1 and SOX9 usually resulted in a poor survival prognosis of patients with lung cancer. In addition, SOX9 was identified as a transcription factor of FOXA1 in lung cancer and involved in affecting the expression of FOXA1 targeted genes—Bcl2, CDKN1B, RPRM and NKX2. What's more, SOX9 can rescue the declining tumorigenic ability of lung cancer cells caused by FOXA1 silenced. This study indicates that SOX9 dependent FOXA1 expression promotes tumorigenic ability of lung cancer cells.

Defective Tmprss3-Associated Hair Cell Degeneration in Inner Ear Organoids.

SummaryMutations in the gene encoding the type II transmembrane protease 3 (TMPRSS3) cause human hearing loss, although the underlying mechanisms that result in TMPRSS3-related hearing loss are still unclear. We combined the use of stem cell-derived inner ear organoids with single-cell RNA sequencing to investigate the role of TMPRSS3. Defective Tmprss3 leads to hair cell apoptosis without altering the development of hair cells and the formation of the mechanotransduction apparatus. Prior to degeneration, Tmprss3-KO hair cells demonstrate reduced numbers of BK channels and lower expressions of genes encoding calcium ion-binding proteins, suggesting a disruption in intracellular homeostasis. A proteolytically active TMPRSS3 was detected on cell membranes in addition to ER of cells in inner ear organoids. Our in vitro model recapitulated salient features of genetically associated inner ear abnormalities and will serve as a powerful tool for studying inner ear disorders.

IMMUNOHISTOCHEMICAL ANALYSIS OF SOX2, FGF-10 AND WNT-1 IN BENIGN EPITHELIAL ODONTOGENIC LESIONS

Objectives This study evaluated the immunoexpression of SOX2, FGF-10 and Wnt-1 in 20 cases of odontogenic keratocyst (OKC), 20 solid ameloblastoma (AM), 20 adenomatoid odontogenic tumor (AOT), 10 calcifying epithelial odontogenic tumor (CEOT) and 5 dental germs. Findings The analysis of SOX2 immunoexpression revealed positivity in most cases of the lesions. The immunostaining score for SOX2 revealed a statistically significant difference between the groups of lesions, with a higher frequency in OKC and CEOT (p Conclusions The expression pattern of SOX2, FGF-10 and Wnt-1 in dental germs and odontogenic lesions evaluated here confirms the participation of these proteins in the tooth development as well as in the development of benign epithelial odontogenic lesions.

Prognostic impact of SOX9 in stage II colon cancer: Results from a large nationwide cohort.

e15165 Background: Up-regulation of the transcription factor SOX9 has been described in colon cancer, and it has been argued that differences in the expression levels dictates cell proliferation. The aim of the present study was to analyze the prognostic impact of SOX9 in stage II colon cancer. Methods: Individual patient data and formalin fixed paraffin embedded tumor tissue were collected from a large unbiased, population-based cohort, representing all patients operated for stage II colon cancer in Denmark in 2002 and 2003. The SOX9 expression was evaluated by immunohistochemistry on whole tumor sections. Patients were classified into three groups dependent on the SOX9 expression gradient in the tumor: luminal, peripheral, and uniform for comparison with the clinical data. The endpoint was disease free survival (DFS). Results: A total of 1,153 patients were included. We detected an expression-dependent relationship between SOX9 and prognoses. Patients with tumors exhibiting a luminal expression pattern (N = 267, increasing SOX9 expression towards the luminal compartment of the tumor) were characterized by a significantly better DFS compared to the uniform (N = 846) and peripheral (N = 40) patterns, p = 0.0070. The five-year DFS rates were 74%, 67%, and 56%, respectively. Multiple Cox regression analysis, adjusted for all standard high risk factors, confirmed an independent prognostic advantage of the luminal SOX9 expression pattern, as compared to the uniform pattern, hazard ratio (HR) 0.7211 (95% confidence interval (CI) 0.5561-0.9351), p = 0.0137, whereas the possible disadvantage of the periphery pattern could not be verified, HR 1.4393 (95% CI 0.8869-2.3660), p = 0.1405. Conclusions: The present results support a prognostic impact of SOX9 in stage II colon cancer. The consequence of an altered SOX9 expression seems to differ between intra-tumoral compartments, and we propose that a gradient-dependent evaluation should be applied to provide the most clinically relevant information about this transcription factor.

TGF-\u03b21-SOX9 axis-inducible COL10A1 promotes invasion and metastasis in gastric cancer via epithelial-to-mesenchymal transition

Molecular biomarkers that predict disease progression might promote drug development and therapeutic strategies in aggressive cancers, such as gastric cancer (GC). High-throughput mRNA sequencing (RNA-seq) revealed that collagen type X alpha 1 (COL10A1) is a disease progression-associated gene. Analysis of 103 GC patients showed that high COL10A1 mRNA expression was associated with GC metastasis and reduced survival. We analyzed the COL10A1 promoter using the UCSC genome website and JASPAR database, and we found potential SOX9 binding site. Here, we demonstrated that SOX9 and COL10A1 were both up-regulated in GC. We observed a positive correlation between the expression patterns of SOX9 and COL10A1 in GC cells and tissues. The results of electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP) assay and promoter reporter indicated that SOX9 could directly bind to the COL10A1 gene promoter and activate its transcription. Biological function experiments showed that COL10A1 regulated the migration and invasion of GC cells. Knockdown COL10A1 inhibited lung and abdominal cavity metastasis in a nude mouse model. Moreover, transforming growth factor-β1 (TGF-β1) treatment up-regulated the phosphorylation of Smad2 and increased SOX9 and COL10A1 expression. COL10A1 was confirmed to be a potential inducer of epithelial-to-mesenchymal transition (EMT). SOX9 was essential for COL10A1-mediated EMT, and cell migration, invasion and metastasis. Co-expression of SOX9 and COL10A1 was associated with tumor progression and was strongly predictive of overall survival in GC patients. In summary, this study elucidated the mechanistic link between COL10A1 and the TGF-β1-SOX9 axis. These findings indicated that COL10A1 might play a crucial role in GC progression and serve as a potential biomarker and therapeutic target in GC patients.

SOX9 is a proliferation and stem cell factor in hepatocellular carcinoma and possess widespread prognostic significance in different cancer types.

SOX9 has been previously shown to be involved in hepatocellular carcinoma (HCC) and other types of cancer. However, prognostic studies so far involved rather small cohorts or lack external validation and experimental data. In this study, we firstly determined the histological expression pattern of SOX9 in human HCC by immunohistochemistry (n = 84) and evaluated its prognostic value. External cohorts of publicly available datasets were used to validate its prognostic relevance in HCC (n = 359) and other types of cancer including breast (n = 3951), ovarian (n = 1306), lung (n = 1926) and gastric cancer (n = 876). Functional SOX9 knock-down studies using siRNA and cancer stem cell models were generated in a panel of liver and breast cancer cell lines. High level of SOX9 was associated with poor survival even after adjustment for other prognostic factors in multivariate analysis (HR = 2.103, 95%CI = 1.064 to 4.156, p = 0.021). SOX9 prevailed a poor prognostic factor in all cancer validation cohorts (p<0.05). Reduced SOX9 expression by siRNA decreased the growth of liver cancer cells (p<0.05). SOX9 expression was associated with stem cell features in all tested cell lines (p<0.05). In conclusion, this study demonstrated in a large number of patients from multiple cohorts that high levels of SOX9 are a consistent negative prognostic factor.

Spatial and temporal expression of Sox9 during murine incisor development.

Mouse incisors are capable of continuously growing due to the renewal of dental epithelium stem cells and mesenchymal stem cells residing at the proximal ends. The transcription factor Sox9 plays important roles in maintaining the stem cells of hair follicles, retinal progenitor cells and neural crest stem cells. Whether Sox9 is involved during mouse incisor development is not reported yet. In this study, we examined the expression pattern of Sox9 during mouse incisor development by in situ hybridization and immunohistochemistry. Sox9 mRNA and protein showed similar expression pattern from embryonic day (E) 13.5 to postnatal (PN) day10. At E13.5 and E14.5, Sox9 was strongly expressed in the dental epithelium. At E16.5, Sox9 started to be detected in the mesenchymal cells within the dental pulp, especially the dental pulp cells that adjacent to the labial cervical loop. Similarly with E14.5, Sox9 was strongly detected in the labial cervical loop, including the basal epithelium, the stellate reticulum and the outer enamel epithelium from E16.5 to PN10. The mesenchyme adjacent to the labial cervical loop also showed strong signal of Sox9. The spatiotemporal expression of Sox9 suggested its possible involvement during mouse incisor development.

Altered SOX9 genital tubercle enhancer region in hypospadias

Human mutations in the SOX9 gene or its regulatory region can disrupt testicular development, leading to disorders of sex development (DSDs). Our previous work involving the genomic analysis of isolated DSD patients revealed a 78kb minimal sex determining region (RevSex) far upstream of SOX9 that was duplicated in 46,XX and deleted in 46,XY DSDs. It was postulated that RevSex contains a gonadal enhancer. However, the most highly conserved sub-region within RevSex, called SR4, was neither responsive to sex determining factors in vitro nor active in the gonads of transgenic mice, suggesting that SR4 may not be functioning as a testicular enhancer. Interestingly, SR4 transgenic mice showed reporter activity in the genital tubercle, the primordium of the penis and clitoris, a previously unreported domain of Sox9 expression. SOX9 protein was detected in the genital tubercle, notably in the urethral plate epithelium, preputial glands, ventral surface ectoderm and corpus cavernosa. SR4 may therefore function as a Sox9 genital tubercle enhancer, mutations of which could possibly lead to hypospadias, a birth defect seen in the DSD patients in the RevSex study. SR4 activity and the observed SOX9 expression pattern suggest that SR4 may function as a Sox9 genital tubercle enhancer. However, conditional ablation of Sox9 in the genital tubercle using Shh-Cre/+;Sox9flox/flox mice revealed no genital tubercle abnormalities, possibly due to compensation by similar Sox factors. To conclude, we have identified a novel regulatory enhancer driving Sox9 expression during external genitalia development.

Foxo1 Is Required for Normal Somatotrope Differentiation.

The etiology for half of congenital hypopituitarism cases is unknown. Our long-term goal is to expand the molecular diagnoses for congenital hypopituitarism by identifying genes that contribute to this condition. We have previously shown that the forkhead box transcription factor, FOXO1, is present in approximately half of somatotropes at embryonic day (e) 18.5, suggesting it may have a role in somatotrope differentiation or function. To elucidate the role of FOXO1 in somatotrope differentiation and function, Foxo1 was conditionally deleted from the anterior pituitary (Foxo1Δpit). Uncommitted progenitor cells are maintained and able to commit to the somatotrope lineage normally based on the expression patterns of Sox2, a marker of uncommitted pituitary progenitors, and Pou1f1 (also known as Pit1), which marks committed progenitors. Interestingly, Foxo1Δpit embryonic mice exhibit delayed somatotrope differentiation as evidenced by an almost complete absence of GH immunoreactivity at e16.5 and reduced expression of Gh at e18.5 and postnatal day (P) 3. Consistent with this conclusion, expression of GHRH receptor, a marker of terminally differentiated somatotropes, is significantly reduced at e18.5 and P3 in the absence of FOXO1. The mechanism of FOXO1 regulation of somatotrope differentiation may involve the basic helix-loop-helix transcription factor, Neurod4, which has been implicated in somatotrope differentiation and is significantly reduced in Foxo1Δpit mice. Foxo1Δpit mice do not exhibit growth defects, and at P21 their pituitary glands exhibit a normal distribution of somatotropes. These studies demonstrate that FOXO1 is important for initial somatotrope specification embryonically but is dispensable for postnatal somatotrope expansion and growth.