Sox-positive cell population in the adult cerebellum increases upon tissue degeneration.

Adult neurogenesis is well-described in the subventricular and subgranular zones of the mammalian brain. Recent observations that resident glia express stem cell markers in some areas of the brain not traditionally associated with neurogenesis hint to a possible role in tissue repair. The Bergmann glia (BG) population in the cerebellum displays markers and in vitro features associated with neural stem cells (NSC), however the physiological relevance of this phenotypic overlap remains unclear in the absence of established in vivo evidence of tissue regeneration in the adult cerebellum. Here, this BG population was analysed in the adult cerebellum of different species and showed conservation of NSC-associated marker expression including Sox1, Sox2 and Sox9, in chick, primate and mouse cerebellum tissue. NSC-like cells isolated from adult mouse cerebellum showed slower growth when compared to lateral ventricle NSC, as well as differences upon differentiation. In a mouse model of cerebellar degeneration, progressive Purkinje cell loss was linked to cerebellar cortex disorganisation and a significant increase in Sox-positive cells compared to matching controls. These results show that this Sox-positive population responds to cerebellar tissue disruption, suggesting it may represent a mobilisable cellular resource for targeted strategies to promote tissue repair.

SOX19b regulates the premature neuronal differentiation of neural stem cells through EZH2-mediated histone methylation in neural tube development of zebrafish.

OBJECTIVE: Neural tube defects (NTDs) are the most serious and common birth defects in the clinic. The SRY-related HMG box B1 (SoxB1) gene family has been implicated in different processes of early embryogenesis. Sox19b is a maternally expressed gene in the SoxB1 family that is found in the region of the presumptive central nervous system (CNS), but its role and mechanism in embryonic neural stem cells (NSCs) during neural tube development have not yet been explored. Considering that Sox19b is specific to bony fish, we intended to investigate the role and mechanism of Sox19b in neural tube development in zebrafish embryos. MATERIAL AND METHODS: Morpholino (MO) antisense oligonucleotides were used to construct a Sox19b loss-of-function zebrafish model. The phenotype and the expression of related genes were analysed by in situ hybridization and immunolabelling. Epigenetic modifications were detected by western blot and chromatin immunoprecipitation. RESULTS: In this study, we found that zebrafish embryos exhibited a reduced or even deleted forebrain phenotype after the expression of the Sox19b gene was inhibited. Moreover, we found for the first time that knockdown of Sox19b reduced the proliferation of NSCs; increased the transcription levels of Ngn1, Ascl1, HuC, Islet1, and cyclin-dependent kinase (CDK) inhibitors; and led to premature differentiation of NSCs. Finally, we found that knockdown of Sox19b decreased the levels of EZH2/H3K27me3 and decreased the level of H3K27me3 at the promoters of Ngn1 and ascl1a. CONCLUSION: Together, our data demonstrate that Sox19b plays an essential role in early NSC proliferation and differentiation through EZH2-mediated histone methylation in neural tube development. This study established the role of transcription factor Sox19b and epigenetic factor EZH2 regulatory network on NSC development, which provides new clues and theoretical guidance for the clinical treatment of neural tube defects.

SOX30 specially prevents Wnt-signaling to suppress metastasis and improve prognosis of lung adenocarcinoma patients.

BACKGROUND: Different histological subtypes of non-small cell lung cancer (NSCLC) show different molecular characteristics and responses to therapeutic strategy. Identification of specific gene, clarification of its special roles and molecular mechanisms are crucial for developing new therapeutic approach for particular subtype patients. METHODS: Surgical specimens of 540 NSCLC patients were recruited. Immunohistochemistry was used to detect SOX30 expression, and correlations with clinical parameters were analyzed. Functional experiments and gene ontology analysis were performed to investigate roles of SOX30. Network analysis, TOP/FOP-Flash assays, luciferase reporter assays and ChIP-PCR assays were performed to determine the mechanism. Survival analyses were calculated by Kaplan-Meier and Cox regression. Recovery experiment was investigated the importance of the target of SOX30. RESULTS: SOX30 expression is closely associated with histological types of NSCLC, and metastasis of adenocarcinoma (ADC) patients but not of squamous cell carcinoma (SCC) patients. SOX30 strongly inhibits cancer cell migration and invasion in ADC cell lines, whrereas not affects cell migration and invasion in SCC cell lines. The genes associated with SOX30 preferentially enrich in metastasis process and Wnt-signaling in only ADC patients. Consistently, SOX30 is negatively associated with the expression of Wnt-signaling and metastasis-related gene CTNNB1 (ß-catenin) in ADC, but not in SCC. At the molecular level, SOX30 represses Wnt-signaling by directly transcriptional inhibition of CTNNB1 in ADC, and also not in SCC. In the clinical, SOX30 is a favorable and independent prognostic factor in ADC patients, whereas is an unfavorable and independent prognostic factor in SCC patients. Moreover, SOX30 expression is a double face early-stage prognostic biomarker in ADC and SCC patients. In addition, forcible restoration of CTNNB1 indeed can inhibit the anti-metastatic role of SOX30 in ADC patients. CONCLUSIONS: In early-stage ADC patients, elevated SOX30 expression inhibits tumor-metastasis by directly binding to CTNNB1 promoter resulting in a favorable prognosis of these patients. However, in early-stage SCC patients, SOX30 has no inhibitory role on tumor-metastasis due to not binding to CTNNB1 promoter leading to an unfavorable prognosis of the patients. This study highlights a special role and prognostic value of SOX30 in ADC, providing a novel therapeutic target for particular subtype NSCLC patients.

Different expressions of miR-125b and SOX30 in malignant lymphomas and their significance.

PURPOSE: To explore the roles of micro RNAs (miRs)-125b and SOX30 in malignant lymphomas. METHODS: The correction of miR-125b targeting SOX30 was examined by the luciferase reporter assay. The expression levels of miR-125b and SOX30 were tested by in situ hybridization and immunohistochemistry. RESULTS: miR-125b was able to bind to the 3'UTR of SOX30 gene and was negatively associated with SOX30. As compared with the reactive hyperplasia lymphatic samples, the higher (miR-125b) and lower (SOX30) rate was expressed positively in the malignant lymphomas, which was related to the stage and grade of malignancy. CONCLUSIONS: miR-125b can regulate SOX30 by binding to its 3'UTR. miR-125b and SOX30 act as diagnostic and therapeutic markers for malignant lymphoma.

5-Aminolevulinic acid-mediated photodynamic therapy can target human glioma stem-like cells refractory to antineoplastic agents.

BACKGROUND: Glioblastoma (GBM) is a highly malignant lethal brain cancer. Accumulated evidence suggests that elevated resistance of GBM to both chemo- and radio-therapy is, at least in part, due to the presence of a small population of glioma stem cells (GSC). In the present study, we aimed to determine the sensitivity of GSCs to 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT). METHODS: For this purpose, we established GSC-enriched cell cultures (termed glioma stem-like cells or GSLCs) from A172 human GBM cell line. Under our cultivation conditions, GSLCs formed floating spheroid clusters that contained increased population of CD133/Sox2 expressing cells. Firstly, to compare the activity of protoporphyrin IX (PpIX) biosynthesis in the GSLCs and the parental A172 glioma cells, we examined the expression levels of biosynthesis enzymes and transporters for PpIX using qRT-PCR, and investigated the intracellular levels of PpIX with use of flow cytometry analysis. Then, we evaluated the sensitivity of these cells to ALA-PDT in vitro. Finally, to confirm the therapeutic impact of ALA-PDT on GSLCs with more clinically relevant model, we performed the same experiment using three different patient-derived glioma sphere lines, which cultivated them either in stem cell media or under differentiation conditions in the presence of serum. RESULTS AND CONCLUSION: GSLCs expressed higher mRNA levels of PpIX biosynthesis enzymes and its transporters PEPT1/2 and ABCB6, when compared to the parental A172 glioma cells. Consistently, flow cytometry analysis revealed that upon incubation with ALA, GSLCs accumulate a higher level of PpIX. Finally, we showed that GSLCs were more sensitive to ALA-PDT than the original A172 cells, and confirmed that all patient-derived glioma sphere lines also showed significantly increased sensitivity to ALA-PDT if cultivated under the pro-stem cell condition. Our data indicate that ALA-PDT has potential as a novel clinically useful treatment that might eliminate GBM stem cells that are highly resistant to current chemo- and radio-therapy.

Expression profiles of Sox transcription factors within the postnatal rodent testes.

SRY-related box (Sox) transcription factors are conserved among vertebrate species. These proteins regulate multiple processes including sex determination and testis differentiation of the male embryo. Members of the Sox family have been identified in pre- and postnatal testis and are known to play an important role in sex determination (Sry, Sox9), male gonadal development, and fertility (Sox4, Sox8, Sox30). However, their expression profiles per cell types remain elusive. The objectives of this research were to characterize the expression profiles of Sox family members within adult testes using publically available datasets and to determine whether these findings are consistent with literature as well as immunofluorescence and in situ hybridization results. We have found that Sox4, Sox8, Sox9, and Sox12 are highly expressed in Sertoli cells, whereas Sox5, Sox6, and Sox30 were typically expressed in spermatocytes and spermatids. Spermatogonia were characterized by the expressions of Sox3, Sox4, Sox12, Sox13, and Sox18. Hence, these results suggest that Sox transcription factors may play different roles according to cell types of the adult mammalian testis.

sox2 and sox3 Play unique roles in development of hair cells and neurons in the zebrafish inner ear.

Formation of neural and sensory progenitors in the inner ear requires Sox2 in mammals, and in other species is thought to rely on both Sox2 and Sox3. How Sox2 and/or Sox3 promote different fates is poorly understood. Our mutant analysis in zebrafish showed that sox2 is uniquely required for sensory development while sox3 is uniquely required for neurogenesis. Moderate misexpression of sox2 during placodal stages led to development of otic vesicles with expanded sensory and reduced neurogenic domains. However, high-level misexpression of sox2 or sox3 expanded both sensory and neurogenic domains to fill the medial and lateral halves of the otic vesicle, respectively. Disruption of medial factor pax2a eliminated the ability of sox2/3 misexpression to expand sensory but not neurogenic domains. Additionally, mild misexpression of fgf8 during placodal development was sufficient to specifically expand the zone of prosensory competence. Later, cross-repression between atoh1a and neurog1 helps maintain the sensory-neural boundary, but unlike mouse this does not require Notch activity. Together, these data show that sox2 and sox3 exhibit intrinsic differences in promoting sensory vs. neural competence, but at high levels these factors can mimic each other to enhance both states. Regional cofactors like pax2a and fgf8 also modify sox2/3 functions.

Morphine delays neural stem cells differentiation by facilitating Nestin overexpression.

BACKGROUND: Morphine is used as an analgesic although it causes important secondary effects. These effects are triggered by several mechanisms leading to the dysregulation of gene expression. Here we aimed to study these alterations on neural stem cells (NSC) during CNS development. METHODS: AB strain and tg nestin:GFP zebrafish embryos, zebrafish primary neuron culture and mouse embryonic stem cells were used to assess the effect of morphine by qPCR, time lapse microscopy and western blot. ChIP-qPCR and bisulfite conversion assay were performed to determine the changes exerted by morphine in a Nestin candidate enhancer. RESULTS: Morphine increases GFP in nestin:GFP embryos and overexpresses the NSC marker Nestin. Morphine also exerts a hyperacetylation effect on H3K27 and decreases DNA methylation within a region located 18 Kb upstream nestin transcription starting site. Here, a binding site for the transcription factor complex Sox2/Oct4/Nanog was predicted. These factors are also upregulated by morphine. Besides, morphine increases the histone acetyl transferase p300. The inhibition of p300 activity decreases Nestin. CONCLUSIONS: Morphine facilitates Nestin increase by several mechanisms which include hyperacetylation of H3K27, decreased DNA methylation and the overexpression of the transcription factors sox2, oct4 and nanog. It has also been demonstrated that nestin levels depend on p300 activity. The facilitated Nestin expression delays the normal differentiation of neural stem cells. GENERAL SIGNIFICANCE: The present work provides novel evidence of the effects induced by morphine in the normal differentiation of NSCs, altering Nestin through changes on p300, H3K27ac, DNA methylation and Oct4, Sox2, and Nanog.

Spatio-temporal expression of Sox genes in murine palatogenesis.

Members of the Sox gene family play critical roles in many biological processes including organogenesis. We carried out comparative in situ hybridisation analysis of seventeen Sox genes (Sox1-14, 17, 18 and 21) during murine palatogenesis from initiation to fusion of the palatal shelves above the dorsal side of the tongue. At palatal shelf initiation (E12.5), the localized expression of six Sox genes (Sox2, 5, 6, 9, 12 and 13) was observed in the shelves, whereas Sox4 and Sox11 showed ubiquitious expression. During the down-growth of palatal shelves (E13.5), Sox4, Sox5, and Sox9 exhibited restricted expression to the interior side of the palatal shelves facing the tongue. Following elevation of the palatal shelves (E14.5), Sox2, Sox11 and Sox21 expression was present in the midline epithelial seam. We thus identify dynamic spatio-temporal expression of Sox gene family during the process of palatogenesis.

Osteochondral tissue regeneration through polymeric delivery of DNA encoding for the SOX trio and RUNX2.

Native osteochondral repair is often inadequate owing to the inherent properties of the tissue, and current clinical repair strategies can result in healing with a limited lifespan and donor site morbidity. This work investigates the use of polymeric gene therapy to address this problem by delivering DNA encoding for transcription factors complexed with the branched poly(ethylenimine)-hyaluronic acid (bPEI-HA) delivery vector via a porous oligo[poly(ethylene glycol) fumarate] hydrogel scaffold. To evaluate the potential of this approach, a bilayered scaffold mimicking native osteochondral tissue organization was loaded with DNA/bPEI-HA complexes. Next, bilayered implants either unloaded or loaded in a spatial fashion with bPEI-HA and DNA encoding for either Runt-related transcription factor 2 (RUNX2) or SRY (sex determining region Y)-box 5, 6, and 9 (the SOX trio), to generate bone and cartilage tissues respectively, were fabricated and implanted in a rat osteochondral defect. At 6weeks post-implantation, micro-computed tomography analysis and histological scoring were performed on the explants to evaluate the quality and quantity of tissue repair in each group. The incorporation of DNA encoding for RUNX2 in the bone layer of these scaffolds significantly increased bone growth. Additionally, a spatially loaded combination of RUNX2 and SOX trio DNA loading significantly improved healing relative to empty hydrogels or either factor alone. Finally, the results of this study suggest that subchondral bone formation is necessary for correct cartilage healing.

The sox gene Dichaete is expressed in local interneurons and functions in development of the Drosophila adult olfactory circuit.

In insects, the primary sites of integration for olfactory sensory input are the glomeruli in the antennal lobes. Here, axons of olfactory receptor neurons synapse with dendrites of the projection neurons that relay olfactory input to higher brain centers, such as the mushroom bodies and lateral horn. Interactions between olfactory receptor neurons and projection neurons are modulated by excitatory and inhibitory input from a group of local interneurons. While significant insight has been gleaned into the differentiation of olfactory receptor and projection neurons, much less is known about the development and function of the local interneurons. We have found that Dichaete, a conserved Sox HMG box gene, is strongly expressed in a cluster of LAAL cells located adjacent to each antennal lobe in the adult brain. Within these clusters, Dichaete protein expression is detected in both cholinergic and GABAergic local interneurons. In contrast, Dichaete expression is not detected in mature or developing projection neurons, or developing olfactory receptor neurons. Analysis of novel viable Dichaete mutant alleles revealed misrouting of specific projection neuron dendrites and axons, and alterations in glomeruli organization. These results suggest noncell autonomous functions of Dichaete in projection neuron differentiation as well as a potential role for Dichaete-expressing local interneurons in development of the adult olfactory circuitry.

Effect of chilling on sox2, sox3 and sox19a gene expression in zebrafish (Danio rerio) embryos.

Zebrafish embryos have not been cryopreserved due to their structural limitations. Although embryo survival rates have been used as the measured outcome for most of the cryopreservation protocols studied, there are very limited data available at the molecular level. This study focused on the effect of chilling and subsequent warming on gene expression of sox2, sox3 and sox19a which play vital roles in the development of zebrafish embryos. A quantitative RT-PCR approach was used to investigate gene expression following chilling at 0°C for up to 180 min. The effect on gene expression was also studied during a 180 min warming period after chilling for 30 or 60 min. There were significant decreases in sox2 (up to 4-fold) and sox3 (up to 3-fold) expressions following chilling. Significant increases in gene expressions of sox2 (up to 2-fold), sox3 (up to 33-fold) and sox19a (up to 25-fold) were observed during warming in the embryos that had been chilled for 30 min. Similarly, significant increases were observed in sox2 (up to 3-fold) and sox3 (up to 2-fold) during warming in embryos that had been chilled for 60 min. These increases may be explained by compensation for the suppression observed during chilling and/or to activate repair mechanisms or maintain homeostasis.

Developmental changes in the expression of Sox2 in the zebrafish brain.

The family of B1 Sox transcription factors plays critical roles in the early stages of development, including the central nervous system. It was demonstrated that Sox2 is expressed in repressed neural stem cells. Therefore, we decided to investigate the expression of Sox2 in the brain of zebrafish at different ages to identify potential neurogenic areas, and to establish the developmental changes they undergo. The brains were assessed by qRT-PCR, western blot, and immunohistochemistry. The maximal expression of Sox2 was found at 15 dpf progressively decreases up to 30 dpf, then increases up to 40 dpf and remains unchanged up to 180 dpf. By western blot three protein bands of 28 kDa, 34 kDa (main band), and 38 kDa were detected in the brain of 180 dpf animals. The immunolocalization of Sox2 revealed that by 15 dpf Sox2 was detected in cells of the olfactory bulb, the walls of the telencephalic and diencephalic ventricles, several nucleus in the diencephalons, and the tectum opticum; by 25-50 dpf the Sox2 positive areas were the same as above, and in the rhombencephalic ventricle and cerebellum. In adult animals Sox2 was restricted to the olfactory bulb and to cells of the telencephalic ventricle walls. Taken together present results demonstrate that the potential neurogenic areas in the brain of zebrafish are widespread than in mammals and change with development, but they are primarily concentrated around the ventricles and olfactory bulb in adults, following a similar localization as in mammals.