Sox2-expressing Cells Research Articles

Continuous cell supply from a Sox9-expressing progenitor zone in adult liver, exocrine pancreas and intestine

The liver and exocrine pancreas share a common structure, with functioning units (hepatic plates and pancreatic acini) connected to the ductal tree. Here we show that Sox9 is expressed throughout the biliary and pancreatic ductal epithelia, which are connected to the intestinal stem-cell zone. Cre-based lineage tracing showed that adult intestinal cells, hepatocytes and pancreatic acinar cells are supplied physiologically from Sox9-expressing progenitors. Combination of lineage analysis and hepatic injury experiments showed involvement of Sox9-positive precursors in liver regeneration. Embryonic pancreatic Sox9-expressing cells differentiate into all types of mature cells, but their capacity for endocrine differentiation diminishes shortly after birth, when endocrine cells detach from the epithelial lining of the ducts and form the islets of Langerhans. We observed a developmental switch in the hepatic progenitor cell type from Sox9-negative to Sox9-positive progenitors as the biliary tree develops. These results suggest interdependence between the structure and homeostasis of endodermal organs, with Sox9 expression being linked to progenitor status.

Sox9‐expressing precursors are the cellular origin of the cruciate ligament of the knee joint and the limb tendons

Sox9 expression defines cell progenitors in a variety of tissues during mouse embryogenesis. To establish a genetic tool for cell-lineage tracing and gene-function analysis, we generated mice in which the CreERT2 gene was targeted to the endogenous mouse Sox9 locus. In Sox9(CreERT2/+) ;R26R embryos, tamoxifen activated Cre recombinase exclusively in Sox9-expressing tissues. To determine the suitability of this mouse line for developmental stage-specific gene recombination, we investigated the cellular origins of the cruciate ligaments of the knee joint and the limb tendons, in which precursor cells have not been defined. The cells in these tissues were labeled after tamoxifen treatment before or at the stage of chondrogenic mesenchymal condensation, indicating that ligament and tendon cells originated from Sox9-expressing cells and that cell fate determination occurred at mesenchymal condensation. This mouse line is a valuable tool for the temporal genetic tracing of the progeny of, and inducible gene modification in Sox9-expressing cells.

Expression of a Hoechst 33342 Efflux Phenomenon and Common Characteristics Of Pluripotent Stem Cells in a Side Population of Amniotic Fluid Cells

The aim of this study was to verify the existence of a side population (SP) of cells in second-trimester amniotic fluid. Amniotic fluid samples (n = 35) were obtained, and the number and size of viable amniotic fluid cells (AFCs) were analyzed. Small AFCs (SAFCs) and large AFCs (LAFCs) were isolated using a sterile 10-microm pore size strainer. Hoechst 33342 dye exclusion assay, flow cytometry analysis, reverse transcriptase polymerase chain reaction and immunocytochemistry were used to analyze the characteristics of SAFCs and LAFCs. The mean concentration of viable AFCs from 16 to 21 weeks of gestation was 0.3 x 10(5), 0.8 x 10(5), 1.1 x 10(5), 1.3 x 10(5), 1.0 x 10(5) and 1.0 x 10(5) cells/mL respectively. The mean percentage of SAFCs from 16 to 21 weeks of gestation was 27.3%, 40.5%, 49.7%, 60.2%, 41.0% and 58.2%, respectively. The Hoechst 33342 efflux phenomenon was obvious among SAFCs but was rare in the LAFC population. Flow cytometry analyses showed that cell surface antigen expression on LAFCs and SAFCs were positive for CD29, CD44, CD73, CD90, CD166 and HLA-I, but negative for CD31, CD34, CD45, CD117 and HLA-II. Importantly, Nanog, Oct-4, ABCG2 and SOX2 expression in cells was easily detectable among the SAFC population. Expression of Nanog and ABCG2 was not observed among LAFCs. Amniotic fluid contains a SP that was found mostly among the SAFCs. Enriched SP cells isolated by the efflux of Hoechst 33342 could be a novel and promising source of pluripotent-like amniotic derived stem cells for cellular therapy in the near future.

Expression and role of Oct3/4, Nanog and Sox2 in regeneration of rat tracheal epithelium

To explore the role of Oct3/4, Nanog and Sox2 in regeneration of rat tracheal epithelium. An ex vivo model of rat tracheal epithelial regeneration using 5-fluorouracil (5-FU) was developed, to induce injury. Expression levels of Oct3/4, Nanog and Sox2 were examined using Western blot analysis, RT-PCR or microscopically observed immunofluorescence, and cell morphological changes were observed using HE staining, during the recovery process. Oct3/4, Nanog and Sox2 were not detectable in normal tracheal epithelium. After treatment with 5-FU, the normally proliferating tracheal epithelium desquamated and only a few cells in G0 phase of the cell cycle were left on the basement membrane and Oct3/4, Nanog and Sox2 could be observed at this time. Thereafter, the number of Oct3/4-, Nanog- and Sox2-positive cells increased gradually. When the cells differentiated into ciliate cells, mucous cells or basal cells, and restored pseudostratified mucociliary epithelium, the number of Oct3/4-, Nanog- and Sox2-positive cells decreased and gradually disappeared. G0 phase cells with resistance to 5-FU damage expressed Oct3/4, Nanog and Sox2. This indicated that these cells were undifferentiated, but had the ability to terminally differentiate into downstream-type cells. They possessed stem cell properties. The results are consistent with Oct3/4, Nanog and Sox2-expressing cells being considered as tracheal stem cells.

Defining the hair follicle stem cell (Part I)

Defining the hair follicle stem cell (Part I)

The Role of Foxg1 in the Development of Neural Stem Cells of the Olfactory Epithelium

The olfactory epithelium (OE) of the mouse is an excellent model system for studying principles of neural stem cell biology because of its well-defined neuronal lineage and its ability to regenerate throughout life. To approach the molecular mechanisms of stem cell regulation in the OE, we have focused on Foxg1, also known as brain factor 1, which is a member of the Forkhead transcription factor family. Foxg1(-/-) mice show major defects in the OE at birth, suggesting that Foxg1 plays an important role in OE development. We find that Foxg1 is expressed in cells within the basal compartment of the OE, the location where OE stem and progenitor cells are known to reside. Since FoxG1 is known to regulate proliferation of neuronal progenitor cells during telencephalon development, we performed bromodeoxyuridine pulse-chase labeling of Sox2-expressing neural stem cells during primary OE neurogenesis. We found the percentage of Sox2-expressing cells that retained bromodeoxyuridine was twice as high in Foxg1(-/-) OE cells as in the wild type, suggesting that these cells are delayed and/or halted in their development in the absence of Foxg1. Our findings suggest that the proliferation and/or subsequent differentiation of Sox2-expressing neural stem cells in the OE is regulated by Foxg1.

Expression of Sox11 in adult neurogenic niches suggests a stage‐specific role in adult neurogenesis

In the mammalian brain, neural stem and progenitor cells in the subventricular zone of the lateral ventricles and the subgranular zone of the dentate gyrus generate new neurons throughout adulthood. The generation of new functional neurons is a complex process that is tightly controlled by extrinsic signals and that is characterized by stage-specific gene expression programs and cell biological processes. The transcription factors regulating such stage-specific developmental steps in adult neurogenesis are largely unknown. Here we report that Sox11, a member of the group C Sox transcription factor family, is prominently expressed in the neurogenic areas of the adult brain. Further analysis revealed that Sox11 expression is strictly confined to doublecortin-expressing neuronally committed precursors and immature neurons but that Sox11 is not expressed in non-committed Sox2-expressing precursor cells and mature neurons of the adult neurogenic lineage. Finally, overexpression of Sox11 promotes the generation of doublecortin-positive immature neurons from adult neural stem cells in vitro. These data indicate that Sox11 is involved in the transcriptional regulation of specific gene expression programs in adult neurogenesis at the stage of the immature neuron.

SOX9 is not required for the cellular events of testicular organogenesis in XX mole ovotestes

Mammalian sex determination is the genetic process that commits the undifferentiated bipotential gonads to develop as either testes or ovaries. The differentiation of SOX9-expressing Sertoli cells is assumed to be necessary to initiate testis development. Insectivorous moles of the genus Talpa represent a unique case of generalized true hermaphroditism, as XX female moles constitutively develop two ovotestes instead of normal ovaries. In this work, we have investigated the expression patterns of a number of genes known to play key roles in gonad organogenesis, throughout the entire process of ovotestis development in female moles. Molecular and morphological evidence are provided that these ovotestes contain primary medullary testis-like cords, Leydig cells, peritubular myoid cells, and a testis-specific vasculature, but no Sertoli cells. Our results show for the first time that SOX9 is not required for the formation of the primary testis cords, but it is necessary for the maintenance and subsequent development of these cords. In addition, the expression pattern of WNT4 in male and female moles indicates that this gene inhibits Leydig cell differentiation and, contrary to the proposed scenario in the mouse, it is not required for the colonization and survival of primordial germ cells. According to our data, mole ovotestes result from a process of PDGFRalpha-mediated mesonephric cell migration, which occurs simultaneously in both sexes. The fact that FST remains inactive during the critical stages of female gonad development, explains the lack of migration inhibition, and may be a consequence of improper WNT4 signalling in the mole.

A Sox10 Expression Screen Identifies an Amino Acid Essential for Erbb3 Function

The neural crest (NC) is a population of embryonic stem cells that gives rise to numerous cell types, including the glia and neurons of the peripheral and enteric nervous systems and the melanocytes of the skin and hair. Mutations in genes and genetic pathways regulating NC development lead to a wide spectrum of human developmental disorders collectively called neurocristopathies. To identify molecular pathways regulating NC development and to understand how alterations in these processes lead to disease, we established an N-ethyl-N-nitrosourea (ENU) mutagenesis screen utilizing a mouse model sensitized for NC defects, Sox10LacZ/+. Out of 71 pedigrees analyzed, we identified and mapped four heritable loci, called modifier of Sox10 expression pattern 1–4 (msp1–4), which show altered NC patterning. In homozygous msp1 embryos, Sox10LacZ expression is absent in cranial ganglia, cranial nerves, and the sympathetic chain; however, the development of other Sox10-expressing cells appears unaffected by the mutation. Linkage analysis, sequencing, and complementation testing confirmed that msp1 is a new allele of the receptor tyrosine kinase Erbb3, Erbb3msp1, that carries a single amino acid substitution in the extracellular region of the protein. The ENU-induced mutation does not alter protein expression, however, it is sufficient to impair ERBB3 signaling such that the embryonic defects observed in msp1 resemble those of Erbb3 null alleles. Biochemical analysis of the mutant protein showed that ERBB3 is expressed on the cell surface, but its ligand-induced phosphorylation is dramatically reduced by the msp1 mutation. These findings highlight the importance of the mutated residue for ERBB3 receptor function and activation. This study underscores the utility of using an ENU mutagenesis to identify genetic pathways regulating NC development and to dissect the roles of discrete protein domains, both of which contribute to a better understanding of gene function in a cellular and developmental setting.

Identification of Neural Crest and Glial Enhancers at the Mouse Sox10 Locus through Transgenesis in Zebrafish

Sox10 is a dynamically regulated transcription factor gene that is essential for the development of neural crest–derived and oligodendroglial populations. Developmental genes often require multiple regulatory sequences that integrate discrete and overlapping functions to coordinate their expression. To identify Sox10 cis-regulatory elements, we integrated multiple model systems, including cell-based screens and transposon-mediated transgensis in zebrafish, to scrutinize mammalian conserved, noncoding genomic segments at the mouse Sox10 locus. We demonstrate that eight of 11 Sox10 genomic elements direct reporter gene expression in transgenic zebrafish similar to patterns observed in transgenic mice, despite an absence of observable sequence conservation between mice and zebrafish. Multiple segments direct expression in overlapping populations of neural crest derivatives and glial cells, ranging from pan-Sox10 and pan-neural crest regulatory control to the modulation of expression in subpopulations of Sox10-expressing cells, including developing melanocytes and Schwann cells. Several sequences demonstrate overlapping spatial control, yet direct expression in incompletely overlapping developmental intervals. We were able to partially explain neural crest expression patterns by the presence of head to head SoxE family binding sites within two of the elements. Moreover, we were able to use this transcription factor binding site signature to identify the corresponding zebrafish enhancers in the absence of overall sequence homology. We demonstrate the utility of zebrafish transgenesis as a high-fidelity surrogate in the dissection of mammalian gene regulation, especially those with dynamically controlled developmental expression.

Expression Profiles of SRY and SOX9 in Rabbit Gonads: The Classical Model of Mammalian Sex Differentiation

Sex determination in mammalian gonads depends on the concerted action of SRY and SOX at the genital ridge. Although most research on the mechanisms involved in sex determination has been done in mice, the study of non-model organisms may indicate the extent of the generalizations currently based on model systems. The present study investigated the correlation between SRY/SOX9 expression patterns and the process of morphogenesis in the developing gonad of the rabbit. In males, the onset of SRY/SOX9 expression closely followed the establishment of undifferentiated genital ridges at 13 to 14 dpc. In contrast to mouse, real-time PCR in the rabbit revealed that levels of SRY/ SOX9 peak after the onset of seminiferous cord formation, while in mouse this occurs before. Furthermore, rabbit gonads maintain low levels of SRY and SOX9 expression in male and female gonads, respectively. In situ hybridization suggests that cells of the mesonephric Bowman capsules, which do not express SRY, may become SOX9-expressing pre-Sertoli cells during the long period of seminiferous cord formation in the rabbit. In contrast to mouse, current results indicate that the patterns of SRY/SOX9 expression associated with the process of gonadal morphogenesis in rabbit appear similar to those of other mammals, including humans.

Sox2 Plays a Role in the Induction of Amacrine and Müller Glial Cells in Mouse Retinal Progenitor Cells

The transcription factor Sox2 plays important roles in both human and mouse retinal development. Although loss-of-function mutations in Sox2 have been studied in mice, gain-of-function experiments in the neural retina have been lacking. The detailed expression pattern of Sox2 in the developing mouse retina was examined by immunohistochemistry. Then, Sox2 was expressed in a retinal explant culture prepared from E17 mouse embryos by retrovirus-mediated gene transfer to examine its role in retinal development. In addition, shRNA was used to suppress Sox2 in a retinal explant using a retrovirus-mediated system. Sox2 was expressed throughout the neuroblastic layer in the embryonic retina, but only in the inner nuclear layer in the mature retina. Double immunostaining revealed that Sox2 was expressed in Müller glial cells and in a subset of amacrine cells. Forced expression of Sox2 in a mouse retinal explant culture resulted in the dramatic accumulation of amacrine cells in the inner nuclear layer; in addition, cells expressing amacrine cell markers were also found on the innermost side of the outer nuclear layer. The expression of Pax6, which plays an important role in amacrine cell differentiation, was observed in the Sox2-expressing cells, and Sox2 activated the Pax6 promoter to drive luciferase expression in Y79 cells. A decrease in retinal progenitor cell proliferation accompanied these effects. The suppression of Sox2 expression by shRNA resulted in a decreased number of cells in the inner nuclear layer. Therefore, ectopic Sox2 expression can induce amacrine cells in the mouse retina from stage E17 onward, possibly by facilitating cell cycle exit.

Gene expression reveals unique skeletal patterning in the limb of the direct‐developing frog, Eleutherodactylus coqui

The growing field of skeletal developmental biology provides new molecular markers for the cellular precursors of cartilage and bone. These markers enable resolution of early features of skeletal development that are otherwise undetectable through conventional staining techniques. This study investigates mRNA distributions of skeletal regulators runx2 and sox9 along with the cartilage-dominant collagen 2(alpha)1 (col2a1) in embryonic limbs of the direct-developing anuran, Eleutherodactylus coqui. To date, distributions of these genes in the limb have only been examined in studies of the two primary amniote models, mouse and chicken. In E. coqui, expression of transcription factors runx2 and sox9 precedes that of col2a1 by 0.5-1 developmental stage (approximately 12-24 h). Limb buds of E. coqui contain unique distal populations of both runx2- and sox9-expressing cells, which appear before formation of the primary limb axis and do not express col2a1. The subsequent distribution of col2a1 reveals a primary limb axis similar to that described for Xenopus laevis. Precocious expression of both runx2 and sox9 in the distal limb bud represents a departure from the conserved pattern of proximodistal formation of the limb skeleton that is central to prevailing models of vertebrate limb morphogenesis. Additionally, runx2 is expressed in the early joint capsule perichondria of the autopod and in the perichondria of long bones well before periosteum formation. The respective distributions of sox9 and col2a1 do not reveal the joint perichondria but instead are expressed in the fibrocartilage that fills each presumptive joint capsule. These distinct patterns of runx2- and sox9-expressing cells reveal precursors of chondrocyte and osteoblast lineages well before the appearance of mature cartilage and bone.

Hair Follicle Stem Cells Are Specified and Function in Early Skin Morphogenesis

In adult skin, epithelial hair follicle stem cells (SCs) reside in a quiescent niche and are essential for cyclic bouts of hair growth. Niche architecture becomes pronounced postnatally at the start of the first hair cycle. Whether SCs exist or function earlier is unknown. Here we show that slow-cycling cells appear early in skin development, express SC markers, and later give rise to the adult SC population. To test whether these early slow-cycling cells function as SCs, we use Sox9-Cre for genetic marking and K14-Cre to embryonically ablate Sox9, an essential adult SC gene. We find that the progeny of Sox9-expressing cells contribute to all skin epithelial lineages and Sox9 is required for SC specification. In the absence of early SCs, hair follicle and sebaceous gland morphogenesis is blocked, and epidermal wound repair is compromised. These findings establish the existence of early hair follicle SCs and reveal their physiological importance in tissue morphogenesis.

SOX2-expressing progenitor cells generate all of the major cell types in the adult mouse pituitary gland

The pituitary gland adapts the proportion of each of its endocrine cell types to meet differing hormonal demands throughout life. There is circumstantial evidence that multipotent adult progenitor cells contribute to this plasticity, but these cells have not been identified. Here, we describe a small (<0.05%) population of progenitor cells in the adult pituitary gland. We show that these cells express SOX2, a marker of several early embryonic progenitor and stem cell types, and form "pituispheres" in culture, which can grow, form secondary spheres, and differentiate to all of the pituitary endocrine cell types, as well as folliculostellate cells. Differentiation of cells in the pituispheres was associated with the expression of nestin, SOX9, and S100. Cells expressing SOX2 and E-cadherin are found throughout Rathke's pouch (RP) in embryos but persist in the adult gland, mostly in a narrow zone lining the pituitary cleft, but also are scattered throughout the pituitary. However, unlike in embryonic RP, most of these SOX2(+) cells in the adult gland also express SOX9 and S100. We suggest that this SOX2(+)/SOX9(+) population represents transit-amplifying cells, whereas the SOX2(+)/SOX9(-) cells we identify are multipotent progenitor/stem cells persisting in the adult pituitary.

Forced expression of Sox2 or Nanog in human bone marrow derived mesenchymal stem cells maintains their expansion and differentiation capabilities

Mesenchymal stem cells (MSCs) derived from human bone marrow have capability to differentiate into cells of mesenchymal lineage. The cells have already been applied in various clinical situations because of their expansion and differentiation capabilities. The cells lose their capabilities after several passages, however. With the aim of conferring higher capability on human bone marrow MSCs, we introduced the Sox2 or Nanog gene into the cells. Sox2 and Nanog are not only essential for pluripotency and self-renewal of embryonic stem cells, but also expressed in somatic stem cells that have superior expansion and differentiation potentials. We found that Sox2-expressing MSCs showed consistent proliferation and osteogenic capability in culture media containing basic fibroblast growth factor (bFGF) compared to control cells. Significantly, in the presence of bFGF in culture media, most of the Sox2-expressing cells were small, whereas the control cells were elongated in shape. We also found that Nanog-expressing cells even in the absence of bFGF had much higher capabilities for expansion and osteogenesis than control cells. These results demonstrate not only an effective way to maintain proliferation and differentiation potentials of MSCs but also an important implication about the function of bFGF for self-renewal of stem cells including MSCs.

The High-mobility Group Transcription Factor Sox10 Interacts with the N-myc-interacting Protein Nmi

The high-mobility group transcription factor Sox10 exerts many different roles during development of the neural crest and nervous system. To unravel its complex transcriptional functions, we have started to look for interaction partners. Here, we identify an association of Sox10 with the N-myc interactor Nmi, which was mediated by the high-mobility group of Sox10 and the central region of Nmi. In vivo relevance of this interaction is indicated by the fact that both proteins were co-expressed in glial cells, gliomas and in the spinal cord. Additionally, subcellular localization of Nmi in C6 glioma depended on the presence of Sox10 such that nuclear Nmi was more frequent in Sox10-expressing cells. Importantly, Nmi modulated the transcriptional activity of Sox10 in reporter gene assays. Nmi effects varied between different Sox10 target gene promoters, indicating that Nmi function in vivo may be promoter-specific.

Sox10 overexpression induces neural crest‐like cells from all dorsoventral levels of the neural tube but inhibits differentiation

SoxE genes (Sox8, Sox9, and Sox10) are early response genes to neural crest induction. Although the early role of Sox9 has been examined in chick and frog, later roles in neural crest migration and differentiation remain largely unexplored. We first examined which SoxE genes were expressed in trunk neural crest cells and then investigated their function using in ovo electroporation. The results of this analysis reveal that Sox10 is present in migrating neural crest cells, whereas other SoxE genes are only expressed transiently after induction. Ectopic expression of Sox10 in the neural tube at trunk level induced expression of HNK-1 in neuroepithelial cells followed by extensive emigration from all levels of the dorsoventral neuraxis, including the floor plate. Sox10-expressing cells failed to express neuronal, Schwann, or melanocyte markers up to 6 days posttransfection (E8), suggesting these cells were maintained in an undifferentiated state. Overexpression of Sox8 or Sox9 had similar but not identical effects on neuroepithelial cells. These results show that high levels of Sox10, Sox9, or Sox8 expression in the neural tube are capable of inducing a migratory neural crest-like phenotype even in the absence of dorsal signals and can maintain these cells in an undifferentiated state.

Potential role of Sox9 in patterning tracheal cartilage ring formation in an embryonic mouse model.

To identify genes expressed early in the formation of the mouse trachea that control patterning of tracheal cartilaginous rings. The mouse larynx and trachea begin as an outpouching from the ventral foregut endoderm at embryonic day (E) 9. Digoxigenin-labeled RNA probes to putative tracheal patterning genes were generated by in vitro transcription. Embryos ranging in age from E9 to E16 were then subjected to whole-mount in situ hybridization using these labeled RNA probes. The RNA probes were then localized using antidigoxigenin antibodies tagged with a reporter molecule. In this manner, the 3-dimensional spatial and temporal expression of putative tracheal patterning genes was examined. Subjects F/VBN mice. In the developing mouse trachea, the expression of Sox9 messenger RNA preceded cartilage ring formation. Sox9 was expressed as 2 distinct longitudinal stripes along the posterolateral aspect of the trachea as early as E9, when the developing trachea is first identified. Collagen 2A1, a cartilage-specific protein, was subsequently expressed in the same longitudinal pattern as Sox9, consistent with the early commitment of Sox9-expressing cells to the cartilage program. As cartilage rings formed, Sox9 and collagen 2A1 was expressed over the lateral and anterior aspects of the trachea. We have developed a system to study the early expression of genes that may pattern the formation of the trachea. We have identified a gene (Sox9) with a known role in chondrocyte differentiation that is expressed in a highly specific temporal and spatial pattern in the developing upper respiratory tract.

Cell aggregation precedes the onset of Sox9-expressing preSertoli cells in the genital ridge of mouse

Sox9 is expressed at the onset of the genital ridge formation in both sexes. It is assumed that Sry, the testis determining gene, turns Sox9 on in male embryos because it is turned off in female embryos. Spatial expression of Sry follows a cranio-caudal pattern. Here, we asked if Sox9 is expressed in the same cell lineage and with a similar pattern as Sry. A correlative study between the structural changes in the genital ridge and the immunocytochemical localization of Sox9-positive cells was undertaken. We used a transgenic strain expressing the green fluorescent protein (GFP) that considerably enhanced the cell context where the first Sox9-positive cells appear. Although Sox9-positive cells are located among loose mesenchymal cells by stages of 8–14 tail somites (ts) in both sexes, they are absent in the thickening coelomic epithelium of females. At 15 ts the first Sox9-positive cells appear within the core of the condensed cells only in male genital ridges. At 17 ts, a gradient of Sox9-positive cells in males is apparent, closely following the cranio-caudal pattern of cell aggregation seen in genital ridges of both sexes. Hence, our results suggest that Sox9 is expressed only in loose mesenchymal cells in both sexes and that expression of Sox9 in males requires the prior aggregation of cells in the genital ridges. The correspondence of Sox9 and Sry pattern of expression supports that both genes are expressed in the preSertoli cell lineage in the core of the genital ridges.