Mol. Cells 2015; 38(6): 548-561
Published online May 27, 2015
https://doi.org/10.14348/molcells.2015.0044
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: mgkim@inha.ac.kr
By combining conventional single cell analysis with flow cytometry and public database searches with bioinformatics tools, we extended the expression profiling of thymic stromal cotransporter (TSCOT), Slc46A2/Ly110, that was shown to be expressed in bipotent precursor and cortical thymic epithelial cells. Genome scale analysis verified
Keywords Ly110, SLC46A2, stem cell, thymic epithelial cell, TSCOT, tumor suppressor
Thymus produces educated T cells that can react with peptide antigen loaded on a self major histocompatibility complex (MHC) but not with self antigens. Developing thymocytes are guided and selected in the microenvironment of thymic stromal cells. Among the stromal cells, thymic epithelial cells (TECs) are major components that plays important roles of thymocyte differentiation in the separate compartments, the cortex and the medulla.
TECs also play critical roles during thymic organogenesis as shown in Foxn1 mutant mice, in which early TEC differentiation is abrogated, functional thymus lacks and, therefore, no T cell is present (Nehls et al., 1996). In mice, initial thymic structure begins to form with the TEC precursor cells originated from the third pharyngeal pouch around fetal day 10.5 (Blackburn and Manley, 2004; Gill et al., 2003; Rodewald, 2008; Su et al., 2001). At this stage, fetal thymus does not show clear medullary compartmentalization yet although the cells of medullary thymic epithelial cells (mTECs) in nature are found (Roberts et al., 2012). Fetal thymus begins to express the cortex-specific markers such as CDR1 in addition to general epithelial markers, EpCAM and MHCII (Ahn et al., 2008; Boehm, 2008; Lee et al., 2012; Yang et al., 2005). Later, thymus undergoes atrophy by aging after puberty and/or by damaging insults such as radiation or stress hormones (Blackburn et al., 2002; Cheng et al., 2010; Gill et al., 2003). However, thymus can also be rejuvenated by removing steroid sex hormones or removing organs that produce sex hormones (Berzins et al., 2002; Lynch et al., 2009; Sutherland et al., 2005). The functional thymic epithelial stem cell (sTEC) in the adult or aged animals were identified (Blackburn et al., 2002; Rodewald et al., 2001; Swann and Boehm, 2007; Ucar et al., 2014; Wong et al., 2014). It is important to identify the molecular marker present in the sTEC to understand the mechanism of thymic regeneration and to translate into the clinic for the recovery of important cellular immunity.
There has been much evidence that cortical TEC (cTEC) and mTEC are derived from the single precursor TECs (pTEC) or sTEC (Bleul et al., 2006; Rossi et al., 2006). While pTEC can be bipotent or specific lineage- committed (Park et al., 2013; Ucar et al., 2014), TEC development may be more progressive without instant commitment to a specific lineage (Alves et al., 2014). The original specific antibodies used for the identification of sTECs are MTS24 and MTS20 (Bennett et al., 2002; Gill et al., 2002). These TEC stem cells were located in the small medullary islets of very young thymus or corticomedullary junction of the adult thymus (Rodewald et al., 2001). The cytokeratin K5 and K8 are also important molecules for the identification of sTECs (Klug et al., 1998; 2002). It was also proposed that TEC stem cells reside in the MTS10+ cells in the medullary area.
It has been considered that
However, the presence of sTEC without
Expression of
In this study, we took advantage of public database and bioinformatics tools and performed genetic profiling in addition to classical methodologies. We show
The data sets were obtained from Gene Expression Omnibus (
The mouse lines TDLacZ (Ahn et al., 2008), 3.1T-EGFP (Chen et al., 2000), and 9.1T-NE (Lee et al., 2012) were maintained in the Laboratory of Molecular and Cellular Immunology Animal Facility of Inha University, Korea. All animal studies are in compliance with the Use of Laboratory Animals under the proper protocols. The protocols were approved by the Committees on the Ethics of Animal Experiments of NIH (LCMI Protocol 8) and Inha University (Protocol LMCI-2). Fetal mice were obtained from timed mating. The presence of a vaginal plug was considered at E0.5.
For genotyping, tail samples were extracted and used for a polymerase chain reaction with primers for the TDLacZ locus: Neo primer (ACCGCTATCAGGACATAGCGTTGG), 1C12 F1 (TTACTCAAAGTGATGCTGGACTGG), 1C12 B2 (CCGAGGGTTCCTTGGTACATTC), and the EGFP locus: EGFP-F (GCCACAAGTTCAGCGTGTCC), EGFP-R (GCTTCTGTTGGGGTCTTTGC), using the red Extract-N-Amp Tissue PCR kit (Sigma).
A fluorescence-based, automatic cell counter (Luna-FL, Logos Biosystems) was used to measure accurately the numbers of cells including thymic epithelial cells. The contamination from red blood cells could be automatically excluded because this system enumerates only nucleated cells.
A single cell suspension was prepared as described (Lee et al., 2012). Briefly, thymic tissues or deoxyguanosine treated fetal thymic organ culture were treated with 0.25% trypsin (Invitrogen) for about 20 min, in the presence of DNase I (Sigma), and washed with phosphate buffered saline (PBS) containing 10% fetal bovine serum (FBS). For further purification of TEC, the single cell suspension was isolated using magnetic bead cell sorting after incubating with anti-Fc mAb 2.4G2 and anti-mouse CD45 microbeads (Milteny Biotec) for 20 min at 4°C.
Monoclonal antibodies used in the staining of cells include anti-MHCII (I-Ab), anti-CD45 (Ly-5), and anti-Sca-1. The antibodies were purchased from Caltag or from BD PharMingen. Anti-aminopeptidase A (CDR-1) and anti-EpCAM (G8.8) were prepared in the Custom Antibody Services Facility, NIAID, NIH. Biotinylated UEA-1 was purchased from Vector Laboratories.
Cells were washed in cold FACS buffer (PBS + 1% BSA), subsequently stained on ice with the primary and the secondary antibodies, then analyzed on FACSCalibur or FACSAriaII with two lasers in the presence of 1?2 μg/ml of propidium iodide (PI). Anti-Fc, 2.4G2 antibody was included in all flow cytometry staining to block Fc receptor. For side population analysis, a 1 × 106 dissociated single cell suspension of fetal thymic organ culture (FD14.5) were incubated for an hour at 4°C in the presence of 5 μg/ml Hoechst 33342 dissolved in Hanks balanced salt solution. For verification of the side population, verapamil 0.25 mM was included. After washing at 4°C, cells were resuspended and examined by a flow cytometer equipped with a UV laser (FACSAriaII). For multicolor staining with SP analysis, cells were prestained with selected antibodies including homemade mAb CLVE (Yang et al., 2005). Negative control of TSCOT staining was carried out with all the same combination of antibodies except mAb CLVE. Analyses were done using the FlowJo program (
Sorted 1000 cells were used for RNA preparation. cDNA was generated with Superscript III and RT-PCR was carried out with the primers for TSCOT: F84 (5-CAGTCTTCCAATAACCTGCTTTGGCCT-3) and B83 (5-CGATTCCATGTGCCCCATTG-3) to amplify a 310 bp fragment and for GAPDH (Ahn et al., 2008;Kim et al., 2000). The primers for TSCOT are located in the separate exons with one intron and RT? control sampled did not show any band in the gel.
The immunofluorescence and X-gal staining the sections is described (Lee et al., 2012). An isolated thymus was washed in PBS and fixed in 1% para-formaldehyde, 0.2% glutaraldehyde, 0.02% NP-40, 1 mM MgCl2 in PBS for 1 or 2 h on ice and was embedded in Tissue Freezing Medium (Triangle Biomedical Sciences, USA). The 4 μm sections were fixed for 2 min in 1% formaldehyde, 0.2% glutaraldehyde, 0.02% NP-40 1 mM NaCl, then incubated with X-gal solution (1 part X-gal 40 μg/ml in dimethyl formamide, in 40 parts 2 mM MgCl2, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide in PBS) at 37°C for 48 h. For the detection of EGFP for fetal thymus sections, confocal microscopy was performed on the frozen sections in NIAID confocal facility (Leica SP2).
In order to study the expression pattern of
Expression profiles of
The expression of
From these analyses, it was concluded that
Next, we investigated the expression of TSCOT and reporters at the fetal stages in the different mouse models that we have previously characterized for the postnatal stages. The β-galactosidase reporter expression in TDLacZ thymus is restricted in the thymus as two dots at FD11 (Ahn et al., 2008). Figure 2A shows the β-galactosidase expression in the thymic sections at FD14.5. Expression of β-galactosidase is evenly distributed in the whole thymus, indicating most, if not all, the thymic epithelial cells at this stage express β-galactosidase. Another reporter mouse line, 3.1T-EGFP, which expresses EGFP in all TECs at the newborn stage (Park et al., 2013), showed EGFP expression earlier during fetal stages (Fig. 2B). At FD14 and 17, EGFP expression is also evenly distributed in the whole thymus. These results are consistent with the conclusion we previously described in which TSCOT is expressed in the pTEC stage (Park et al., 2013).
Fetal thymic stromal cells from normal C57BL/6 mouse (FD14) were analyzed for TSCOT expression with specific mAb CLVE (Yang et al., 2005). At this stage, EpCAM+ cells were all TSCOT+ (data not shown). When CD45? stromal cells were displayed for CD31 as an endothelial lineage marker along with MHCII, it became clear that TSCOT expression is present in all MHCII+ cells and CD31? MHCII? cells (Fig. 2C). Only CD31+MHCII? cells of endothelial lineage were TSCOT?. From these results, it is concluded that endothelial cells either lost TSCOT expression due to lineage commitment from the common stem cell or originated from other type of precursor cells that do not express TSCOT.
By using the TEC preparation from the deoxyguanosine treated FTOC of FD14.5, the presence of SP was tested with Hoechst 33342. In Fig. 3A, SP, which is ABC transporter sensitive, is clearly visible. When the inhibitor Verapamil was included, SP had decreased to 0.21% from 1.45%. Side population analyses were also applied with the TEC preparation using the same type culture of fetal thymus from 9.1T-NE mouse that shows EGFP expression patterns in the same way as endogenous TSCOT (Lee et al., 2012). As shown in Fig. 3B, a portion of the SP of 9.1T-NE TECs expresses EGFP when compared with that of normal C57BL/6 TEC preparation. In contrast, the side population of 3.1T-EGFP TEC population did not show any EGFP expression (data not shown).
When SP analysis was carried out along with antibody staining, TSCOT expression in SP and the major population (MP) were also clear. SP, either MHCII negative or positive, showed specific TSCOT expression with mAb CLVE. In addition, 84% of the SP cells and 95% of the MP cells were TSCOT+ (Fig. 3C). The next experiment was to verify TSCOT expression at the RNA level using a sorted side population of TECs prepared from normal C57BL/6. RT-PCR using the RNA prepared from the sorted cells clearly showed TSCOT expression in SP and in MP (Fig. 3D).
Many different types of stem cells express Sca-1 marker and a recent report mentioned that a TEC progenitor population is Sca-1+ (Golebiewska et al., 2011). We also tested expression of Sca-1 in fetal TEC preparation (Fig. 3E). Sca-1+ populations are present in both EpCAM+MHCII+ and EpCAM?MHCII? populations and most of them are TSCOT+. From these results, fetal TEC contains significant portion of sTECs that are TSCOT+.
We searched the available data on
The expression profile of
When the data from various side populations were specifically searched for, SP of mouse mammary gland cells showed an increased
From the results above, we concluded that
Because FOXN1 was considered as a putative TEC key transcription factor, we searched for
In order to find the putative regulatory factors, differential
These results suggest a regulatory mechanism for
We also researched
In Fig. 6B, the cluster analysis of 20 genes coexpressed in the same fashion as TSCOT is shown in Table 3. To our surprise,
Using bioinformatics approaches and conventional molecular and cellular methods, we showed that
During last several years, gene expression profiling at the genome scale are accumulated in the databases and accessible to the public. We took advantage of this advancement for the study of thymic organogenesis and TEC lineage differentiation using TSCOT as a lineage cell type specific marker and other known genes. From this approach, we learned that our previous studies are verified and we can get a lot more information than conventional experiments that are difficult to perform due to the limitation of small numbers of cells present in the actual organ.
In our previous studies, we asserted that TSCOT is TEC lineage specific and expressed in the cTEC and bipotent pTEC (Ahn et al., 2008; Kim et al., 2000; Park et al., 2013). Tissue specificity in thymus and expression in the limited TEC lineages are verified by the genome scale data analysis of expression profiling (Figs. 1, 4, and 5). Furthermore, we learned that more tissues such as skin and lung also express
To further investigate cells at earlier stages than pTEC expression, we first utilized a functional SP analysis and showed that SP of fetal TEC preparation expresses
Given the concept that
A schematic model in Figure 7 summarizes the findings of the expression profile during organogenesis.
By cluster analyses with selected transcription and soluble factors that are shown to be involved during the thymic organogenesis, we were able to identify multiple putative positive or negative regulators. IRF6 and GRHL3 are putative positive regulators for
Besides the structural features of a transporter that appeared containing primary amino acid sequences (Kim et al., 2000), it is still unclear what biological and biochemical functions that TSCOT plays. TSCOT is a member of Slc46A, and another member, Slc46A1, has been characterized as a proton coupled folate transporter (Diop-Bove et al., 2013). The heavily hydrophobic nature of the TSCOT amino acid composition and a simple twelve membrane spanning feature, with the presence of a central inner loop in the absence of ATP binding domain, suggests that TSCOT may transport small hydrophobic molecules. We proposed earlier that it may function in the survival of TECs based on the expression (Kim et al., 2000).
It is exciting to find that
. List of Selected Gene Probe IDs used in the bioinformatics analyses
GPL* | Gene symbol | Probe ID | GenBank access number |
---|---|---|---|
GPL570 | 208090_s_at | NM_000658 | |
211518_s_at | D30751 | ||
219025_at | NM_020404 | ||
214135_at | BE551219 | ||
219866_at | NM_016929 | ||
235720_at | AI042209 | ||
221204_s_at | NM_018058 | ||
1555497_a_at | AY151049 | ||
214608_s_at | AJ000098 | ||
205782_at | NM_002009 | ||
208449_s_at | NM_006119 | ||
206018_at | NM_005249 | ||
207683_at | NM_003593 | ||
238222_at | AI821357 | ||
232116_at | AL137763 | ||
208604_s_at | NM_030661 | ||
219832_s_at | NM_017410 | ||
205700_at | NM_003725 | ||
221165_s_at | NM_020525 | ||
205207_at | NM_000600 | ||
206693_at | NM_000880 | ||
1552478_a_at | NM_006147 | ||
206104_at | NM_002202 | ||
205266_at | NM_002309 | ||
229584_at | AK026776 | ||
203238_s_at | NM_000435 | ||
230170_at | AI079327 | ||
1553492_a_at | NM_006192 | ||
207059_at | NM_006194 | ||
227848_at | AI218954 | ||
206311_s_at | NM_000928 | ||
215454_x_at | AI831055 | ||
207586_at | NM_000193 | ||
205817_at | NM_005982 | ||
223816_at | AF242557 | ||
228038_at | AI669815 | ||
234310_s_at | AK026431 | ||
207662_at | NM_005992 | ||
232122_s_at | AK022666 | ||
208606_s_at | NM_030761 | ||
205990_s_at | NM_003392 | ||
221029_s_at | NM_030775 | ||
GPL1261 | 1419241_a_at | NM_009646 | |
1422938_at | NM_009741 | ||
1422912_at | NM_007554 | ||
1417439_at | NM_054042 | ||
1451410_a_at | AF367970 | ||
1420498_a_at | NM_023118 | ||
1417312_at | AK004853 | ||
1421727_at | NM_010164 | ||
1422243_at | NM_008008 | ||
1451882_a_at | U18673 | ||
1418357_at | NM_008241 | ||
1450508_at | NM_008238 | ||
1416855_at | BB550400 | ||
1452421_at | BB496114 | ||
1425874_at | AF193796 | ||
1450297_at | NM_031168 | ||
1422080_at | NM_008371 | ||
1418301_at | NM_016851 | ||
1422720_at | BQ176915 | ||
1450160_at | AF065917 | ||
1449328_at | NM_013825 | ||
1443260_at | BB055155 | ||
1421964_at | NM_008716 | ||
1438767_at | BB237825 | ||
1449359_at | NM_008780 | ||
1421246_at | BC005794 | ||
1449542_at | NM_008783 | ||
1453150_at | BG069341 | ||
1436869_at | AV304616 | ||
1427277_at | BB137929 | ||
1456862_at | AI893638 | ||
1423476_at | BB329435 | ||
1416967_at | U31967 | ||
1425779_a_at | AF326960 | ||
1450254_at | NM_009354 | ||
1450782_at | NM_009523 | ||
1436791_at | BB067079 | ||
1422602_a_at | NM_009525 | ||
GPL2987 | hCG31797.3 | NM_003593.2 | |
hCG1640627.4 | NM_153632.1, NM_030661.3, NM_153631.1 | ||
hCG20991.2 | NM_006194.1 | ||
hCG29190.4 | NM_033051.2 | ||
GPL8217 | HSG00201177 (ROSETTAGENE MODEL_ID) | NM_006015 | |
HSG00314123 (ROSETTAGENE MODEL_ID) | NM_002309 | ||
HSG00282340 (ROSETTAGENE MODEL_ID) | NM_030775 | ||
HSG00262163 (ROSETTAGENE MODEL_ID) | NM_033051 |
*GPL, GEO platform accession number
. List of genes used in expression profiling during organogenesis
Gene* name | Full name | Function | Reference | TSCOT expression from GEO data |
---|---|---|---|---|
Autoimmune regulator | Regulate mTEC development and differentiation, Transcription factor | Gordon and Manley, 2011; Sun et al., 2013 | ||
Growth Arrest-Specific 1 | Antiapoptotic gene | Wong et al., 2014 | ||
Bone morphogenic protein 4 | Essential for thymus and parathyroid morphogenesis prior to Foxn1 | Gordon et al., 2010; Gordon and Manley, 2011 | Higher TSCOT level in BMP4 treated 10T1/2 stem cells (GDS3025/GSE5921) (P: 0.4685) | |
CD248 Molecule, Endosialin | Required for postnatal thymic growth and regeneration following infection-dependent thymic atrophy | Liu et al., 2014 | ||
Cystein-Rich Protein 3 (Thymus Lim Protein) | Appears to have a role in normal thymus development | Kirchner et al., 2001 | ||
Mitogen-Responsive Phosphoprotein, Homolog | Wnt-inhibitors, Control proliferation and differentiation of stem cells into lineage-restricted cells | Wong et al., 2014 | ||
Dickkopf WNT Signaling Pathway Inhibitor 3 | Wnt-inhibitors, Control proliferation and differentiation of stem cells into lineage-restricted cells | Wong et al., 2014 | ||
Eyes absent 1 homolog | Necessary for 3rd pouch development | Wei and Condie, 2011; Gordon and Manley, 2011 | ||
Keratinocyte growth factor | Induces mature and immature TECs and promotes differentiation of immature TECs | Rossi et al., 2006 | ||
Fibroblast growth factor 8 | Indirectly influence TECs by regulating neural crest cells survival and differentiation, relate to early pouch formation | Gordon and Manley, 2011; Sun et al., 2013 | ||
Forkhead Box G1 | May play a role in the regulation of TEC differentiation during fetal and postnatal stages, Transcription factor | Wei and Condie, 2011 | ||
Forkhead Box N1 | Necessary for the development of immature TEC progenitor cells into cTECs and mTECs, Transcription factor | Blackburn et al., 1996; Bennett et al., 2002; Gordon and Manley, 2011; Bredenkamp et al., 2014 | ||
Growth Arrest-Specific 1 | Cell-cycle suppressor gene | Wong et al., 2014 | ||
Grainyhead-Like 3 | Ancient mediator of epithelial integrity, Transcription factor | Yu et al., 2008; de la Garza et al., 2012 | Reduced TSCOT level in Get-1 KO skin (GDS2629/GSE7381) (P: 0.0042**) | |
Homeobox A3 | Early pouch patterning and initial organ formation, Transcription factor | Manley and Capecchi, 1995; Su et al., 2001; Gordon and Manley, 2011 | ||
Homeobox C13 | Mediates transcriptional regulation of Foxn1, Transcription factor | Potter et al., 2010 | ||
Interleukin 22 | Leads to regeneration of supporting epithelial microenvironment for enhanced thymopoiesis after thymic injury | Dudakov et al., 2012 | Reduced TSCOT level of IL22 treated epidermal keratinocytes (GDS2611/ GSE7216) (p < 0.0001****) | |
Interleukin 6 | Associated with thymic involution | Chinn et al., 2012 | ||
Interleukin 7 | Cofactor for V(D)J rearrangement of the T cell receptor beta during early T cell development | Huang and Muegge, 2001; Zamisch et al., 2005 | ||
Interferon regulatory factor 6 | Key determinant of keratinocyte proliferation-differentiation switch, Transcription factor | Richardson et al., 2006 | Reduced TSCOT level in IRF6 KO skin (GDS2359/GSE5800) (P< 0.0001****) | |
ISL LIM Homeobox 1 | May play a role in the regulation of TEC differentiation during fetal and postnatal stages, Transcription factor | Wei and Condie, 2011 | ||
Leukemia inhibitory factor | Maintenance mouse ES cell pluripotency, Associated with thymic involution | Shen and Leder, 1992; Graf et al., 2011; Chinn et al., 2012 | Increased TSCOT level in murine CGR8 ES cells treated LIF (GDS3729/ GSE6689) (P: 0.1181) | |
Lymphocyte antigen 75 | Contribute to antigen presentation, Marker of cTEC in adult thymus | Jiang et al., 1995; Shakib et al., 2009 | ||
Myeloid ecotropic viral integration site 1 | Functional and physical partners of Pbx1 and Hoxa3, Required for maintenance of the postnatal thymic microenvironment, Transcription factor | Hirayama et al., 2014 | ||
Notch homolog protein 3 | Regulate murine T cell differentiation and leukemogenesis | Bellavia et al., 2008 | ||
Oncostatin M | Plays an inhibitory role in normal and malignant mammary epithelial cell growth in vitro, Associated with thymic involution | Liu et al., 1998; Chinn et al., 2012 | ||
Paired Box 1 | Early pouch formation and parathyroid development, minor role in thymus size, Transcription factor | Wallin et al., 1996; Gordon and Manley, 2011 | ||
Paired Box 9 | Pouch and initial organ formation, TEC differentiation, Transcription factor | Hetzer-Egger et al., 2002; Gordon and Manley, 2011 | ||
Pre-B-cell leukemia homeobox | Required for embryonic thymic organogenesis, Transcription factor | Hirayama et al., 2014 | ||
Proteasome (prosome, macropain) subunit, beta type, 11 | Positive selection of CD8+ T cells, cTEC specific proteosome subunit | Murata et al., 2007; Shakib et al., 2009 | ||
Sonic hedgehog | Regulate pharyngeal region development | Moore-Scott and Manley, 2005; Gordon and Manley, 2011 | Increased TSCOT level in SHH treated human fibroblasts (GDS4512/ GSE29316) (P: 0.1122) | |
Sine oculis-related homeobox 1/4 | Necessary for 3rd pouch development, Transcription factor | Wei and Condie, 2011; Gordon and Manley, 2011 | ||
SRY (sex determining region Y)-box 2 | Regulate self-renewal of the mouse and human ESCs, important for the maintenance of stem cells in multiple adult tissue, establish induced pluripotent stem cells, Transcription factor | Cimpean et al., 2011; Liu et al., 2013 | Higher TSCOT level in SOX2+ follicle dermal cells (GDS3753/ GSE18690) (P: 0.0015**) | |
T-box transcription factor | Pouch formation and patterning, might establish parathyroid fate, Transcription factor | Jerome and Papaioannou, 2001; Hollander et al., 2006; Gordon and Manley, 2011 | ||
Telomerase Reverse Transcriptase | Telomerase reverse transcriptase | Wong et al., 2014 | ||
Wingless-type MMTV integration site family, member 4 | Controls thymopoiesis and thymus size by regulating TEC, thymocyte and their progenitor proliferation, regulate Foxn1 expression in TECs | Sun et al., 2013 | ||
Wingless-type MMTV integration site family, 5A | Regulate the survival of αβ lineage thymocytes, regulator of cell growth in hematopoietic tissue | Liang et al., 2007 | ||
Wingless-type MMTV integration site family, 5B | Produced by TECs and thymocytes, regulate Foxn1 expression in TECs | Gordon and Manley, 2011; Sun et al., 2013 |
*Gene names are listed in alphabetical order.
. List of genes down-regulated along with TSCOT during lung cancer development
Gene name* | Full name | Relation with cancer | Reference |
---|---|---|---|
Claudin-18 | CLDN18 splice variant 2 is frequent Ectopic activation in pancreatic, Esophageal, ovarian, and lung tumors | Sahin et al., 2008 | |
Cartilage acidic protein 1 | Copy number alteration in CRTAC1 gene have been observed in neurofibromatosis Type 1-associated glomus tumors | Brems et al., 2009 | |
Cytochrome P450, Family 4, Subfamily B, Polypeptide 1 | High expression of CYP4B1 increases the risk of bladder tumor by activation of carcinogenic aromatic amines | Imaoka et al., 2000 | |
Gastrokine-2 | Gastrointestinal tract specific gene GKN2 might inhibit gastric cancer growth in a TFF1 dependent manner | Chu et al., 2012 | |
Leucine-rich repeat serine | LRRK2 G2019S mutations are associated with an increased cancer risk in Pakinson’s disease | Saunders-Pullman et al., 2010 | |
Sushi domain-containing protein 2 | SUSD2 increases the invasion of breast cancer cells and contributes to a potential immune evasion | Watson et al., 2013 |
*Gene names are listed in alphabetical order
Mol. Cells 2015; 38(6): 548-561
Published online June 30, 2015 https://doi.org/10.14348/molcells.2015.0044
Copyright © The Korean Society for Molecular and Cellular Biology.
Ki Yeon Kim1, Gwanghee Lee2,4, Minsang Yoon1, Eun Hye Cho1, Chan-Sik Park3, and Moon Gyo Kim1,*
1Department of Biological Sciences, Inha University, Incheon 402-720, Korea, 2Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, MO 63110, USA, 3Department of Pathology, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736, Korea, 4Present address: Therapeutic strategic unit, Asia Pacific R&D, Sanofi, Daejeon 302-120, Korea
Correspondence to:*Correspondence: mgkim@inha.ac.kr
By combining conventional single cell analysis with flow cytometry and public database searches with bioinformatics tools, we extended the expression profiling of thymic stromal cotransporter (TSCOT), Slc46A2/Ly110, that was shown to be expressed in bipotent precursor and cortical thymic epithelial cells. Genome scale analysis verified
Keywords: Ly110, SLC46A2, stem cell, thymic epithelial cell, TSCOT, tumor suppressor
Thymus produces educated T cells that can react with peptide antigen loaded on a self major histocompatibility complex (MHC) but not with self antigens. Developing thymocytes are guided and selected in the microenvironment of thymic stromal cells. Among the stromal cells, thymic epithelial cells (TECs) are major components that plays important roles of thymocyte differentiation in the separate compartments, the cortex and the medulla.
TECs also play critical roles during thymic organogenesis as shown in Foxn1 mutant mice, in which early TEC differentiation is abrogated, functional thymus lacks and, therefore, no T cell is present (Nehls et al., 1996). In mice, initial thymic structure begins to form with the TEC precursor cells originated from the third pharyngeal pouch around fetal day 10.5 (Blackburn and Manley, 2004; Gill et al., 2003; Rodewald, 2008; Su et al., 2001). At this stage, fetal thymus does not show clear medullary compartmentalization yet although the cells of medullary thymic epithelial cells (mTECs) in nature are found (Roberts et al., 2012). Fetal thymus begins to express the cortex-specific markers such as CDR1 in addition to general epithelial markers, EpCAM and MHCII (Ahn et al., 2008; Boehm, 2008; Lee et al., 2012; Yang et al., 2005). Later, thymus undergoes atrophy by aging after puberty and/or by damaging insults such as radiation or stress hormones (Blackburn et al., 2002; Cheng et al., 2010; Gill et al., 2003). However, thymus can also be rejuvenated by removing steroid sex hormones or removing organs that produce sex hormones (Berzins et al., 2002; Lynch et al., 2009; Sutherland et al., 2005). The functional thymic epithelial stem cell (sTEC) in the adult or aged animals were identified (Blackburn et al., 2002; Rodewald et al., 2001; Swann and Boehm, 2007; Ucar et al., 2014; Wong et al., 2014). It is important to identify the molecular marker present in the sTEC to understand the mechanism of thymic regeneration and to translate into the clinic for the recovery of important cellular immunity.
There has been much evidence that cortical TEC (cTEC) and mTEC are derived from the single precursor TECs (pTEC) or sTEC (Bleul et al., 2006; Rossi et al., 2006). While pTEC can be bipotent or specific lineage- committed (Park et al., 2013; Ucar et al., 2014), TEC development may be more progressive without instant commitment to a specific lineage (Alves et al., 2014). The original specific antibodies used for the identification of sTECs are MTS24 and MTS20 (Bennett et al., 2002; Gill et al., 2002). These TEC stem cells were located in the small medullary islets of very young thymus or corticomedullary junction of the adult thymus (Rodewald et al., 2001). The cytokeratin K5 and K8 are also important molecules for the identification of sTECs (Klug et al., 1998; 2002). It was also proposed that TEC stem cells reside in the MTS10+ cells in the medullary area.
It has been considered that
However, the presence of sTEC without
Expression of
In this study, we took advantage of public database and bioinformatics tools and performed genetic profiling in addition to classical methodologies. We show
The data sets were obtained from Gene Expression Omnibus (
The mouse lines TDLacZ (Ahn et al., 2008), 3.1T-EGFP (Chen et al., 2000), and 9.1T-NE (Lee et al., 2012) were maintained in the Laboratory of Molecular and Cellular Immunology Animal Facility of Inha University, Korea. All animal studies are in compliance with the Use of Laboratory Animals under the proper protocols. The protocols were approved by the Committees on the Ethics of Animal Experiments of NIH (LCMI Protocol 8) and Inha University (Protocol LMCI-2). Fetal mice were obtained from timed mating. The presence of a vaginal plug was considered at E0.5.
For genotyping, tail samples were extracted and used for a polymerase chain reaction with primers for the TDLacZ locus: Neo primer (ACCGCTATCAGGACATAGCGTTGG), 1C12 F1 (TTACTCAAAGTGATGCTGGACTGG), 1C12 B2 (CCGAGGGTTCCTTGGTACATTC), and the EGFP locus: EGFP-F (GCCACAAGTTCAGCGTGTCC), EGFP-R (GCTTCTGTTGGGGTCTTTGC), using the red Extract-N-Amp Tissue PCR kit (Sigma).
A fluorescence-based, automatic cell counter (Luna-FL, Logos Biosystems) was used to measure accurately the numbers of cells including thymic epithelial cells. The contamination from red blood cells could be automatically excluded because this system enumerates only nucleated cells.
A single cell suspension was prepared as described (Lee et al., 2012). Briefly, thymic tissues or deoxyguanosine treated fetal thymic organ culture were treated with 0.25% trypsin (Invitrogen) for about 20 min, in the presence of DNase I (Sigma), and washed with phosphate buffered saline (PBS) containing 10% fetal bovine serum (FBS). For further purification of TEC, the single cell suspension was isolated using magnetic bead cell sorting after incubating with anti-Fc mAb 2.4G2 and anti-mouse CD45 microbeads (Milteny Biotec) for 20 min at 4°C.
Monoclonal antibodies used in the staining of cells include anti-MHCII (I-Ab), anti-CD45 (Ly-5), and anti-Sca-1. The antibodies were purchased from Caltag or from BD PharMingen. Anti-aminopeptidase A (CDR-1) and anti-EpCAM (G8.8) were prepared in the Custom Antibody Services Facility, NIAID, NIH. Biotinylated UEA-1 was purchased from Vector Laboratories.
Cells were washed in cold FACS buffer (PBS + 1% BSA), subsequently stained on ice with the primary and the secondary antibodies, then analyzed on FACSCalibur or FACSAriaII with two lasers in the presence of 1?2 μg/ml of propidium iodide (PI). Anti-Fc, 2.4G2 antibody was included in all flow cytometry staining to block Fc receptor. For side population analysis, a 1 × 106 dissociated single cell suspension of fetal thymic organ culture (FD14.5) were incubated for an hour at 4°C in the presence of 5 μg/ml Hoechst 33342 dissolved in Hanks balanced salt solution. For verification of the side population, verapamil 0.25 mM was included. After washing at 4°C, cells were resuspended and examined by a flow cytometer equipped with a UV laser (FACSAriaII). For multicolor staining with SP analysis, cells were prestained with selected antibodies including homemade mAb CLVE (Yang et al., 2005). Negative control of TSCOT staining was carried out with all the same combination of antibodies except mAb CLVE. Analyses were done using the FlowJo program (
Sorted 1000 cells were used for RNA preparation. cDNA was generated with Superscript III and RT-PCR was carried out with the primers for TSCOT: F84 (5-CAGTCTTCCAATAACCTGCTTTGGCCT-3) and B83 (5-CGATTCCATGTGCCCCATTG-3) to amplify a 310 bp fragment and for GAPDH (Ahn et al., 2008;Kim et al., 2000). The primers for TSCOT are located in the separate exons with one intron and RT? control sampled did not show any band in the gel.
The immunofluorescence and X-gal staining the sections is described (Lee et al., 2012). An isolated thymus was washed in PBS and fixed in 1% para-formaldehyde, 0.2% glutaraldehyde, 0.02% NP-40, 1 mM MgCl2 in PBS for 1 or 2 h on ice and was embedded in Tissue Freezing Medium (Triangle Biomedical Sciences, USA). The 4 μm sections were fixed for 2 min in 1% formaldehyde, 0.2% glutaraldehyde, 0.02% NP-40 1 mM NaCl, then incubated with X-gal solution (1 part X-gal 40 μg/ml in dimethyl formamide, in 40 parts 2 mM MgCl2, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide in PBS) at 37°C for 48 h. For the detection of EGFP for fetal thymus sections, confocal microscopy was performed on the frozen sections in NIAID confocal facility (Leica SP2).
In order to study the expression pattern of
Expression profiles of
The expression of
From these analyses, it was concluded that
Next, we investigated the expression of TSCOT and reporters at the fetal stages in the different mouse models that we have previously characterized for the postnatal stages. The β-galactosidase reporter expression in TDLacZ thymus is restricted in the thymus as two dots at FD11 (Ahn et al., 2008). Figure 2A shows the β-galactosidase expression in the thymic sections at FD14.5. Expression of β-galactosidase is evenly distributed in the whole thymus, indicating most, if not all, the thymic epithelial cells at this stage express β-galactosidase. Another reporter mouse line, 3.1T-EGFP, which expresses EGFP in all TECs at the newborn stage (Park et al., 2013), showed EGFP expression earlier during fetal stages (Fig. 2B). At FD14 and 17, EGFP expression is also evenly distributed in the whole thymus. These results are consistent with the conclusion we previously described in which TSCOT is expressed in the pTEC stage (Park et al., 2013).
Fetal thymic stromal cells from normal C57BL/6 mouse (FD14) were analyzed for TSCOT expression with specific mAb CLVE (Yang et al., 2005). At this stage, EpCAM+ cells were all TSCOT+ (data not shown). When CD45? stromal cells were displayed for CD31 as an endothelial lineage marker along with MHCII, it became clear that TSCOT expression is present in all MHCII+ cells and CD31? MHCII? cells (Fig. 2C). Only CD31+MHCII? cells of endothelial lineage were TSCOT?. From these results, it is concluded that endothelial cells either lost TSCOT expression due to lineage commitment from the common stem cell or originated from other type of precursor cells that do not express TSCOT.
By using the TEC preparation from the deoxyguanosine treated FTOC of FD14.5, the presence of SP was tested with Hoechst 33342. In Fig. 3A, SP, which is ABC transporter sensitive, is clearly visible. When the inhibitor Verapamil was included, SP had decreased to 0.21% from 1.45%. Side population analyses were also applied with the TEC preparation using the same type culture of fetal thymus from 9.1T-NE mouse that shows EGFP expression patterns in the same way as endogenous TSCOT (Lee et al., 2012). As shown in Fig. 3B, a portion of the SP of 9.1T-NE TECs expresses EGFP when compared with that of normal C57BL/6 TEC preparation. In contrast, the side population of 3.1T-EGFP TEC population did not show any EGFP expression (data not shown).
When SP analysis was carried out along with antibody staining, TSCOT expression in SP and the major population (MP) were also clear. SP, either MHCII negative or positive, showed specific TSCOT expression with mAb CLVE. In addition, 84% of the SP cells and 95% of the MP cells were TSCOT+ (Fig. 3C). The next experiment was to verify TSCOT expression at the RNA level using a sorted side population of TECs prepared from normal C57BL/6. RT-PCR using the RNA prepared from the sorted cells clearly showed TSCOT expression in SP and in MP (Fig. 3D).
Many different types of stem cells express Sca-1 marker and a recent report mentioned that a TEC progenitor population is Sca-1+ (Golebiewska et al., 2011). We also tested expression of Sca-1 in fetal TEC preparation (Fig. 3E). Sca-1+ populations are present in both EpCAM+MHCII+ and EpCAM?MHCII? populations and most of them are TSCOT+. From these results, fetal TEC contains significant portion of sTECs that are TSCOT+.
We searched the available data on
The expression profile of
When the data from various side populations were specifically searched for, SP of mouse mammary gland cells showed an increased
From the results above, we concluded that
Because FOXN1 was considered as a putative TEC key transcription factor, we searched for
In order to find the putative regulatory factors, differential
These results suggest a regulatory mechanism for
We also researched
In Fig. 6B, the cluster analysis of 20 genes coexpressed in the same fashion as TSCOT is shown in Table 3. To our surprise,
Using bioinformatics approaches and conventional molecular and cellular methods, we showed that
During last several years, gene expression profiling at the genome scale are accumulated in the databases and accessible to the public. We took advantage of this advancement for the study of thymic organogenesis and TEC lineage differentiation using TSCOT as a lineage cell type specific marker and other known genes. From this approach, we learned that our previous studies are verified and we can get a lot more information than conventional experiments that are difficult to perform due to the limitation of small numbers of cells present in the actual organ.
In our previous studies, we asserted that TSCOT is TEC lineage specific and expressed in the cTEC and bipotent pTEC (Ahn et al., 2008; Kim et al., 2000; Park et al., 2013). Tissue specificity in thymus and expression in the limited TEC lineages are verified by the genome scale data analysis of expression profiling (Figs. 1, 4, and 5). Furthermore, we learned that more tissues such as skin and lung also express
To further investigate cells at earlier stages than pTEC expression, we first utilized a functional SP analysis and showed that SP of fetal TEC preparation expresses
Given the concept that
A schematic model in Figure 7 summarizes the findings of the expression profile during organogenesis.
By cluster analyses with selected transcription and soluble factors that are shown to be involved during the thymic organogenesis, we were able to identify multiple putative positive or negative regulators. IRF6 and GRHL3 are putative positive regulators for
Besides the structural features of a transporter that appeared containing primary amino acid sequences (Kim et al., 2000), it is still unclear what biological and biochemical functions that TSCOT plays. TSCOT is a member of Slc46A, and another member, Slc46A1, has been characterized as a proton coupled folate transporter (Diop-Bove et al., 2013). The heavily hydrophobic nature of the TSCOT amino acid composition and a simple twelve membrane spanning feature, with the presence of a central inner loop in the absence of ATP binding domain, suggests that TSCOT may transport small hydrophobic molecules. We proposed earlier that it may function in the survival of TECs based on the expression (Kim et al., 2000).
It is exciting to find that
. List of Selected Gene Probe IDs used in the bioinformatics analyses.
GPL* | Gene symbol | Probe ID | GenBank access number |
---|---|---|---|
GPL570 | 208090_s_at | NM_000658 | |
211518_s_at | D30751 | ||
219025_at | NM_020404 | ||
214135_at | BE551219 | ||
219866_at | NM_016929 | ||
235720_at | AI042209 | ||
221204_s_at | NM_018058 | ||
1555497_a_at | AY151049 | ||
214608_s_at | AJ000098 | ||
205782_at | NM_002009 | ||
208449_s_at | NM_006119 | ||
206018_at | NM_005249 | ||
207683_at | NM_003593 | ||
238222_at | AI821357 | ||
232116_at | AL137763 | ||
208604_s_at | NM_030661 | ||
219832_s_at | NM_017410 | ||
205700_at | NM_003725 | ||
221165_s_at | NM_020525 | ||
205207_at | NM_000600 | ||
206693_at | NM_000880 | ||
1552478_a_at | NM_006147 | ||
206104_at | NM_002202 | ||
205266_at | NM_002309 | ||
229584_at | AK026776 | ||
203238_s_at | NM_000435 | ||
230170_at | AI079327 | ||
1553492_a_at | NM_006192 | ||
207059_at | NM_006194 | ||
227848_at | AI218954 | ||
206311_s_at | NM_000928 | ||
215454_x_at | AI831055 | ||
207586_at | NM_000193 | ||
205817_at | NM_005982 | ||
223816_at | AF242557 | ||
228038_at | AI669815 | ||
234310_s_at | AK026431 | ||
207662_at | NM_005992 | ||
232122_s_at | AK022666 | ||
208606_s_at | NM_030761 | ||
205990_s_at | NM_003392 | ||
221029_s_at | NM_030775 | ||
GPL1261 | 1419241_a_at | NM_009646 | |
1422938_at | NM_009741 | ||
1422912_at | NM_007554 | ||
1417439_at | NM_054042 | ||
1451410_a_at | AF367970 | ||
1420498_a_at | NM_023118 | ||
1417312_at | AK004853 | ||
1421727_at | NM_010164 | ||
1422243_at | NM_008008 | ||
1451882_a_at | U18673 | ||
1418357_at | NM_008241 | ||
1450508_at | NM_008238 | ||
1416855_at | BB550400 | ||
1452421_at | BB496114 | ||
1425874_at | AF193796 | ||
1450297_at | NM_031168 | ||
1422080_at | NM_008371 | ||
1418301_at | NM_016851 | ||
1422720_at | BQ176915 | ||
1450160_at | AF065917 | ||
1449328_at | NM_013825 | ||
1443260_at | BB055155 | ||
1421964_at | NM_008716 | ||
1438767_at | BB237825 | ||
1449359_at | NM_008780 | ||
1421246_at | BC005794 | ||
1449542_at | NM_008783 | ||
1453150_at | BG069341 | ||
1436869_at | AV304616 | ||
1427277_at | BB137929 | ||
1456862_at | AI893638 | ||
1423476_at | BB329435 | ||
1416967_at | U31967 | ||
1425779_a_at | AF326960 | ||
1450254_at | NM_009354 | ||
1450782_at | NM_009523 | ||
1436791_at | BB067079 | ||
1422602_a_at | NM_009525 | ||
GPL2987 | hCG31797.3 | NM_003593.2 | |
hCG1640627.4 | NM_153632.1, NM_030661.3, NM_153631.1 | ||
hCG20991.2 | NM_006194.1 | ||
hCG29190.4 | NM_033051.2 | ||
GPL8217 | HSG00201177 (ROSETTAGENE MODEL_ID) | NM_006015 | |
HSG00314123 (ROSETTAGENE MODEL_ID) | NM_002309 | ||
HSG00282340 (ROSETTAGENE MODEL_ID) | NM_030775 | ||
HSG00262163 (ROSETTAGENE MODEL_ID) | NM_033051 |
*GPL, GEO platform accession number
. List of genes used in expression profiling during organogenesis.
Gene* name | Full name | Function | Reference | TSCOT expression from GEO data |
---|---|---|---|---|
Autoimmune regulator | Regulate mTEC development and differentiation, Transcription factor | Gordon and Manley, 2011; Sun et al., 2013 | ||
Growth Arrest-Specific 1 | Antiapoptotic gene | Wong et al., 2014 | ||
Bone morphogenic protein 4 | Essential for thymus and parathyroid morphogenesis prior to Foxn1 | Gordon et al., 2010; Gordon and Manley, 2011 | Higher TSCOT level in BMP4 treated 10T1/2 stem cells (GDS3025/GSE5921) (P: 0.4685) | |
CD248 Molecule, Endosialin | Required for postnatal thymic growth and regeneration following infection-dependent thymic atrophy | Liu et al., 2014 | ||
Cystein-Rich Protein 3 (Thymus Lim Protein) | Appears to have a role in normal thymus development | Kirchner et al., 2001 | ||
Mitogen-Responsive Phosphoprotein, Homolog | Wnt-inhibitors, Control proliferation and differentiation of stem cells into lineage-restricted cells | Wong et al., 2014 | ||
Dickkopf WNT Signaling Pathway Inhibitor 3 | Wnt-inhibitors, Control proliferation and differentiation of stem cells into lineage-restricted cells | Wong et al., 2014 | ||
Eyes absent 1 homolog | Necessary for 3rd pouch development | Wei and Condie, 2011; Gordon and Manley, 2011 | ||
Keratinocyte growth factor | Induces mature and immature TECs and promotes differentiation of immature TECs | Rossi et al., 2006 | ||
Fibroblast growth factor 8 | Indirectly influence TECs by regulating neural crest cells survival and differentiation, relate to early pouch formation | Gordon and Manley, 2011; Sun et al., 2013 | ||
Forkhead Box G1 | May play a role in the regulation of TEC differentiation during fetal and postnatal stages, Transcription factor | Wei and Condie, 2011 | ||
Forkhead Box N1 | Necessary for the development of immature TEC progenitor cells into cTECs and mTECs, Transcription factor | Blackburn et al., 1996; Bennett et al., 2002; Gordon and Manley, 2011; Bredenkamp et al., 2014 | ||
Growth Arrest-Specific 1 | Cell-cycle suppressor gene | Wong et al., 2014 | ||
Grainyhead-Like 3 | Ancient mediator of epithelial integrity, Transcription factor | Yu et al., 2008; de la Garza et al., 2012 | Reduced TSCOT level in Get-1 KO skin (GDS2629/GSE7381) (P: 0.0042**) | |
Homeobox A3 | Early pouch patterning and initial organ formation, Transcription factor | Manley and Capecchi, 1995; Su et al., 2001; Gordon and Manley, 2011 | ||
Homeobox C13 | Mediates transcriptional regulation of Foxn1, Transcription factor | Potter et al., 2010 | ||
Interleukin 22 | Leads to regeneration of supporting epithelial microenvironment for enhanced thymopoiesis after thymic injury | Dudakov et al., 2012 | Reduced TSCOT level of IL22 treated epidermal keratinocytes (GDS2611/ GSE7216) (p < 0.0001****) | |
Interleukin 6 | Associated with thymic involution | Chinn et al., 2012 | ||
Interleukin 7 | Cofactor for V(D)J rearrangement of the T cell receptor beta during early T cell development | Huang and Muegge, 2001; Zamisch et al., 2005 | ||
Interferon regulatory factor 6 | Key determinant of keratinocyte proliferation-differentiation switch, Transcription factor | Richardson et al., 2006 | Reduced TSCOT level in IRF6 KO skin (GDS2359/GSE5800) (P< 0.0001****) | |
ISL LIM Homeobox 1 | May play a role in the regulation of TEC differentiation during fetal and postnatal stages, Transcription factor | Wei and Condie, 2011 | ||
Leukemia inhibitory factor | Maintenance mouse ES cell pluripotency, Associated with thymic involution | Shen and Leder, 1992; Graf et al., 2011; Chinn et al., 2012 | Increased TSCOT level in murine CGR8 ES cells treated LIF (GDS3729/ GSE6689) (P: 0.1181) | |
Lymphocyte antigen 75 | Contribute to antigen presentation, Marker of cTEC in adult thymus | Jiang et al., 1995; Shakib et al., 2009 | ||
Myeloid ecotropic viral integration site 1 | Functional and physical partners of Pbx1 and Hoxa3, Required for maintenance of the postnatal thymic microenvironment, Transcription factor | Hirayama et al., 2014 | ||
Notch homolog protein 3 | Regulate murine T cell differentiation and leukemogenesis | Bellavia et al., 2008 | ||
Oncostatin M | Plays an inhibitory role in normal and malignant mammary epithelial cell growth in vitro, Associated with thymic involution | Liu et al., 1998; Chinn et al., 2012 | ||
Paired Box 1 | Early pouch formation and parathyroid development, minor role in thymus size, Transcription factor | Wallin et al., 1996; Gordon and Manley, 2011 | ||
Paired Box 9 | Pouch and initial organ formation, TEC differentiation, Transcription factor | Hetzer-Egger et al., 2002; Gordon and Manley, 2011 | ||
Pre-B-cell leukemia homeobox | Required for embryonic thymic organogenesis, Transcription factor | Hirayama et al., 2014 | ||
Proteasome (prosome, macropain) subunit, beta type, 11 | Positive selection of CD8+ T cells, cTEC specific proteosome subunit | Murata et al., 2007; Shakib et al., 2009 | ||
Sonic hedgehog | Regulate pharyngeal region development | Moore-Scott and Manley, 2005; Gordon and Manley, 2011 | Increased TSCOT level in SHH treated human fibroblasts (GDS4512/ GSE29316) (P: 0.1122) | |
Sine oculis-related homeobox 1/4 | Necessary for 3rd pouch development, Transcription factor | Wei and Condie, 2011; Gordon and Manley, 2011 | ||
SRY (sex determining region Y)-box 2 | Regulate self-renewal of the mouse and human ESCs, important for the maintenance of stem cells in multiple adult tissue, establish induced pluripotent stem cells, Transcription factor | Cimpean et al., 2011; Liu et al., 2013 | Higher TSCOT level in SOX2+ follicle dermal cells (GDS3753/ GSE18690) (P: 0.0015**) | |
T-box transcription factor | Pouch formation and patterning, might establish parathyroid fate, Transcription factor | Jerome and Papaioannou, 2001; Hollander et al., 2006; Gordon and Manley, 2011 | ||
Telomerase Reverse Transcriptase | Telomerase reverse transcriptase | Wong et al., 2014 | ||
Wingless-type MMTV integration site family, member 4 | Controls thymopoiesis and thymus size by regulating TEC, thymocyte and their progenitor proliferation, regulate Foxn1 expression in TECs | Sun et al., 2013 | ||
Wingless-type MMTV integration site family, 5A | Regulate the survival of αβ lineage thymocytes, regulator of cell growth in hematopoietic tissue | Liang et al., 2007 | ||
Wingless-type MMTV integration site family, 5B | Produced by TECs and thymocytes, regulate Foxn1 expression in TECs | Gordon and Manley, 2011; Sun et al., 2013 |
*Gene names are listed in alphabetical order.
. List of genes down-regulated along with TSCOT during lung cancer development.
Gene name* | Full name | Relation with cancer | Reference |
---|---|---|---|
Claudin-18 | CLDN18 splice variant 2 is frequent Ectopic activation in pancreatic, Esophageal, ovarian, and lung tumors | Sahin et al., 2008 | |
Cartilage acidic protein 1 | Copy number alteration in CRTAC1 gene have been observed in neurofibromatosis Type 1-associated glomus tumors | Brems et al., 2009 | |
Cytochrome P450, Family 4, Subfamily B, Polypeptide 1 | High expression of CYP4B1 increases the risk of bladder tumor by activation of carcinogenic aromatic amines | Imaoka et al., 2000 | |
Gastrokine-2 | Gastrointestinal tract specific gene GKN2 might inhibit gastric cancer growth in a TFF1 dependent manner | Chu et al., 2012 | |
Leucine-rich repeat serine | LRRK2 G2019S mutations are associated with an increased cancer risk in Pakinson’s disease | Saunders-Pullman et al., 2010 | |
Sushi domain-containing protein 2 | SUSD2 increases the invasion of breast cancer cells and contributes to a potential immune evasion | Watson et al., 2013 |
*Gene names are listed in alphabetical order
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