Fig 1.
Anaysis of transcriptomes obtained from jejunal and colonic PDGFRα+ cells.
(A) Identification of PDGFRα+ cells in the intestinal smooth muscle with eGFP and a PDGFRA antibody. A z-stack image, obtained through confocal microscopy, of whole-mount jejunum muscularis showing PDGFRα+ cells expressing eGFP in the nucleus (left). Immunohistochemistry of PDGFRα+ cells using a PDGFRA antibody (middle). Merged images of eGFP and PDGFRA (right). (B) Primary eGFP+ PDGFRα+ cells from jejunum (left) and colon (right) identified (circled) by flow cytometry. (C) Venn diagram showing the number of genes identifed in jejunal and colonic PDGFRα+ cells (JPαC anc CPαC) by RNA-seq. (D) Comparison of expression levels of genes in JPαC and CPαC. (E) Gene ontologies reported in JPαC and CPαC. The gene ontology (GO: function, process, and component) of PαC-specific genes was analyzed, and key GO terms were compared using normalized expression (FPKM) percentile.
Fig 2.
Comparison of ion channel and transporter isoform genes expressed in PDGFRα+ cells.
(A) Comparison of expression levels of ion channel isoforms in JPαC and CPαC. (B) Comparison of expression levels of ion transproter isoforms in JPαC and CPαC. (C) Ca2+ channel isoforms enriched in JPαC and CPαC. (D) PαC-specific Ca2+ channel isoforms. Cell specificity was determined by comparative analysis of gene expression profiles among PαC, SMC, and ICC: PαCexpression level (FPKM)/[SMCexpression level (FPKM) + ICCexpression level (FPKM)]. (E and F) Voltage-dependent Ca2+ channel isoforms (L-type: Cacna1c & d, T-type: Cacna1h & g, R-type: Cacna1e, P/Q-type: Cacna11) expressed in JPαC (E) and CPαC (F).
Fig 3.
Identification of mutiple Cacna1g trancritional variants.
(A) A genomic map view of Cacna1g variants expressed in JPαC and CPαC. Seven alternative initial exons (V1-7) are circled in red and six differentially spliced exons (E8, E14, E25, E30, E34, and E37) are boxed in blue. (B) Expression (FPKM) levels of total Cacna1g mRNAs in JPαC and CPαC. (C) Expression levels of Cacna1g transcriptional vaiants in JPαC and CPαC. (D) A topological map of CACNA1G variants. Each circle denotes a single amino acid. Colors on amino acid sequence show distinct regions and domains. Red represents missing, or inserted, peptides from differentially spliced exons. Green represents start codons found in differentially spliced variants. Six transmembrane domains (S1-6) and a pore region are shown.
Fig 4.
Increased PDGFRα+ cells in hypertrophic smooth muscle.
Hypertrophic tissue was surgically induced for ~2 weeks by placing a small silicone ring on the distal ileum of transgenic PDGFRα-eGFP mice to partially obstruct normal peristaltic movement. (A) Gross images of GI tract in sham and obstruction surgeries. (B) Representative H&E staining of jejunal cross sections from sham control and partially obstructed mice. Hypertrophied jejunum contained significantly thicker circular and longitudinal muscle layers compared to a sham control. Scale bar: 50 μm. (C) Representative confocal laser scanning images of jejunal cross sections from sham operation control and partial obstruction mice showing nuclear eGFP expression in PDGFRα+ cells and DAPI (blue) counterstained in the cells. Scale bar: 50 μm. (D) Two populations (eGFPhigh and eGFPlow) of primary PDGFRα+ cells from hypertrophic jejunum identified by flow cytometry. Note that eGFPhigh PDGFRα+ cells are significantly increased in partial obstruction smooth muscle. (E) A z-stack image, obtained through confocal microscopy, of whole-mount jejunum muscularis from the partial obstruction (bottom) and sham operation control (top) showing eGFPhigh (arrow heads) and eGFPlow (arrows) PDGFRα+ cells. Scale bar: 50 μm.
Fig 5.
Identification of PDGFRα+ cell subpopulations dedifferentiated in hypertrophic smooth muscle.
(A-C) Confocal section images of PDGFRα+ (Pdgfra-eGFP+) cell subpopulations identified in the subserosal layer (SS), myenteric region (MY), and deep muscular plexus (DMP) with PDGFRA antibody (A), eGFP (B) and merged (C) in jejunum. (D and E) Cross section images of PDGFRα+ cell subpopulations (green) in sham operation (SO, D) and partial obstruction (PO, E). Proliferating PDGFRα+ cells are marked by arrows. LM, lonitudinal muscle; CM, circular muscle; SM, submucosa; M, mucosa. All scale bars are 50 μm.
Fig 6.
Induced expression of Cacna1g mRNAs and protein in hypertrophic smooth muscle.
(A) Expression of Cacna1g exons with alternative transcriptional start sites (ATSS) and/or alternative spliced sites (ASS) in sham operation (SO) and partial obstruction (PO) examined by RT-PCR. NTC is non-template control. (B) Detection of Cacna1g mRNAs in isolated eGFPhigh and eGFPlow PDGFRα+ cells from sham operation and partial obstruction opertaion by RT-PCR. PCR products were analyzed with a DNA size marker on 1.5% agarose gels. Note expression of Cacna1g is increased in eGFPhigh and eGFPlow PDGFRα+ cells. (C) Quantification of Cacna1g mRNAs by qPCR. * p ≤ 0.05 and ** p ≤ 0.01, SO versus PO. (D) Western blot analysis using N-terminal and C-terminal CACNA1G antibodies (CACNA1G-N and CACNA1G-C), showing that the protein has significantly higher expression levels in hypertrophic tissue induced by partial obstruction. (E) Detection of serosal PDGFRα+ cells expressing CACNA1G in partial obstruction models by CACNA1G-C antibody. Scale bars are 50 μm (F) Confocal cross section images of serosal PDGFRα+ cells in sham operation and partial obstruction screened with CACNA1G-N and CACNA1G-C antibodies co-labeled with PDGFRA and PDGFRB antibodies. Scale bars are 50 μm.
Fig 7.
Induced expression of CACNA1G in human GI diseases.
(A) Anatomical cross-sections of colorectal cancer (CRC), Crohn’s disease (CD) small intestine and diverticulitis (DIV) colon tissue. Marginal colon tissue away from diverticula was used as healthy colon tissue. LM: longitudinal smooth muscle, CM: circular smooth muscle, Mu: mucosa. Scale bars are 100 μm. (B) Inflamed CRC compared to healthy colon tissue. (C) Detection of human CACNA1G mRNAs in diseased GI tissues (n = 2). Two independent regions of CACNA1G exons (7–8, and 28–29) were amplified by RT-PCR and PCR products were analyzed with a DNA size marker on 1.5% agarose gels. GAPDH gene was used as an endogenous control. (D) Western blot analysis of diseased GI tissues (n = 2) using CACNA1G antibody (N-terminal). (E) Confocal cross section images of PDGFRα+ cells screened with CACNA1G antibody (N-terminal) co-labeled with PDGFRA, PDGFRB, and ACTA2 antibodies in CRC and healthy tissues. Scale bars are 50 μm.