Desert Hedgehog mediates stem Leydig cell differentiation through Ptch2/Gli1/Sf1 signaling axis
- Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City, Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, China
Figures
Figure 1 with 2 supplements
Mutation of dhh resulted in testicular developmental disorders.
(A–B) Morphological analysis of the testes from wild-type (WT) (dhh+/+) and dhh-/- XY fish at 90 days after hatching (dah). Arrows indicate the location of the testes. (C–D’) Histological analysis of testicular sections from WT and dhh-/- XY fish at 90 dah. Sg, spermatogonia; Sc, spermatocyte. (E–J’) Representative immunofluorescence images showing the expression of germ cell marker Vasa (E–F’), meiosis cell marker Sycp3 (G–H’), and Leydig cell marker Cyp11c1 (I–J’). (K) Gonadosomatic index (GSI) of the testes from WT and dhh-/- XY fish at 90 dah (n=5 fish/genotype). (L–N) Quantification of the percentage of cells positive for Vasa (E–F’), Sycp3 (G–H’), and Cyp11c1 (I–J’) among all DAPI-positive cells in the WT and dhh-/- testes (n=5 fish/genotype). (O) Serum 11-KT level of WT and dhh-/- XY fish at 90 dah (n=6 fish/genotype). Values were presented as mean ± SD. Differences were determined by two-tailed independent Student’s t-test. **, p<0.01. Scale bars: (A–B), 1 cm; (C–J), 50 μm.
Figure 1—figure supplement 1
Sequence analysis and mRNA expression profile of Nile tilapia dhh.
(A) Dhh amino acid sequence alignment. Amino acids are numbered in the right margin. Deletions are indicated by dashes; shaded areas indicate shared sequences. The amino-terminal hedge domain is underlined by a dotted line, with the signal peptide region in the front and the autocatalytic carboxy-terminal domain (hog) in the back. The box indicates the autocatalytic site of an absolutely conserved Gly-Cys-Phe tripeptide. At the end of the alignment are percentage identity values of the full-length and hedge domain of tilapia Dhh to the orthologue from other species. (B) Phylogenetic analysis of Dhh. The phylogenetic tree was constructed using the neighbor-joining method within the MEGA7.0 program. Node values represent percent bootstrap confidence derived from 1000 replicates. (C–D) The RPKM (reads per kb per million reads) values of tilapia dhh in various adult tissues (C) and XY and XX gonads at 5, 7, 20, 30, 90, 180, 300 dah (days after hatching) (D) in transcriptome sequencing data which were sequenced using Illumina 2000 HiSeq technology in our previous study (Tao et al., 2013).
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Figure 1—figure supplement 1—source data 1
Transcriptomic expression data of dhh in adult tissues and developing gonads.
- https://cdn.elifesciences.org/articles/109979/elife-109979-fig1-figsupp1-data1-v1.xlsx
Figure 1—figure supplement 2
Establishment of the Nile tilapia dhh mutant line by CRISPR/Cas9.
(A) Schematic representation of guide RNA (gRNA) targeting the Nile tilapia dhh locus. The gRNA was designed to target exon 1. The translation start codon ATG and stop codon TAA were indicated by arrows. The PAM (protospacer adjacent motif) site was marked by a green box. DNA sequence alignment of the three mutant lines with wild-type (WT). The added sequences were marked by red letters, and the deleted sequences were indicated in red dotted lines. One had a 4 bp addition, one had an 8 bp deletion, and the other had a 13 bp deletion. (B) Schematic diagram showing the breeding plans of dhh F0 to F2 fish. (C) Homozygous mutants of F2 fish were screened by polyacrylamide gel electrophoresis (PAGE). The first lane is DNA marker, the second lane is dhh+/+ fish, the third lane is dhh +/- fish, and the fourth lane is dhh-/- fish. (D) Sanger sequencing results of dhh genes from WT and the 13 bp deletion homozygous mutant fishes. (E) The dhh ORF sequence in WT was indicated in black and the frameshift-altered sequence for the 13 bp deletion dhh ORF was indicated in red. (F) The relative mRNA expression of dhh in WT and the 13 bp deletion homozygous mutant fishes by RT-qPCR. Values were presented as mean ± SD (n=3). Differences were determined by two-tailed independent Student’s t-test. **, p<0.01.
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Figure 1—figure supplement 2—source data 1
Original uncropped polyacrylamide gel electrophoresis (PAGE) images for screening of dhh mutants.
- https://cdn.elifesciences.org/articles/109979/elife-109979-fig1-figsupp2-data1-v1.zip
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Figure 1—figure supplement 2—source data 2
Labeled polyacrylamide gel electrophoresis (PAGE) images for screening of dhh mutants.
- https://cdn.elifesciences.org/articles/109979/elife-109979-fig1-figsupp2-data2-v1.zip
Figure 2 with 1 supplement
Rescue of testicular development in dhh-/- XY Nile tilapia by 11-ketotestosterone (11-KT) and SAG.
dhh-/- XY Nile tilapias at 30 days after hatching (dah) were treated either with 11-KT via water immersion (with water changed every 2 days) or with 10 mg/kg SAG via intraperitoneal injection (with supplemental injection every 7 days). WT controls received 11-KT, and dhh-/- controls received an equivalent volume of the DMSO. Then, at 90 dah, morphological and histological experiments were conducted. (A–D’) Histological analysis of testicular sections from 90 dah XY fish subjected to different treatments as indicated. Sg, spermatogonia; Sc, spermatocyte. Scale bars: 50 μm. (E–P’) Representative immunofluorescence images showing the expression of Vasa (E–H’), Sycp3 (I–L’), and Cyp11c1 (M–P’). Scale bars: 50 μm. (Q) Gonadosomatic index (GSI) of testes from the different treatment groups at 90 dah (n=5 fish per group). (R–T) Quantification of the percentage of cells positive for Vasa (E–H’), Sycp3 (I–L’), and Cyp11c1 (M–P’) among all DAPI-positive cells in the testes (n=3–5 fish per group). Values were presented as mean ± SD. Different letters above the error bar indicate statistical differences at p<0.05 as determined by one-way ANOVA followed by Tukey’s test.
Figure 2—figure supplement 1
The expression levels of Cyp11c1 in the testis and the levels of 11-ketotestosterone (11-KT) in tissue fluid from wild-type (WT) and dhh-/- XY fish at 5, 10, 20, and 30 days after hatching (dah).
(A–E) Representative immunofluorescence images showing Cyp11c1 expression (green) in testis sections from WT fish at 5, 10, 20, 30 dah and dhh-/- mutants at 30 dah. Scale bars: 4 μm. (F) Tissue fluid 11-KT levels in WT and dhh-/- XY fish at indicated time points (n=6 fish per group). Values were presented as mean ± SD. Differences were determined by two-tailed independent Student’s t-test. NS, no significant difference.
Figure 3
Dhh is required for stem Leydig cell (SLC) differentiation in vivo.
TSL, Dhh-overexpressing TSL (TSL-OnDhh), or 0.5 μM SAG-treated TSL (TSL+SAG) cells were labeled with PKH26 and transplanted into the testes of 90 days after hatching (dah) wild-type (WT) or dhh-/- recipient fish. Analyses were performed 10 days post-transplantation. (A) Schematic diagram of the experimental design for SLCs transplantation and analysis. (B1–E3) Representative immunofluorescence images of testicular sections from recipient fish, showing the localization of transplanted PKH26-labeled SLCs (red) and the expression of Cyp11c1 (green). Nuclei are stained with DAPI (blue). Scale bars: 4 μm. (F) Quantification of the percentage of Cyp11c1-positive cells among the transplanted PKH26-positive SLCs for each treatment group (n=5 fish per group). (G) Serum 11-KT level in recipient fish following SLC transplantation (n=5 fish per group). Values were presented as mean ± SD. Different letters above the error bar indicate statistical differences at p<0.05 as determined by one-way ANOVA followed by Tukey’s test.
Figure 4 with 4 supplements
Ptch2 mediates Dhh signaling in stem Leydig cells (SLCs).
(A) Co-localization of Cyp11c1 (green, by immunofluorescence) and ptch1 or ptch2 mRNA (red, by RNA-FISH) in adult (90 days after hatching [dah]) testis sections. Dashed lines outline representative Leydig cells. Scale bars: 4 μm. (B) Schematic illustration of the experimental setup for the luciferase reporter assays shown in panels C and D. (C) Luciferase activity in TSL cells co-transfected with a Gli-responsive reporter (8×GLI) and overexpression of tilapia Dhh (OnDhh). A plasmid lacking Gli-binding sites (pGL4.23) served as a negative control (n=3). (D) Luciferase activity in TSL-WT, TSL-ptch1-/-, and TSL-ptch2-/- cells transfected with the 8×GLI reporter with or without OnDhh (n=4). (E–G’) Histological analysis of testis sections from the indicated genotypes at 90 dah. Sg, spermatogonia; Sc, spermatocyte. Scale bars: 50 μm. (H–P’) Immunofluorescence analysis of Vasa (H–J’), Sycp3 (K–M’), and Cyp11c1 (N–P’) in testis sections from the indicated genotypes. Scale bars: 50 μm. (Q) Gonadosomatic index (GSI) of the testes from the indicated genotypes at 90 dah (n=4 fish per group). (R–T) Quantification of the percentage of Vasa (H–J’), Sycp3 (K–M’), and Cyp11c1 (N–P’) positive cells among DAPI-positive cells (n=6 fish per group). (U) Serum 11-ketotestosterone (11-KT) levels in fish of the indicated genotypes at 90 dah (n=6 fish per group). Statistical significance was determined by one-way ANOVA followed by Tukey’s test (C, Q–U, different letters above the error bar indicate statistical differences at p<0.05) or Student’s t-test (D) (*, p<0.05; **, p<0.01; NS, no significant difference).
Figure 4—figure supplement 1
RT-PCR analyses of Hh pathway genes in TSL cells.
β-aActin served as a loading control, and RNAs from TSL cells were used as templates for negative controls. The numbers in parentheses indicate the PCR cycles.
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Figure 4—figure supplement 1—source data 1
Original agarose gel electrophoresis images for RT-PCR products of Hh pathway genes in TSL cells.
- https://cdn.elifesciences.org/articles/109979/elife-109979-fig4-figsupp1-data1-v1.zip
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Figure 4—figure supplement 1—source data 2
Labeled agarose gel electrophoresis images for RT-PCR products of Hh pathway genes in TSL cells.
- https://cdn.elifesciences.org/articles/109979/elife-109979-fig4-figsupp1-data2-v1.zip
Figure 4—figure supplement 2
Establishment of the TSL ptch1, ptch2, gli1, gli2, gli3, and sf1 mutant lines.
(A–F) Schematic representation of guide RNAs (gRNAs) targeting the ptch1, ptch2, gli1, gli2, gli3, and sf1 locus. The gRNAs were designed to target ORF (open reading frame). The PAM (protospacer adjacent motif) sites were marked by red letters. Sanger sequencing of ptch1, ptch2, gli1, gli2, gli3, and sf1 single mutant alleles in single-cell clones. PCR amplicons from DNA templates of the six cell clones were directly used for sequencing.
Figure 4—figure supplement 3
Establishment of the Nile tilapia ptch2 mutant line by CRISPR/Cas9 system.
(A) Schematic representation of guide RNA (gRNA) targeting the Nile tilapia ptch2 locus. The gRNA was designed to target exon 1. The translation start codon ATG and stop codon TGA were indicated by arrows. The PAM site was marked by a green box. DNA sequence alignment of the five mutant lines with wild-type (WT). The added sequences were marked by red letters, and the deleted sequences were indicated in red dotted lines. (B) Schematic diagram showing the breeding plans of ptch2 F0 to F2 fish. (C) DNA sequencing showed the 25 bp deletion within the ptch2 ORF in the homozygous mutant compared to the WT. (D) Schematic of the prediction of the intact Ptch2 protein in the WT and the truncated Ptch2 protein in the homozygous mutants. (E) Homozygous mutants of F2 fish were screened by polyacrylamide gel electrophoresis (PAGE). The first lane is DNA marker, the second lane is ptch2+/+ fish, the third lane is ptch2+/-fish, and the fourth lane is ptch2-/- fish. ‘*’ indicates heteroduplex, and arrows indicate homoduplex.
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Figure 4—figure supplement 3—source data 1
Original uncropped polyacrylamide gel electrophoresis (PAGE) images for screening of ptch2 mutants.
- https://cdn.elifesciences.org/articles/109979/elife-109979-fig4-figsupp3-data1-v1.zip
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Figure 4—figure supplement 3—source data 2
Labeled polyacrylamide gel electrophoresis (PAGE) images for screening of ptch2 mutants.
- https://cdn.elifesciences.org/articles/109979/elife-109979-fig4-figsupp3-data2-v1.zip
Figure 4—figure supplement 4
Expression analysis of ptch1 in wild-type (WT) and mutant XY testes.
Relative mRNA expression levels of ptch1 in XY testes from WT, ptch2-/-, and dhh-/-;ptch2-/- fish at 90 days after hatching (dah) by RT-qPCR. Values were presented as mean ± SD (n=3 fish per group). Statistical significance was determined by one-way ANOVA followed by Tukey’s test. NS, no significant difference.
Figure 5 with 1 supplement
Gli1 transactivates sf1 to drive stem Leydig cell (SLC) differentiation.
(A) Co-localization of Cyp11c1 (green) and gli1, gli2, or gli3 mRNA (red) in adult (90 days after hatching [dah]) testis sections by immunofluorescence and RNA-FISH. Dashed lines outline representative Leydig cells. Scale bars: 4 μm. (B) Luciferase activity in TSL-WT, TSL-gli1-/-, TSL-gli2-/-, and TSL-gli3-/- cells transfected with the 8×GLI reporter with or without OnDhh (n=4). (C) Volcano plots of transcriptomic changes in TSL-OnDhh, TSL-OnGli1, and TSL+SAG, compared to TSL-WT. Red and blue dots represent significantly up- and downregulated genes, respectively. (D) Schematic of the tilapia sf1 gene promoter, indicating the two predicted Gli1 binding sites (B1 and B2). (E) Transcriptional activation of the sf1 promoter by Gli1. HEK293 cells were co-transfected with pRL-TK, pGL3, pcDNA3.1, pGL3-sf1, pcDNA3.1-OnGli1, and the indicated cold probe constructs, and luciferase activity was measured 48 hr post-transfection. Competition assays were performed using unlabeled cold probe (Cold probe, GACCACCCA, 10/100/250 ng/mL) or mutant unlabeled cold probe (mutant Cold probe, TTAATTAAA, 10/100/250 ng/mL) (n=4). ‘+’ indicates the addition of the corresponding substance, while ‘-’ indicates no addition, and the number represents the amount added (ng/mL). (F–G) Representative immunofluorescence images of testicular sections from recipient fish transplanted with sf1-deficient (TSL-sf1-/-, F) or Sf1-overexpressing (TSL-OnSf1, G) SLCs, stained for Cyp11c1 (green) and PKH26 (red). Nuclei are stained with DAPI (blue). Scale bars: 4 μm. (H) Quantification of the percentage of Cyp11c1-positive cells among the transplanted PKH26-positive SLCs in the two transplantation groups (n=5 fish per group). Statistical significance was determined by one-way ANOVA followed by Tukey’s test (E, different letters above the error bar indicate statistical differences at p<0.05) or Student’s t-test (B, H) (*, p<0.05; **, p<0.01; NS, no significant difference).
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Figure 5—source data 1
Transcriptomic expression data for TSL cells under Dhh pathway activation conditions.
- https://cdn.elifesciences.org/articles/109979/elife-109979-fig5-data1-v1.xls
Figure 5—figure supplement 1
Transcriptome data analyses and verification by RT-qPCR.
(A) Hierarchical clustering analysis of global gene expression patterns in TSL-WT, TSL-OnDhh, TSL-OnGli1, and TSL+0.5 μM SAG. Blue indicates decreased expression, and red indicates increased expression. (B) Heatmap showing the expression patterns of upregulated differentially expressed genes (DEGs) identified in Figure 5C. The FPKM value for each gene in each sample is indicated within the squares. The color gradient from blue to red reflects low to high expression levels per row (gene). (C) qRT-PCR validation. Values were presented as mean ± SD (n=3).
Additional files
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Supplementary file 1
List of primers used in this study.
- https://cdn.elifesciences.org/articles/109979/elife-109979-supp1-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/109979/elife-109979-mdarchecklist1-v1.docx
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Desert Hedgehog mediates stem Leydig cell differentiation through Ptch2/Gli1/Sf1 signaling axis
eLife 15:RP109979.
https://doi.org/10.7554/eLife.109979.3
