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. 2021 Jun 4;10:e62917. doi: 10.7554/eLife.62917

Figure 1. Gli1 labels mesenchymal progenitors in mouse meniscus during development.

(A) Schematic graph of the study protocol. Male Gli1ER/Td mice were treated with Tam at 1, 2, 4, 8, and 12 weeks of age and analyzed at 24 hr (pulse) or 6 weeks (tracing) after the last Tam dosing. (B) Schematic cartoon of meniscus shows sectioning sites. M: meniscus; ACL: anterior cruciate ligament; PCL: posterior cruciate ligament. (C) Representative fluorescence images of meniscus sections at indicated ages and sectioning sites. n = 3 mice/age/sectioning site. Scale bars, 200 μm. F: femur; T: tibia; A: anterior; P: posterior; M: medial meniscus; L: lateral meniscus; Med: medial; Ant: anterior; Post: posterior. Red: Td; Blue: DAPI. (D) Representative fluorescence images of meniscus body at coronal (a) and sagittal (b) planes from 12-week-old Gli1ER/Td mice. Meniscus were harvested at 24 hr after the last Tam injection. n = 3 mice/sectioning site. Scale bars, 200 μm. Boxed areas in a and b are shown at high magnification as c and d, respectively. Dashed lines outline meniscus. (E) qRT-PCR analysis of Hh signaling component genes in mouse meniscus tissues at 1, 4, 8 weeks of age. n = 4 independent experiments. (F) Top panel is a schematic representation of the study protocol. Gli1ER/Td mice were injected with EdU at P3-6 and Tam at P25-29. Joints were harvested 24 hr later. Representative confocal images of coronal sections of mouse knee joints are presented at the bottom panel. Boxed area in a (Scale bars, 200 μm) is shown at high magnification in b (Scale bars, 50 μm). Green: EdU. (G) The percentage of EdU+ cells within Gli1+ or Gli1- meniscus cells was quantified. n = 6 mice/group. (H) Gli1ER/Td mice were treated with Tam at 24 or 48 weeks of age and analyzed 24 hr later. Representative fluorescence images of sagittal (a, b) and coronal (c–f) sections of knee joints are presented. Scale bars, 200 μm. Statistical analysis was performed using unpaired two-tailed t-test and one-way ANOVA with Tukey-Kramer post-hoc test. Data presented as mean ± s.e.m. *p<0.05, **p<0.01, ***p<0.001.

Figure 1—source data 1. Raw data for Figure 1G and E.

Figure 1.

Figure 1—figure supplement 1. Mouse meniscal morphogenesis during development.

Figure 1—figure supplement 1.

(A) The morphological overview of meniscus at 1, 2, 3, 4, 8, 12, 24, 48 weeks of age. Scale bars, 1 mm. (B) The meniscal perimeter was quantified. n = 3 mice/age.
Figure 1—figure supplement 2. The density of Gli1+ cells along meniscus surface was measured in mice at different ages.

Figure 1—figure supplement 2.

n = 5 mice/age.
Figure 1—figure supplement 3. Meniscal enthesis and ligamental enthesis regions in joint are enriched with Gli1+ cells.

Figure 1—figure supplement 3.

Gli1ER/Td mice at 12 weeks of age received Tam injections followed by tissue harvest 24 hr later. Knees were sectioned to show meniscal enthesis regions (A) and ligamental enthesis regions (B, C) within the knee joint. MM: medial meniscus; LM: lateral meniscus; ACL: anterior cruciate ligament; PCL: posterior cruciate ligament. Yellow arrows indicate the enthesis regions.
Figure 1—figure supplement 4. Gli1 labels superficial zone cells of mouse and mini-pig meniscal horns.

Figure 1—figure supplement 4.

(A) Immunofluorescence staining of Gli1 (green) on sagittal sections of 12-week-old Gli1ER/Td mouse knee joints. Boxed area in a is enlarged in b. Dashed line indicates the surface of meniscus. Scale bars, 200 μm. F: femur; T: tibia; M: meniscus. Blue: DAPI, Red: Td; Green: Gli1. (B) Representative safranin O/fast green staining (left) and immunohistochemistry staining of Gli1 (right) in the horn area of mini-pig meniscus. Scale bars, 200 μm.