Cell origin, volume and arrangement are drivers of articular cartilage formation, morphogenesis and response to injury in mouse limbs

Abstract

Limb synovial joints are composed of distinct tissues, but it is unclear which progenitors produce those tissues and how articular cartilage acquires its functional postnatal organization characterized by chondrocyte columns, zone-specific cell volumes and anisotropic matrix. Using novel Gdf5^CreERT2 (Gdf5-CE), Prg4-CE and Dkk3-CE mice mated to R26-Confetti or single-color reporters, we found that knee joint progenitors produced small non-migratory progenies and distinct local tissues over prenatal and postnatal time. Stereological imaging and quantification indicated that the columns present in juvenile-adult tibial articular cartilage consisted of non-daughter, partially overlapping lineage cells, likely reflecting cell rearrangement and stacking. Zone-specific increases in cell volume were major drivers of tissue thickening, while cell proliferation or death played minor roles. Second harmonic generation with 2-photon microscopy showed that the collagen matrix went from being isotropic and scattered at young stages to being anisotropic and aligned along the cell stacks in adults. Progenitor tracing at prenatal or juvenile stages showed that joint injury provoked a massive and rapid increase in synovial Prg4+ and CD44+/P75+ cells some of which filling the injury site, while neighboring chondrocytes appeared unresponsive. Our data indicate that local cell populations produce distinct joint tissues and that articular cartilage growth and zonal organization are mainly brought about by cell volume expansion and topographical cell rearrangement. Synovial Prg4+ lineage progenitors are exquisitely responsive to acute injury and may represent pioneers in joint tissue repair.

Keywords: Articular cartilage growth and morphogenesis; Dkk3; Gdf5; Genetic cell tracing; Limb development; Mouse; Prg4; Progenitor cell fate; Synovial joint formation; Tissue repair.

Fig. 1

Postnatal articular cartilage development and growth involve formation of non-daughter cell stacks. (A–E) Two-dimensional (2D) sections and 3D fluorescence images of joints from Prg4-CE/R26-Confetti mice induced with tamoxifen at E17.5 and collected at: P0 (newborn) (A–B) and 2 months of age (C–E). D is the same 3D image in C, but with superimposed second harmonic signal from matrix (blue color). Arrows in D indicate local same-color reporter clusters throughout the tissue. Arrows in E indicate non-daughter cells in stack expressing different reporters. (F–K) 2D and 3D fluorescence images of joints from constitutive Gdf5Cre/Confetti mice at: P0 (F–G); and 2 months of age (H–K). I is the same 3D image in H, but with superimposed second harmonic signal (blue color). Red arrow in H indicates clonal expansion perpendicular to the articular surface, while yellow arrow in I indicates clonal expansion parallel to the articular surface. Arrowhead in J points to occasional dual reporter-positive cell; arrows in K point to examples of non-daughter cell stacks. (L–O) 2D and 3D images of joints from ROSA-CE/R26-Confetti mice induced with tamoxifen at E17.5 and examined at: P0 (L); and 2 months (M–O). (O) Fluorescence image of proximal tibia growth plate from 1 month-old ROSA-CE/R26-Confetti mice induced at E17.5. Arrowheads indicate continuous single reporter-expressing chondrocyte columns made of daughter cells. At least 5 mice were evaluated at each stage. A–B and F–G, scale bar = 50 µm; C–D, H–I and M–N, scale bar = 55 µm; E, J, scale bar = 60 µm; K, scale bar = 40 µm; and L, scale bar = 125 µm.

Fig. 2

Zonal arrangement and thickening of articular cartilage are linked to changes in cellularity and collagen matrix configuration. (A–E) Incipient proximal tibia articular cartilage at P0 consists of small and closely-bound cells (A, bracket) that express Prg4 (C) and TnC (E), but lack Matn1 expression (E) and a SHG-detectable collagen matrix (B). Brackets demarcate the approximate thickness of incipient articular cartilage which overlays Matn1-expressing shaft cartilage destined for secondary ossification (D). (F–J) Articular cartilage at P14 is thicker (F, bracket). Its surface zone (S) retains Prg4 expression (H), while its developing transitional (T) and deep (D) zones display isotropic SHG-detectable collagen fibrils (G). Strong Matn1 and ColX expression depicts underlying secondary ossification center cartilage (I–J). (K–P) At P28 and 2 months, articular cartilage acquires typical zones that include a narrow surface Prg4-expressing zone (M) and intermediate and deep zones with chondrocyte stacks with anisotropic collagen matrix (L and P) with underlying subchondral ColX expressing cells (N). A characteristic tidemark delineates the boundary between deep and calcified zones (K and O, arrowheads). Data are representative of a minimum of 6 mice evaluated at each stage. (Q) Relative sizes of superficial (S), transitional (T), deep (D) and calcified (C) zones at P14, P28, 6 weeks and 2 months were calculated by histological morphometry; n=12 for each time point. (R) Cellularity (right Y axis, red line) versus overall tissue thickness (left Y axis, blue line) from P0 to 2 months were determined by fluorescence microscopy and histomorphometry; n=12 for each time point, mean ± s.d.. Images A–P same scale, bar in A = 75 µm.

Fig. 3

Differential changes in cell proliferation, death and volume subtend tibial articular cartilage growth over age. (A–H) Immunostaining fluorescence images of cell proliferation marker Ki67 (A–D) and TUNEL staining for apoptosis (E–H) indicating that proliferation was maximal at early stages while cell death was maximal at late stages. Arrowheads point to representative positive cells. (I–L) Cell progeny clusters in articular tissue in ROSA-CE/R26-Confetti mice induced once at E13.5, P0, P7 and P14 and examined at 2 months. Images are representative of a minimum of 6 mice evaluated at each stage. Maximal cluster sizes of about 18 cells were observed after E13.5 induction (I), and average sizes dwindled thereafter (J–L). (M–P) 2D and 3D imaging-based quantifications of: average cell cluster size (M); Ki67- and TUNEL-positive cell numbers (N); individual cells for volume quantification (O, P); and calculated average cell volumes (Q) at indicated ages. n=4 mice, minimum of 40 cells measured per mouse at each stage. When present, vertical bars indicate means ± s.d. A–E, bar = 75 µm; I–J, bar = 50 µm; K, bar = 60 µm; L, bar =75 µm; and O–P, bar =8 µm.

Fig. 4

Joint progenitors are topographically static and contribute to diverse tissues over developmental time. (A–F) Metatarsal-phalangeal joints at P0 (A, C, E) and 1 month of age (B, D, F) from Gdf5-ER/R26-zsGreen mice induced once with tamoxifen at E13.5 (A–B), E15.5 (C–D) or E17.5 (E–F) show persistent local distribution of reporter-positive cells over time. Red arrows indicate representative labeled cells in peripheral tissues, and yellow arrowheads indicated representative labeled articular cells. (G–L) Images of knee sections from constitutive Gdf5Cre/GFP mice indicating that Gdf5Cre+ cells constitute most joint tissues over indicated times, including ligaments (arrows), meniscus (double arrowheads) and synovium (arrowhead). (M–T) Knee joint images showing that both Prg4-CE+ and Dkk-CE+ cells in mice induced at E17.5 are present and abundant in tibial articular cartilage at P0 and 2 months (M–N, Q–R). Dkk-CE+ cells are also numerous in posterior cruciate ligament at either age (S–T, arrows), whereas Prg4-CE+ cells are scarce (O–P, arrows). Note that Gdf5Cre+ cells also are abundant in ligaments (K, arrow). (U–Z’’) In mice induced at P28, Prg4-CE+ cells populate the surface layers of articular cartilage at P31 and 2 months (U–V), while Dkk-CE+ cells are rare and only found in the most superficial articular layer (Y–Z, arrow). Prg4-CE+ and Dkk3-CE+ cells are found throughout the posterior cruciate ligament (W–X, Z’–Z’’) at both P31 and 2 months. Images are representative of a minimum of 3 mice evaluated at each stage. Images A, C, E, bar = 75 µm; B, D, F, bar = 125 µm; G, J, bar = 300 µm; and H–Z’’, bar = 100 µm.

Fig. 5

Embryonically labeled joint cell populations respond to articular cartilage injury. (A–D) Safranin-O histological and anatomical images from un-operated and operated 2 month-old adult knee joints and femoropatellar groove showing: typical joint organization in controls (A); operated site immediately after surgery (B, circled area); representative field showing minor synovial hyperplasia 7 days after surgery (C, asterisk); and more severe synovial hyperplasia after surgery (D, double asterisk). Note that cells usually filled the surgical defect by day 7 (C, dashed line), and the hyperplastic synovial tissue was often continuous with cells filling the defect (arrowhead), yet appearing unattached to the adjacent articular cartilage (arrow) (D). (E–H) In adult mice that were induced with tamoxifen at E17.5, Prg4-CE+ and Dkk3-CE+ cells become very numerous within the thickened synovium by day 7 from surgery (F, H, asterisks), but remain few in number and restricted to articular and synovial surfaces in companion controls (E, G, asterisks). (I–K) EdU labeling (green label) revealed that proliferating cells are present within defect site and synovium (I–J), but are rare in adjacent articular cartilage after surgery (K, arrow). Images are representative of a minimum of 6 mice evaluated at each stage. (L) Quantification of (i) Dkk3-CE+ and Prg4-CE+ cell number present in hyperplastic synovium (L, upper histograms) on day 7 after surgery and (ii) proliferative cell number in synovium (SL) and articular cartilage (AC) on day 7 before or after surgery (L, lower histograms) calculated as mean labeled cells/100 cells ± s.d., n=6, *P < 0.01. A, C, D, bar = 200 µm; B, bar = 4 mm; E–H, bar = 75 µm; and I–K, bar = 50 µm

Fig. 6

Prg4-CE+ progeny and real-time Prg4-expressing cells respond differently to acute joint injury. (A–B) After tamoxifen induction at P28, Prg4-CE+cells are restricted to synovial lining (arrowhead) and superficial layer of articular cartilage (arrow). (B) Real-time Prg4mCherry (red) expressing cells remain few in number and limited to articular and synovial surfaces. (C–D) Prg4-CE+ cells become very numerous in the thickened synovium within 7 days from surgery (C, asterisk) and fill the injury defect (dashed line), but real-time Prg4mCherry (red) expressing cells remain few in number and limited to articular and synovial surfaces (D), with some exhibiting progeny plus real-time character (yellow). (E–J) In unoperated knees (E–F), synovial cells exhibit CD44 immunostaining (E, arrowhead), P75 immunostaning (F, arrowheads), and Prg4-CE+ character. After 7 days from surgery, the number of positive cells greatly increases in synovium (G–H) and within defect site (I–J). Images are representative of a minimum of 10 mice evaluated at each stage. A–D, bar = 75 µm; and E–J, bar =15 µm.

Fig. 7

Model of synovial joint formation and postnatal growth and morphogenesis. (A) Mesenchymal Gdf5+ cells constituting the interzone area at incipient joint sites in early embryonic limbs would comprise centrally-located descendants of chondrocytes (red color) and flanking cells recruited from surrounding tissues into the Gdf5 lineage (green color). (B–C) Both populations would increase in number, but would not migrate significantly over time and would give rise to distinct local tissues, with the flanking cells largely producing synovial lining, capsule and peripheral ligaments and central cells generating articular cartilage and intrajoint ligaments. (D–F) Starting at neonatal stages and proceeding into adulthood, knee articular cartilage (ac) would progressively acquire its functional organization and structure that include: superficial layers (sl); intermediate (int.) and deep zones; and a tidemark overlaying the calcified (cal.) cartilage and secondary ossification center (soc). Growth in thickness would be driven by limited proliferation at neonatal stages (D) and by cell volume increases at subsequent stages (E–F). The characteristic cell stacks perpendicular to the articular surface would entail repositioning and intercalation of neighboring chondrocytes, producing stacks of non-daughter (and differently labeled) cells. We should note that as this schematic makes it clear, the model focuses on the central region of articular cartilage such as that occurring in the proximal tibia plateau. Additional and diverse mechanisms may be involved in development, thickening and expansion of articular cartilage in the lateral portions of the joints over postnatal life.