Abstract
Attachment sites of tendons to bones, called entheses, are essential for proper musculoskeletal function. They are formed embryonically by Sox9+ progenitors and undergo a developmental process that continues into the postnatal period and involves Gli1 lineage cells. During bone elongation, some entheses maintain their relative positions by actively migrating along the bone shaft, while others, located at the bone’s extremities, remain stationary. Despite their importance, we lack information on the developmental transition from embryonic to mature enthesis and on the relation between Sox9+ progenitors and Gli1 lineage cells. Here, by performing a series of lineage tracing experiments, we identify the onset of Gli1 lineage contribution to different entheses during embryogenesis. We show that Gli1 expression is regulated by SHH signaling during embryonic development, whereas postnatally it is maintained by IHH signaling. Interestingly, we found that unlike in stationary entheses, where Sox9+ cells differentiate into the Gli1 lineage, in migrating entheses the Sox9 lineage is replaced by Gli1 lineage and do not contribute to the mature enthesis. Moreover, we show that these Gli1+ progenitors are pre-specified embryonically to form the different cellular domains of the mature enthesis.
Overall, these findings demonstrate a developmental strategy whereby one progenitor population establishes a simple, embryonic tissue, whereas another population is responsible for its maturation into a complex structure during its migration. Moreover, they suggest that different cell populations may be considered for cell-based therapy of enthesis injuries.
INTRODUCTION
The proper assembly of the musculoskeletal system is essential for the function, form and stability of the organism. During embryogenesis, an attachment between tendon and bone, known as enthesis, is formed. Thereafter and into the postnatal period, the rudimentary enthesis further develops into a more complex tissue. While some knowledge on enthesis formation and maturation exists, far less is known about the processes that transform the simple embryonic enthesis into the structure of the mature enthesis.
Traditionally, entheses are divided to either fibrous or fibrocartilaginous, according to their composition. Fibrous entheses form at the attachment site of a tendon that is inserted directly into the bone shaft, forming a structure that resembles a root system. This structure, which is composed of a dense connective tissue (Doschak and Zernicke, 2005; Shaw and Benjamin, 2007; Wang et al., 2013), was shown to be regulated by parathyroid hormone-like hormone (PTHLH, also known as PTHrP) (Wang et al., 2013). Fibrocartilage entheses typically form at attachment sites of tendons to the epiphysis or to bone eminences. Relative to fibrous entheses, fibrocartilage entheses are structurally more complex, displaying a cellular gradient that is typically divided into four zones, namely tendon, fibrocartilage, mineralized fibrocartilage and bone. The graded tissue that develops postnatally dissipates the stress that forms at the attachment site, thereby providing the enthesis with the mechanical strength necessary to withstand compression (Benjamin et al., 2006).
Enthesis development is initiated by the specification of a specialized pool of progenitor cells that express both SRY-box 9 (Sox9), a key regulator of chondrogenesis, and the tendon marker scleraxis (Scx) (Akiyama et al., 2005; Blitz et al., 2013; Schweitzer et al., 2001; Sugimoto et al., 2013). Cell lineage studies showed that Sox9-expressing progenitor cells contribute to the formation of the enthesis in neonatal mice (Akiyama et al., 2005; Soeda et al., 2010). Another marker for enthesis cells is GLI-Kruppel family member GLI1 (Gli1), a component of the hedgehog (HH) signaling pathway. Lineage studies revealed that Gli1-expressing cells act as progenitors that contribute to the formation of some adult entheses (Dyment et al., 2015; Schwartz et al., 2015). However, the relation between progenitors of the embryonic enthesis and Gli1 lineage cells, which contribute to the mature enthesis, has not been determined.
The relative position of an enthesis along the bone directly affects its mechanical function and, subsequently, the animal’s mobility (Polly, 2007; Salton and Sargis, 2009). Recently, it was shown that all entheses maintain their relative position during bone elongation (Stern et al., 2015). The mechanism that maintains their positions involves regulation of the relative growth rates at the two epiphyseal plates. Additionally, some entheses migrate through continuous reconstruction to maintain their position, a process known as bone modelling or drift (Benjamin and McGonagle, 2009; Dörfl, 1980a; Dörfl, 1980b). These entheses, referred to in the following as migrating entheses, face a unique developmental challenge. Unlike most organs and tissues, they must develop into a complex graded tissue while constantly drifting. This raises the question of whether the descendants of the embryonic enthesis progenitors continue to serve as the building blocks during maturation of migrating entheses.
In this work, we identify the embryonic stage at which Gli1 lineage is initiated and demonstrate its contribution to the postnatal enthesis. We show that embryonic Gli1 expression is initially under the regulation of SHH. Later during postnatal development, Gli1 expression is maintained by IHH. Moreover, we show that Sox9 lineage does not contribute to postnatal migrating entheses. Instead, Gli1 lineage cells replace the Sox9 lineage cells and populate the enthesis. Finally, we show that embryonic Gli1 lineage cells are pre-determined to contribute to the different layers of the fibrocartilaginous enthesis.
RESULTS
Some entheses undergo cellular and morphological changes while migrating
The development of the rudimentary embryonic attachment site into the complex structure of a mature enthesis has received little attention (Galatz et al., 2007). As mentioned, in addition to substantial cellular and morphological changes, some entheses also migrate considerably along the bone during bone growth (Benjamin and McGonagle, 2009; Dörfl, 1980a; Dörfl, 1980b; Stern et al., 2015). Therefore, to study the transition that migrating entheses undergo during maturation, we documented morphological and molecular changes as well as drifting activity in entheses from embryonic day (E) 14.5 to postnatal day (P) 14. We focused on two migrating entheses, namely the deltoid enthesis (DT), a fibrocartilaginous enthesis that forms between the deltoid tendon and the deltoid tuberosity, and the teres major enthesis (TM), a fibrous enthesis that forms between the teres major tendon and the humeral shaft. Analysis of enthesis positions during development revealed that both DT (0.778±0.026 mm) and TM (1.624±0.171 mm) entheses drifted considerably along the bone shaft during bone elongation (Fig. 1A). Histological sections through wild-type (WT) mouse humeri showed that both embryonic entheses displayed a simple structure of layered cells (Fig. 1B,C(a),D(a)). However, the overall shape of the enthesis dramatically changed through development, as it protruded outwards from the bone shaft and the different enthesis domains became more noticeable (Fig. 1C(a’-a’’’),D(a’-a’’’). Tissue complexity also increased, as a larger variety of cells, such as fibrocartilage cells and osteoblasts (Benjamin and McGonagle, 2009), were identified along with an increase in extracellular matrix (Fig. 1C(d,d’,h,h’), D(d,d’,h,h’)). The increased complexity was also demonstrated by a change in the expression patterns of structural genes, such as bone sialoprotein (Bsp; also known as integrin binding sialoprotein (Ibsp), collagen type 2 alpha 1 (Col2a1), collagen type 12 alpha 1 (Col12a1) and tenascin C (Tnc), and regulatory genes such as Gli1. Furthermore, expression domains correlating to various structural domains emerged, namely Col12a1, Tnc, Gli1 and Col1a1 in tendon and fibrocartilage (Fig. 1C(c,f-h),D(c,f-h), and Col1a1, Gli1 and Bsp in mineralized fibrocartilage and bone (Fig. 1C(c,e,h),D(c,e,h)).
Gli1+ cell lineage contributes to the postnatal enthesis
Previously, lineage tracing experiments showed that Gli1 lineage cells contribute to postnatal enthesis development (Dyment et al., 2015; Schwartz et al., 2015). Yet, the onset of this lineage during embryogenesis and its dynamics in different entheses have been missing. In order to identify the onset of Gli1 lineage, we performed pulse-chase experiments on Gli1-CreERT2 (Ahn & Joyner, 2004) mice crossed with R26R-tdTomato reporter mice (Madisen et al., 2010), which allowed us to mark Gli1 expressing cells at specific time points and follow their descendants. We analyzed three entheses representing different types, namely DT (migratory-fibrocartilaginous), TM (migratory-fibrous), and Achilles (stationary-fibrocartilaginous). Examination of neonatal (P0) TM and DT entheses following tamoxifen administration at E11.5 and E12.5 revealed only a few Gli1 lineage cells. However, administration at E13.5 and E15.5 resulted in extensive labeling in both entheses (Fig. 2A-A’’’,B-B’’’). In the stationary Achilles enthesis, extensive labeling was observed at P0 only after tamoxifen administration at E15.5 (Fig. 3C’’’). The postnatal contribution of Gli1 lineage to migratory entheses was further established by following E13.5 lineage induction to P14 (Fig. 2D,E).
Together, these results indicate that although Gli1 lineage is not induced at the onset of enthesis formation (Blitz et al., 2013; Sugimoto et al., 2013), it contributes differentially to different entheses during embryonic development.
Gli1 expression in migrating entheses is initiated by SHH and maintained by IHH
The Hedgehog (HH) signaling pathway has previously been suggested to play a role in regulating the activity of Gli1-positive enthesis cells (Breidenbach et al., 2015; Dyment et al., 2015; Liu et al., 2013; Schwartz et al., 2015). Thus, our finding that Gli1 expression is initiated at early stages of enthesis development (Fig. 1C(h)D(h)) raised the question of which component of the HH pathway regulates Gli1 expression in enthesis cells and whether it also affects migratory entheses. It was suggested that Indian hedgehog (IHH), an effector of the HH signaling pathway, is a possible regulator of Gli1 lineage cells in adult stationary entheses. In order to examine the possible role of IHH in inducing Gli1 expression in the embryonic enthesis, we blocked Ihh expression in the limb by using Prx1-Cre-Ihh−/- mice. Interestingly, at E14.5 Gli1 was expressed in control and mutant entheses, suggesting that Gli1 expression in embryonic enthesis progenitors is not regulated by Ihh (Fig. 3A,A’). We therefore examined the possible role of another regulator of HH signaling, namely sonic hedgehog (SHH), by analyzing Gli1 expression by embryonic enthesis cells in Shh KO embryos (Shh-GFPCre−/-). Results showed that Gli1 expression dramatically decreased in the mutant entheses compared to the WT (Fig. 3B,B’), suggesting that SHH is necessary for the induction of Gli1 in embryonic enthesis cells.
That result was intriguing, because we observed that Gli1 was constantly expressed by both embryonic and postnatal enthesis cells (Fig. 1A(h-h’’’),B(h-h’’’)), while Shh is expressed in the limb only during embryogenesis (Harfe et al., 2004). This raised the question of how Gli1 expression in the enthesis is maintained after the loss of Shh expression. To address the possibility that Ihh controls Gli1 expression in the postnatal enthesis even though it is not involved in the induction of Gli1 expression in embryonic enthesis progenitors, we analyzed Prx1-Cre-Ihh−/- mice at P10. Results showed that although Gli1 expression was maintained in mutant muscle and bone, in the enthesis it was completely lost (Fig. 3C,C’), indicating that Ihh is indeed required for the maintenance of Gli1 expression in enthesis cells. To identify the source of Ihh in the enthesis, we performed immunofluorescence staining for IHH protein in P6 enthesis sections. As seen in Figure 3 (D,D’), IHH was highly expressed in mineralized fibrocartilage and bone regions at the enthesis center.
Taken together, these results suggest that Gli1 expression by embryonic enthesis progenitors is induced by SHH and later, in the maturing enthesis, maintained by IHH originating in mineralized fibrocartilage and bone.
Sox9 lineage cells of the embryonic enthesis do not contribute to postnatal migrating entheses
The embryonic enthesis originates from Scx/Sox9 double-positive progenitor cells (Blitz et al., 2013; Sugimoto et al., 2013). Yet, the contribution of these progenitors to the postnatal enthesis and their relation to the Gli1 lineage cells have never been studied. To fill this gap, we first examined the contribution of Sox9 lineage to the postnatal enthesis. To that end, we performed a pulse-chase cell lineage experiment using mice that express Cre-ER under control of the Sox9 promoter (Sox9-CreERT2) crossed with R26R-tdTomato reporter mice (Soeda et al., 2010). It was previously demonstrated that tamoxifen administration at E12.5 effectively labels embryonic enthesis cells (Blitz et al., 2013; Soeda et al., 2010). Indeed, examination at P0 following tamoxifen administration at E12.5 showed that the DT, TM and Achilles entheses were populated by tdTomato-positive cells, suggesting that at that stage, the enthesis is populated by Sox9 lineage cells (Fig. 4A(a,b,d)). Yet, surprisingly, at P14 we observed a dramatic decrease in the contribution of tdTomato-expressing cells to the two migrating entheses (Fig. 4A(a’,b’,c), although the cells in the stationary enthesis were still extensively labeled (Fig. 4A(d’). These results suggest that postnatal stationary enthesis cells were descendants of the Sox9-positive embryonic lineage, whereas in migrating entheses this lineage was lost.
The labeled stationary entheses could serve as a positive internal control for the effectiveness of labeling. Nevertheless, to rule out the possibility that tamoxifen was administered at the wrong time point, we repeated the experiment while administering tamoxifen at different time points from E11.5 to E17.5 (Fig. 4B). Examination at P14 showed minimal contribution of Sox9 lineage cells to the postnatal entheses, similar to the results obtained following pulsing at E12.5. Taken together, these results indicate that the embryonic Sox9 lineage contributes poorly to postnatal migrating entheses, suggesting that these entheses are populated by another cell lineage postnatally.
Gli1 lineage cells replace the embryonic Sox9 lineage during enthesis maturation
Our finding that both embryonic Gli1 and Sox9 lineages contribute to the stationary postnatal enthesis suggests that these two genes mark a common cell lineage. Conversely, the finding that embryonic Sox9 lineage contributes to embryonic but not to postnatal migrating enthesis implies that postnatally, embryonic Gli1 lineage replaces the Sox9 lineage to form the mature enthesis.
To study the process of lineage replacement and to follow its dynamics, we traced both lineages throughout enthesis development, from E15.5 to maturation at P14, by pulse-chase experiments. As seen in Figure 5, at E15.5 and P0 in both the DT and TM, Sox9 lineage cells populated the embryonic enthesis. However, from P0 their number decreased dramatically and by P8, the enthesis contained only a limited number of these cells. Concurrently, the number of cells of the Gli1 lineage gradually increased in both entheses and, by P8, Gli1+ cells inhabited most of the enthesis structure. Interestingly, the gradual population of the DT and TM entheses by Gli1 lineage cells correlated with the temporal dynamics of enthesis migration (Fig. 1A). These results support our hypothesis that during early postnatal development of migrating entheses, a new population derived from Gli1-positive progenitors substitutes the Sox9 lineage cells of the embryonic enthesis.
Cell fate of Gli1-positive progenitors is predetermined
The increasing cellular complexity of the developing enthesis and our finding that progenitors of the Gli1 lineage that will contribute to the postnatal enthesis are present already during embryonic development led us to ask how these cells form the graded tissue of the mature enthesis. Specifically, we sought to determine whether Gli1 lineage cells possess a multipotent capacity and, thereby, produce the different cell types of the various enthesis domains, or have predetermined cell fates already at the time of their recruitment to the embryonic enthesis. To decide between these options, we conducted pulse-chase experiments on Gli1-CreERT2 mice crossed with R26R-Confetti mice (Snippert et al., 2010) that, upon recombination, stochastically express GFP, RFP, CFP or YFP, allowing for the identification of different cell clones. Clones derived from multipotent progenitors would be spread among the different domains, whereas predetermined progenitors would produce clones that are confined to specific domains.
We focused on the two main domains of the DT enthesis, namely mineralized and non-mineralized fibrocartilage, and on the border between entheseal mesenchymal cells and bone in the TM enthesis. Examination of P14 limbs following tamoxifen administration at E13.5 revealed multiple clones in both entheses, most of which were restricted to a specific domain (Fig. 6). These results suggest that at E13.5, the cell fates of Gli1 lineage progenitors have already been determined.
DISCUSSION
The transition from the embryonic to the mature enthesis has been understudied. Here, we use genetic lineage tracing to unravel the cellular developmental sequence of fibrous and fibrocartilaginous entheses. We show that although Gli1+ progenitors contribute to entheses of both types, their contribution differ greatly between stationary and migratory entheses. In stationary entheses, Gli1+ progenitors are descendants of Sox9+ progenitors that have established the embryonic enthesis. However, in migrating entheses, a separate, pre-specified population of Gli1+ progenitors is recruited to the embryonic enthesis, co-populates it alongside Sox9+ progenitors and, eventually, replaces them during postnatal development.
As mentioned, fibrous and fibrocartilaginous entheses exhibit marked differences in structure and composition (Benjamin et al., 2002). Notwithstanding the importance of this distinction, the observed differences in cellular origin between stationary and migrating entheses calls for a revision of the traditional classification and suggests that it should be taken into account whether an enthesis is “migratory” or “stationary”.
Organ development can be mediated by several cellular mechanisms. In a linear mechanism, an embryonic set of progenitors forms a primordium and then continues to proliferate and differentiate to form the mature organ. Another mechanism is cell recruitment, during which cells are supplied to the forming organ after primordium establishment. A third mechanism involves template replacement, in which an initial template is formed by one type of cells to be later replaced by another cell population, which will form the mature organ. Interestingly, in the musculoskeletal system there are examples of all these modes of development. Muscles and joints develop through cell recruitment (Buckingham et al., 2003; Shwartz et al., 2016), whereas most of the skeleton develops through template replacement, namely by endochondral ossification (Kronenberg, 2003). Here, we show that stationary entheses develop linearly by embryonic Sox9+ progenitors that form the postnatal enthesis and later upregulate the expression of Gli1. However, we show that migrating entheses develop through template replacement, as Gli1+ progenitors replace the Sox9+ embryonic enthesis cells and form the mature enthesis.
Previous studies have shown not only that Gli1 is a marker for the forming enthesis, but that the HH pathway plays an active role in regulating enthesis maturation and regeneration (Breidenbach et al., 2015; Dyment et al., 2015; Liu et al., 2013; Schwartz et al., 2015; Schwartz et al., 2017). Ihh expression was identified in proximity to Gli1+ progenitor population in stationary entheses (Dyment et al., 2015; Liu et al., 2013; Schwartz et al., 2015). Moreover, loss-of-function of Smo, a componenet of the HH pathway, in Scx-expressing cells resulted in reduced mineralization of the enthesis (Breidenbach et al., 2015; Dyment et al., 2015; Liu et al., 2013; Schwartz et al., 2015). Yet, the question of which ligand of the HH pathway regulates the specification of Gli1+ cells in the embryo and, specifically, in migrating entheses has remained open. Our results clearly suggest that Gli1 expression is regulated by both SHH and IHH during enthesis development. In the embryo, we show that SHH, but not IHH, signaling is essential for Gli1 expression in the enthesis, implicating SHH in Gli1+ progenitor specification. However, during postnatal development, Ihh expression is vital for maintaining Gli1 enthesis expression (Fig. 7A).
During endochondral ossification, IHH regulates hypertrophic chondrocyte differentiation and, thereby, bone elongation (Vortkamp et al., 1996). It is therefore possible that by controling both these processes, IHH coordinates enthesis migration with the concurrent bone elongation to preserve enthesis position along the shaft. However, the signals that govern enthesis positioning and migration along the bone, including the possible effect of IHH on these processes, are yet to be elucidated.
Another key question regards the mechanism that facilitates the replacement between Sox9+ and Gli1+ cell populations. It is possible that the former cells die, while the latter cells proliferate and populate the entire enthesis. A more attractive hypothesis is that during enthesis drift, Sox9+ cells are removed together with eroded bone tissue by osteoclasts or other phagocytic cells. However, the replacement mechanism remains an open question. Moreover, whether the replacement process is the mechanism that allows migrating entheses to maintain their relative position along the bone is yet to be determined. Finally, our finding that Gli1-expressing cells are pre-determined already in the embryo suggests the existence of an earlier, yet unknown player in the specification of these progenitors. The discovery of this early marker gene may enable the identification of multipotent enthesis cells, which may hold therapeutic potential.
To conclude, our findings shed light on developmental linearity in organogenesis. Using the enthesis as a model system, we identify two different strategies of development (Fig. 7). The first, found in stationary entheses, is linear development where embryonic progenitors and their descendants contribute to the entire process of enthesis development, from embryogenesis to maturity. Conversely, in migrating entheses, another developmental strategy was identified where one type of progenitor cells form an embryonic template, only to be later replaced by another cell lineage that contributes to the mature organ.
MATERIALS AND METHODS
Animals
All experiments involving mice were approved by the Institutional Animal Care and Use Committee (IACUC) of the Weizmann Institute. Histology was performed on BL6 mice.
For lineage tracing experiments, Sox9-Cre (Akiyama et al., 2005), Sox9-CreER (Soeda et al., 2010) and Gli1-CreERT2 (Ahn and Joyner, 2004, Jackson laboratories) mice were crossed with R26R-tdTomato mice (B6;129S6-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J, (Madisen et al., 2010) or with R26R-Confetti mice (B6.129P2-Gt(ROSA)26Sortm1(CAG-Brainbow2.1)Cle/J, Snippert et al., 2010).
To create Shh KO mice, mice heterozygous for a mutation in Shh (B6.Cg-Shhtm1(EGFP/cre)Cjt/J; Jackson Laboratory) were intercrossed; heterozygotes or WT littermates were used as a control. The generation of Prx1-Cre (Logan et al., 2002) was previously described. To generate Prx1-Cre-Ihh mutant mice, Ihh-floxed mice (B6N;129S4-Ihhtm1Blan/J; Jackson Laboratory) (Razzaque et al., 2005) were mated with Prx1-Cre mice. As a control, Prx1-Cre-negative animals were used.
For cell lineage experiments, Sox9-CreER T2/+ or Gli1-CreERT2/+ males were crossed with R26R-tdTomato females to produce embryos carrying both the relevant CreERT2 and R26R-tdTomato alleles. For fate mapping, Gli1-CreERT2/+ males were crossed with R26R-Confetti females to produce embryos carrying both the Gli1-CreERT2 and Confetti alleles.
In all timed pregnancies, plug date was defined as E0.5. For harvesting of embryos, timed-pregnant females were sacrificed by cervical dislocation. The gravid uterus was dissected out and suspended in a bath of cold phosphate-buffered saline (PBS) and the embryos were harvested after removal of amnion and placenta. Tail genomic DNA was used for genotyping.
Histological analysis, in situ hybridization and immunofluorescence
For histology and in situ hybridization, embryos were harvested at various ages, dissected, and fixed in 4% paraformaldehyde (PFA)/PBS at 4°C overnight. After fixation, tissues were dehydrated to 70% EtOH and embedded in paraffin. The embedded tissues were cut to generate 7-µm-thick sections and mounted onto slides. Hematoxylin and eosin (H&E) staining was performed following standard protocols. Non-fluorescent and fluorescent in situ hybridizations were performed as previously described using digoxigenin-(DIG) labeled probes (Shwartz and Zelzer, 2014). All probes are available upon request.
For immunofluorescence staining, 10-µm-thick cryosections were air-dried for 1 hour before staining. For IHH staining, sections were washed twice in PBST for 5 minutes and blocked to prevent non-specific binding with 7% goat serum and 1% BSA dissolved in PBST. Then, sections were incubated with rabbit anti-IHH antibody (Abcam, #AB39364, 1:50) at 4C° overnight. The next day, sections were washed twice with PBST and incubated for 1 hour with Cy2 conjugated fluorescent antibody (1:100, Jackson Laboratories). Slides were mounted with Immuno-mount aqueous-based mounting medium (Thermo).
For Gli1 staining, 10-µm-thick cryosections were air dried for 1 hour and fixed in 4% PFA for 10 minutes. Then, sections were washed twice in PBST and endogenous peroxidase was quenched using 3% H2O2 in PBS. Next, antigen retrieval was preformed using 0.3% Triton in PBS. Non-specific binding was blocked using 7% horse serum and 1% BSA dissolved in PBST for 1 hour. Then, sections were incubated with Goat anti-GLI1 antibody (R&D systems, #AF3455, 1:100) overnight at room temperature. The next day, sections were washed twice in PBST and incubated with Biotin anti-goat (1:100, Jackson laboratories, 705065147) for 1 hour and then with streptavidin-HRP (1:200 Perkin Elmer, NEL750001EA) for 1 hour. HRP was developed using TSA amplification kit (1:100, Prekin Elmer) for 20 minutes, counterstained with DAPI and mounted with Immuno-mount aqueous-based mounting medium (Thermo).
Physical position of entheses
For identification of enthesis physical position, 2-4 limb samples at stages E16.5-P14 were scanned ex vivo using iodine contrast agent to allow visualization of the soft tissue. DT and TM positions were identified manually, and their physical position along the bone was calculated as described previously (Stern et al., 2015).
Cell lineage analysis
Tamoxifen (Sigma T-5648) was dissolved in corn oil (Sigma C-8267) at a final concentration of 5 mg/ml. Time-mated R26R-tdTomato females were administered 1 mg of tamoxifen by oral gavage (FST) at designated time points as indicated.
Cell count
For each age, 2-4 limbs from different litters were harvested, embedded in OCT, and sectioned at a thickness of 10 µm. Sections were imaged using Zeiss LSM 780 microscope approximately every 80 µm, capturing red and DAPI channels. Images were then processed in ImageJ as follows: Each image was converted to an RGB stack and the region of interest was manually identified and cropped. Then, image levels were adjusted to improve separation between nuclei. A binary threshold was set automatically and conjoined nuclei were automatically separated using the binary watershed function. Using a home-made Matlab script, the number of nuclei was counted. Each nucleus that was co-localized with a red channel signal was counted as a tdTomato-positive cell. For each section, the percentage of tdTomato-positive cells was calculated. The average percentage of these cells was calculated first for each bone and then for all samples. From each line and for each age group, at least 3 individual bones were sampled, and 9-17 sections per bone were analyzed, depending on bone length. Data are presented as mean ± SD.
Fate mapping Confetti experiment
Tamoxifen (Sigma T-5648) was dissolved in corn oil (Sigma C-8267) at a final concentration of 20 mg/ml. Time-mated R26R-Confetti females were administered 4 mg of tamoxifen by oral gavage at E13.5. Cre-positive pups were sacrificed at P14. 1 hour prior to sacrifice, each pup was injected with Calcein blue (Sigma, #m1255; 30 mg/kg). Limbs were harvested, fixed for 30 minutes in 4% PFA at 4°C, embedded in OCT, and sectioned at a thickness of 10 µm. Sections were imaged using Zeiss LSM 780 microscope approximately every 80 µm.
Microscope settings and image analysis
At least 1024×1024 pixels, 8-bit images were acquired using the X20 lens. A Z-stack of 2-4 images was taken from each section. To detect GFP and YFP, the argon laser 488 nm was used. For RFP detection, a red diode laser emitting at 561 nm was used, and blue mCFP was excited using a laser line at 458 nm. Calcein blue staining was detected using the 405-nm laser line. GFP fluorescence was collected between ~500-598 nm, airy 1; RFP fluorescence was collected between ~606-654nm, airy 1; mCFP fluorescence was collected between ~464-500 nm, airy 1 and Calcein blue was collected between ~410-451. For each image, a corresponding bright field image was captured. The acquired images were processed using Photoshop and ImageJ.
To calculate the percentage of Gli1+ clones found in both mineralized and non-mineralized fibrocartilage, the border between the zones was identified by Calcein blue signal. Clones that crossed the mineral border were manually counted and their percentage was calculated first for each section, then for each limb and, finally, for each type of enthesis. Data are presented as mean ± SD.
Acknowledgments
We thank Nitzan Konstantin for expert editorial assistance. We thank Dr. Patrick Tschopp from the Clifford J. Tabin lab, Harvard Medical School, for his assistance in generating Shh KO mice. Special thanks to all members of the Zelzer laboratory for encouragement and advice.
This study was supported by grants from the National Institutes of Health (grant #R01 AR055580), European Research Council (ERC) (grant #310098), the Jeanne and Joseph Nissim Foundation for Life Sciences Research, the Y. Leon Benoziyo Institute for Molecular Medicine, Beth Rom-Rymer, the Estate of David Levinson, the Jaffe Bernard and Audrey Foundation, Georges Lustgarten Cancer Research Fund, the David and Fela Shapell Family Center for Genetic Disorders, the David and Fela Shapell Family Foundation INCPM Fund for Preclinical Studies, and the Estate of Bernard Bishin for the WIS-Clalit Program.