For the dominant active CaMKII transgene a cassette containing CaMKII-T286D C-terminal fused to eGFP (Taschner et al., 2008) followed at the 3′ end by two SV40polyA sequences was inserted via blunt-end cloning into the BamHI site of a plasmid carrying the Col2a1 promoter–rabbit β globin intron–Col2a1 enhancer element (a gift from Yoshi Yamada; (Nakata et al., 1993)). For the rKIIN transgene, a peptide inhibitor for CaMKII from rat (Chang et al., 1998, 2001) fused with eGFP at its N-terminus (Taschner et al., 2008) was cloned accordingly. The final transgenic constructs (Figures 1A,B) were excised with the restriction enzymes AfllII and SwaI, purified on agarose gel and eluted with DNA microinjection buffer. The linear transgene cassettes were microinjected into the pronucleus of B6CBAF₁ zygotes in the transgene facility of the IMP, Vienna, Austria (Hogan et al., 1994). The zygotes were implanted into pseudo-pregnant mice to obtain transgenic founder lines. To maintain the transgenic lines in a pure genetic background, the founder lines were crossed with C57Bl/6J females and the subsequent generations were backcrossed with C57Bl/6J animals over at least eight generations. Genotyping of transgenic mice and embryos was performed by PCR using ear biopsies and material from the embryonic tail respectively, in combination with transgene-specific primer pairs (listed in Supplementary Table 1). Transgene-copy numbers were determined using genomic DNA from ear biopsies of the different transgenic lines (two independent samples per line) by qPCR and normalized to actin and control genomic DNA from mice carrying one copy of the transgene in the Rosa26 locus (Amara and Hartmann, unpublished; Ballester et al., 2004). Primers are listed in Supplementary Table 2. Mouse experiments were performed in accordance with local, institutional and national regulations under the following licenses (84-02.05.2012.260, 84-02.04.2015.A278, 84-02.05.50.15.022).

For timed pregnancies, heterozygous transgenic mice were interbred overnight and the plug day was designated as embryonic day 0.5 (E0.5). Embryos were harvested at the required stages, dissected and fixed overnight in 4% paraformaldehyde (PFA). For stages E18.5 and older, the skin above the limbs was removed prior to fixation. Fixed limbs were removed, washed in PBS, and tissue was dehydrated using increasing ethanol concentrations (25, 50, 75, 100%).

For in situ hybridization (ISH) and histological staining on sections, limbs were processed into paraffin using the Excelsior ES Vacuum Infiltration Processor (Thermo Scientific), embedded in paraffin and sectioned at 5 μm. Non-radioactive ISH were performed using digoxigenin (DIG)-labeled anti-sense RNA probes as previously described (Murtaugh et al., 2001). Probes for type 2 collagen (Col2a1), indian hedgehog (Ihh), osteocalcin (Ocn), osteopontin (Opn), type 1 collagen (Col1a1), and type 10 collagen (Col10a1) have been published previously (Hill et al., 2005). The Gfp probe was generated using a plasmid containing the eGFP coding region. All probes are available upon request. Histological stainings such as alcian blue, alcian blue/von Kossa, and hematoxylin/eosin were performed as previously described (Houben et al., 2016). Skeletal preparations were performed on 6 day old pups which were sacrificed by decapitation, skinned, eviscerated, fixed in 95% EtOH, and double-stained for alcian blue and alizarin red (McLeod, 1980). The length of isolated humeri and femora was calculated by using Zeiss image analysis software.

The stylo- and zeugopod regions of forelimbs were isolated from embryos at the required stage. Skin and soft tissue was removed and skeletal elements were dispersed using the Polytron PT 1200 E manual disperser with the aggregate PT-DA 03/2 EC-E50 (Kinematica). The RNA was isolated using the QIAGEN RNeasy micro kit according to the manufacturer's instructions. A total of 500 ng RNA was reverse transcribed using PrimeScript RT reagent kit (Takara, #RR037A) with oligo dT primer. For qPCR cDNA was diluted 1:10 in water. 3 μl of diluted cDNA were mixed with 1 μl of primer mix and 10 μl SYBR Premix Ex TaqII (Tli RNaseH plus) (Takara, #RR820Q) in a final volume of 20 μl. Gene expression was monitored using the BioRad CFX96 cycler and the following protocol: 95°C 10 min, 45 × (95°C 30 s, 60°C 30 s, 72°C 45 s + plate read), 72°C 5 min, melting curve: 55°C–99°C in 0.5°C increments for 5 s + plate read. qPCR analysis was performed in duplicates and results were produced from at least three independent samples. Expression values were calculated using the comparative ΔC(t) method and normalized to Gapdh and Actb expression. For primer sequences and product sizes see Supplementary Table 3.

E13.5 appendicular skeletal elements from the stylo- and zeugopod were dissected in PBS supplemented with 1% P/S followed by a digestion with 0.1% type II collagenase (Gibco, #17101-015) and 0.3% dispase (Gibco, #17105-041) in medium (DMEM/F-12; 1% P/S) for a period of 60 min shaking at 100 rpm in a CO₂ incubator. Cells were filtered through a cell strainer, centrifuged and washed with culture medium (DMEM/F-12, 1% P/S, and 10% FCS). Cell pellets were then resuspended in culture medium, plated at 2.5–3 × 10⁴ cells/cm² in tissue culture flasks and grown for 5 days.

Primary chondrocytes were seeded at a density of 3 × 10⁴ cells/well in 48 well plates 1 day prior to transfection. Cells were transfected with the pGL4.23-Mef2-luc (a gift from Eric Olson) or the pGL4.10-Ose2-luc reporter plasmid (a gift from G. Karsenty) in combination with pRL-TK (Promega) as a control and pCDNA3.1-DaCaMKII or pCDNA3.1-rKIIN expression vectors. Total amounts of transfected plasmids (equivalent to 52 fM) in each group were adjusted by adding empty vector (pCDNA3.1+). Transfection was performed using jetPRIME™ (Polyplus transfection) according to manufacturer's instructions. Luciferase activities were measured 48 h after transfection following the protocol by (Hampf and Gossen, 2006). Luciferase measurements were normalized to the corresponding renilla activities for transfection efficiency. Experiments were performed in triplicates and repeated at least three times.

For immunofluorescent staining on cells, primary chondrocytes isolated from P6 C57Bl/6J knee epiphyseal regions were seeded on 0.1% gelatin coated glass coverslips. Cells were transfected with DaCaMKII::eGFP and eGFP expression vectors (equivalent to 0.12 pmol) using jetPRIME™ (Polyplus transfection). Forty-Eight hours after transfection, cells were fixed with 4% PFA/PBS for 20 min, treated with 0.5% TritonX-100 for 5 min and blocked with 10% normal goat serum (NGS) in PBS for 1 h at room temperature (RT). Incubation with β-catenin antibody (1:200 in 2% NGS/PBS, BD Biosciences, #610154, RRID:AB_397555) was performed overnight at 4°C. Coverslips were subsequently incubated with Alexa Fluor 647 goat anti-mouse IgG (Molecular Probes) at 1:200 in 2% NGS/PBS. Nuclei were counterstained with DAPI.

HEK293 cells were co-transfected with Mef2c-Myc and DaCaMKII::eGFP at 4:3 ratio or FLAG-HDAC4 and DaCaMKII::eGFP at 1:3 ratio using CaPO₄ transfection method. Cells were lysed in ice-cold lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40 with proteinase and phosphatase inhibitors). After centrifugation, a total amount of 150 μg of protein from the supernatant was subjected to immunoprecipitation using 25 μl magnetic beads coupled with GFP monoclonal antibody (MBL, #D153-11) under the following conditions: 15 min at 4°C on a rotating wheel (10 rpm), washed 4 times with wash buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40) for 5 min at RT. For the pull-down of FLAG-HDAC4 using GFP-magnetic beads the conditions were as following: 500 μg of protein from the supernatant was subjected to immunoprecipitation using 50 μl magnetic beads and bound for 45 min at 4°C on a rotating wheel (10 rpm), followed by 3 washes (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% NP-40) for 30 s at RT. Magnetic beads were then boiled for 2 min in 25 μl Laemmli's sample buffer and supernatant of the beads was loaded on a 10% SDS-PAGE to separate the immunoprecipitated proteins.

Transfected primary chondrocytes were lysed with ice-cold lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40 with proteinase and phosphatase inhibitors). 50 μg of protein lysates were run on a 10% SDS-PAGE and transferred to a 0.45 μm PVDF-membrane by semi-dry transfer (PerfectBlue™, PeqLab). The membrane was blocked with 5% milk and incubated with the appropriate primary antibodies anti-GFP (1:15000, Abcam, #ab13970, RRID:AB_300798), anti-Myc (1:1000, Cell Signaling Technology, mAb #2276, RRID:AB_331783), pan-CaMKII (1:1000, Cell Signaling Technology, mAb #4436S, RRID:AB_10545451), anti-Flag (1:1000, Sigma-Aldrich, #F1804, RRID:AB_262044), and proteasome 20 (P20) (1:15000, Abcam, #ab3325, RRID:AB_303706), followed by incubation with the respective, species-specific HRP-coupled secondary antibodies (1:1000 and 1:5000). ECL substrate was used for signal development (Amersham, #RPN2106) on X-ray film (Amersham).

Histological images were acquired using Zeiss AxioImager M2 equipped with an AxioCam MRc 6.45 μm color camera (Zeiss, Jena). Images of embryos were acquired using a Zeiss Stereo discovery V8 equipped with Zeiss plan Apo S, 0.63X lens. Immunofluorescent images were acquired using Zeiss AxioImager M2 equipped with an Apotome2 and an AxioCam MRm 6.45 μm monochromatic camera (Zeiss, Jena) using the Zen software (Zeiss, Jena).

All statistical analyses were performed using GraphPad Prism software 6.0 (RRID: SCR_002798). Data are displayed as mean values ± standard error of the mean (SEM). Statistical significance of differences (P-value) was determined by the two-tailed, unpaired Student's t-test.

Based on previous studies in chicken, potential molecular mechanisms of how CaMKII signaling regulates chondrocyte hypertrophy have been suggested (Li et al., 2011). These included increased nuclear localization of β-catenin and Runx2 in chondrocytes with activated CaMKII signaling (Li et al., 2011). Thus, we investigated whether the molecular mechanism in mouse chondrocytes may be similar. Mouse chondrocytes, like chicken chondrocytes, express all four CaMKII isoforms, and also here the CaMKIIβ isoform was expressed at lower levels than the other three isoforms (Figure 1A, see also Taschner et al., 2008; Li et al., 2011). Similar to what was observed in chicken, β-catenin was localized in the nucleus in mouse chondrocytes transfected with a DaCaMKII::GFP expression plasmid (Figure 1B, white arrows). This was not observed in the GFP-transfected control cultures (Figure 1B, left panel). We also analyzed whether Runx2 transcriptional activity was altered by the presence of DaCaMKII or rKIIN in mouse chondrocytes. For this, the Runx2-dependent OSE2-luc reporter (Ducy and Karsenty, 1995) was co-transfected into primary chondrocytes with increasing amounts of either DaCaMKII or rKIIN expression plasmid. OSE2-luc reporter activity was upregulated in response to increasing amounts of DaCaMKII, while expression of rKIIN led to a downregulation of the reporter activity in a concentration independent manner (Figure 1C). To rule out the possiblity that modulation of CaMKII activity affects the expression levels of Runx2 itself, we examined the Runx2 expression levels in the OSE2-luc assay by qPCR. Here, Runx2 expression was not affected in cells transfected with the DaCaMKII or rKIIN expression plasmids (Figure 1D).

Another transcription factor promoting hypertrophy is Mef2c (Arnold et al., 2007). The MEF2-luc reporter (Naya et al., 1999) was activated over 50-fold by DaCaMKII at the highest concentration used (Figure 1E). An increase in CaMKII protein levels corresponding to the increased amount of transfected expression plasmid was demonstrated by western blot (Figure 1E, lower panel). The DaCaMKII expression levels were also determined by qPCR using a primer that detects the mouse as well as the transgenic rat Camk2a transcript. Here, the Camk2a expression levels were normalized to the endogenous Camk2a expression levels in control-transfected chondrocytes. This quantitative approach revealed an over 100-fold increase in Camk2a levels the cells transfected with 30 fM DaCaMKII (Supplementary Figure 1). Overexpression of rKIIN on the other hand, led, with the exception of the highest concentration, to a downregulation of the MEF2-luc reporter activity independent of the concentration (Figure 1E). To our surprise, at the highest concentration even a two-fold activation was observed (Figure 1E). Again, the endogenous Mef2c expression levels were not significantly affected by either DaCaMKII or rKIIN expression in the transfected primary chondrocytes (Figure 1F). Given the strong effect on Mef2c activity we tested whether CaMKII may directly interact with Mef2c. Co-immunopre-cipitation assays in HEK293 cells using tagged proteins revealed that the activated form of CaMKII physically interacts with Mef2c (Figure 2A). Here we used GFP-magnetic beads and pulled on the DaCaMKII::GFP fusion protein and detected bound Mef2c::Myc (Figure 2A). Conversely, using Myc-magnetic beads and pulling on Mef2c::Myc the DaCaMKII::GFP fusion protein was detected by immunoblot in the bound fraction (data not shown).

Given previous findings that (a) the CaMKIIδ isoform can interact with and influence histone deacetylase 4 (Hdac4) activity in cardiomyocyte hypertrophy (Backs et al., 2006, 2009), (b) Hdac4 controls chondrocyte hypertrophy by interacting with and inhibiting the activity of Runx2 and Mef2c (Vega et al., 2004; Arnold et al., 2007), and (c) our observation that DaCaMKII modulates Mef2c as well as Runx2 activity, we addressed whether DaCaMKII also physically interacts with Hdac4 or whether it alters its repressive activity. For this, HEK293 cells were co-transfected with FLAG::Hdac4 and DaCaMKII::GFP. Co-IP studies with GFP-coupled magnetic beads revealed no physical interaction for Hdac4 in the pull-down lysate (Figure 2B). Luciferase assays on primary chondrocytes transfected with MEF2-luc, Mef2c and Hdac4 resulted in the inhibition of Mef2 activity. Yet, this inhibitory effect was not altered by the addition of increasing amounts of DaCaMKII (Figure 2C). This suggests that the DaCaMKII does not modulate Hdac4 activity. In essence, our results show that the activated form of CaMKII positively influences the activity of the transcription factors Mef2c and to a lesser extent Runx2 activity and that this effect is probably not mediated via an inhibitory effect on Hdac4. Furthermore, CaMKII physically interacts with Mef2c suggesting that it may possibly modulate its transcriptional activity by phosphorylation.

In order to determine the in vivo role of CaMKII modulation in mouse endochondral ossification, we employed a transgenic approach using the Col2a1 promoter/enhancer element to drive transgene expression in chondrocytes (Figures 3A,B). From previous studies in the chicken we knew that eGFP tagged transgenes are functional (Taschner et al., 2008). Hence, in order to visualize transgene expression and to facilitate the distinction between heterozygous and homozygous embryos, eGFP tagged transgenes were used. For the DaCaMKII::eGFP construct two independent transgenic founder lines (Tg1 and Tg2-DaCaMKII) were established and for the eGFP::rKIIN construct three independent founder lines were obtained (Tg1, Tg2, and Tg3-rKIIN; Figures 3A,B). All founder lines transmitted the transgene to the F1 generation. The transgenic lines differed in the number of transgenes integrated in the genome (Figure 3A). For a first characterization of the transgenic lines, heterozygous transgenic mice of the independent founder lines were intercrossed to isolate embryos at embryonic day (E) 15.5. Contrary to our expectations, no obvious gross morphological differences were detected within the offspring of the various intercrosses (Figures 3C,D). Genotyping of the offspring was performed examining the GFP intensity using a fluorescent stereomicroscope and confirmed by conventional PCR-based genotyping (Figures 3C,D; data not shown). Heterozygous and homozygous transgenic embryos were classified based on the fluorescence intensity (Figures 3C,D). As expected, GFP activity was detected in the cartilaginous regions of the skeletal elements, particularly visible in the skull and limbs at sites corresponding to Col2a1 expression domains (Figures 3C,D; Cheah et al., 1991). This was confirmed by ISH with RNA antisense probes for Col2a1 and Gfp on adjacent sections of transgenic limbs (Figures 3E,F). As expected, no Gfp signal was detected in humeri of non-transgenic (wild-type) littermates (Figures 3E,F). These results demonstrated that the transgene is indeed expressed in a Col2a1-promoter/enhancer-dependent manner. Furthermore, the GFP signal intensity in embryos as well as the Gfp ISH signal on sections allowed us to distinguish heterozygous and homozygous transgenic littermates.

In the chicken a subset of the chondrocytes that expressed DaCaMKII prematurely differentiated into prehypertrophic and subsequently into hypertrophic chondrocytes outside their normal expression domains (Taschner et al., 2008; Li et al., 2011). Hence, we performed ISH on E15.5 sections through the limbs using Col2a1 as a marker for more immature chondrocytes, Ihh as a marker for prehypertrophic cells, and Col10a1 as a marker for hypertrophic cells. Yet, no expression of the maturation markers Ihh or Col10a1 was observed outside their normal expression domains and no obvious differences were observed with respect to the individual domain sizes, distance between the domains, or the expression levels of these three markers comparing non-transgenic with heterozygous and homozygous DaCaMKII-tg littermates (Figures 4A,B). Similar, negative results were obtained performing the analogous analysis on sections of E15.5 humeri from non-transgenic, heterozygous, and homozygous rKIIN-tg littermates (Figures 4C,D). Here, we did not observe an obvious delay in chondrocyte maturation, as one could have expected based on the effect of rKIIN overexpression in chicken.

The final step in endochondral ossification is the remodeling of the cartilage template into bone. Thus, in order to see whether this remodeling process was altered, we analyzed the longitudinal extension of the ossified zone by performing ISH analysis for the osteoblastic markers collagen 1 (Col1a1), osteopontin (Opn), and osteocalcin (Ocn) on sections of E18.5 DaCaMKII- and rKIIN-tg humeri and corresponding non-transgenic littermates. No significant alteration to the longitudinal extension of the ossified zone for either of the three analyzed markers was observed when comparing the humeri of the transgenic embryos expressing either DaCaMKII (Figures 5A,B) or rKIIN with humeri of their respective, non-transgenic controls (Figures 5C,D). Similar to what we had observed at E15.5, no morphological alterations with regard to the shape, organization or appearance of the prehypertrophic and hypertrophic chondrocytes were detected in the growth plates of the E18.5 DaCaMKII- and rKIIN-tg humeri compared to their corresponding non-transgenic littermate controls (Supplementary Figures 2A,B). In addition, the total length, as well as the length of the mineralized region was measured in alician blue/alizarin red stained humeri of 6-day-old pups. Here again, no differences regarding the two parameters were observed in comparison to non-transgenic littermate controls (Supplementary Figure 3).

As there were no obvious effects visible in the older mouse limbs, we addressed whether early steps of skeletogenesis would potentially be affected. In the forelimb the anlagen of humerus, radius and ulna are visible at E11.5. At this stage the cells in the center of the humerus begin to express Ihh (data not shown), while they start to express Col10a1 around E12.5. In humeri of E12.5 DaCaMKII transgenic limbs the marker for prehypertrophic chondrocytes, Ihh, was expressed in a slightly broader domain compared to non-transgenic, control littermates (Figures 6A–C). The effect was more prominent in humeri of homozygous (6/8) than heterozygous (8/11). When we analyzed the expression of Col10a1, as a marker for hypertrophic chondrocyte differentiation, weak Col10a1 expression was first detected in the humerus at E12.5-E13.0 in wild-type limbs. In the transgenic specimens, in which we had observed a broadend Ihh domain we noticed on adjacent sections a more intense staining for Col10a1 in a broader domain (Figure 6D). Again the effect was more obvious in the homozygous specimens. Next we asked whether the increased Col10a1 expression was accompanied by the histological appearance of hypertrophic chondrocytes. For this, we examined E12.5 and E13.5 limbs by alcian blue staining. On alcian blue stained sections, the hypertrophic chondrocytes within the cartilage elements appear lighter in color due to their increase in size and vacuolization. Histological examination of E12.5 humeri (n = 2) revealed no apparent signs of hypertrophic chondrocyte differentiation in the transgenic limbs (Supplementary Figure S4A). In E13.5 DaCaMKII-tg humeri, a moderate size increase of the zone of hypertrophic chondrocytes was detected compared to the non-transgenic control (Figure 6E, n = 3). Consistent with the in vivo ISH results, qPCR analysis of material from E12.5 DaCaMKII transgenic and non-transgenic control limbs revealed an increase in Ihh and Col10a1 expression levels in the transgenic limbs in comparison to the control (Figure 6F). These results suggest that the onset of chondrocyte maturation is slightly accelerated in the transgenic limbs.

We then analyzed rKIIN transgenic limbs at equivalent stages. At E12.5, no obvious differences were detected with respect to the size or intensity of the Ihh expression domains between transgenic and non-transgenic embryos (Supplementary Figure S4B). However, at E13.5 the two expression domains of Ihh were not yet separated in the humeri of homozygous transgenic animals and still closer together in the heterozygous transgenic animals compared to non-transgenic littermate controls (Figure 6I). Furthermore, the Col10a1 expression domain was reduced in size in the humeri of the transgenic animals compared to non-transgenic littermate controls (Figure 6J). Together, his suggests that the onset of chondrocyte maturation is slightly delayed when CaMKII signaling is antagonized. Again, the effect was more pronounced in homozygous (9/9) than in heterozygous transgenic specimens (11/13) compared to non-transgenic specimens (Figures 6I,J). Histological examination of alcian blue stained E13.5 humeri confirmed that the decrease in the size of the Col10a1 expression domain in homo- and heterozygous humeri was associated with reduced hypertrophic chondrocyte differentiation (Figure 6K). Quantification of Ihh and Col10a1 expression levels by qPCR using material from E13.5 forelimbs corroborated the dose-dependent effects of CaMKII-signaling inhibition, which was statistically significant with respect to Col10a1 expression (Figure 6L). Together, our results suggest that modulation of CaMKII signaling in the mouse affects the onset of chondrocyte maturation during the early stages of endochondral ossification.

ISH with a Gfp riboprobe confirmed transgene expression at all stages examined and at least according to the ISH transgene expression was not silenced after E13.5 (Supplementary Figure 5). In order to determine why the expression of the DaCaMKII and rKIIN-transgenes may not lead to a major phenotype at later stages we examined the transgene expression level in the two DaCaMKII-transgenic lines, DaCaMKII-Tg1 (1 copy) and DaCaMKII -Tg2 (19 copies), using qPCR and RNA isolated from the chondrogenic elements of E12.5 limbs. Transgene expression levels were estimated by comparing the DaCaMKII::GFP transgene expression level to the endogenous expression levels of the four CaMKII isoforms and the levels were normalized to the Camk2g expression level. Here, we noticed that the transgene expression levels in the Tg1 line were similar to the Camk2a endogenous levels in the wild-type control. Yet, they were lower than the endogenous expression levels of the γ and δ isoforms. Interestingly, the Camk2a expression levels were increased significantly in the transgenic skeletal elements of the DaCaMKII-Tg1 line. In contrast, transgene expression level in the Tg2 line with 19 copies did not even reach the Camk2a endogenous levels. As such, the transgene copy number did not correlate with the transgene expression levels in the two independent lines (Supplementary Figures 6A,B).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.