Cell-nonautonomous local and systemic responses to cell arrest enable long-bone catch-up growth in developing mice

Catch-up growth after insults to growing organs is paramount to achieving robust body proportions. In fly larvae, injury to individual tissues is followed by local and systemic compensatory mechanisms that allow the damaged tissue to regain normal proportions with other tissues. In vertebrates, local catch-up growth has been described after transient reduction of bone growth, but the underlying cellular responses are controversial. We developed an approach to study catch-up growth in foetal mice in which mosaic expression of the cell cycle suppressor p21 is induced in the cartilage cells (chondrocytes) that drive long-bone elongation. By specifically targeting p21 expression to left hindlimb chondrocytes, the right limb serves as an internal control. Unexpectedly, left–right limb symmetry remained normal, revealing deployment of compensatory mechanisms. Above a certain threshold of insult, an orchestrated response was triggered involving local enhancement of bone growth and systemic growth reduction that ensured that body proportions were maintained. The local response entailed hyperproliferation of spared left limb chondrocytes that was associated with reduced chondrocyte density. The systemic effect involved impaired placental function and IGF signalling, revealing bone–placenta communication. Therefore, vertebrates, like invertebrates, can mount coordinated local and systemic responses to developmental insults that ensure that normal body proportions are maintained.


Introduction
An important question in biology is how cells integrate intrinsic and extrinsic information such that their combined behaviours produce higher-order processes and structures, as seen during organogenesis and tissue repair. In Drosophila larvae, injured imaginal discs can undergo compensatory proliferation [1] as well as secrete an alarm signal that triggers both a systemic developmental delay and reduced growth of the spared imaginal discs [2][3][4][5]. Together, these processes allow the damaged tissue(s) to catch-up with other tissues, but the role of damaged versus undamaged cells remains controversial [6,7]. In vertebrates, systemic growth reduction after injury in a nonessential organ has not been reported. However, systemic catch-up growth has been described after transient impairment of whole-body growth [8][9][10], and local growth compensation can occur after unilateral manipulation of long bones within the limbs [11]. Tight control of inter-limb and limb-body proportions are critical for efficient locomotion and interaction with the environment, and therefore long bones are an excellent model for studies of growth regulation.
Growth of the long bones is driven by a process called endochondral ossification (reviewed in [12,13]). Once mesenchymal cells condense into the template of the skeletal elements, they differentiate into collagen II-expressing chondrocytes that go through sequential differentiation steps from bone ends to centre. Round resting chondrocytes give rise to flat proliferative cells that form columns and, after a few rounds of duplication, cease proliferation and start to differentiate into hypertrophic chondrocytes. These cells increase their volume as they progress towards the centre of the shaft, lay down a collagen X-rich extracellular matrix, and secrete factors that recruit vasculature and bone precursors (osteoblasts) from the perichondrium, a fibrous layer that wraps the cartilage [14,15]. Some hypertrophic chondrocytes die by apoptosis, while others transdifferentiate into osteoblasts [16,17]. Osteoblasts form the primary ossification centre by replacing the original matrix with bone. This process is later repeated at the ends of the bone (epiphyses), forming the secondary ossification centres. The so-called growth plate remains as a cartilage disc between the primary and secondary ossification centres and responds to both intrinsic and extrinsic factors that ultimately regulate bone length. For example, indian hedgehog (IHH), secreted by pre-hypertrophic chondrocytes, and parathyroid hormone-like peptide, secreted by resting chondrocytes, form a negative feedback loop that couples chondrocyte proliferation and differentiation (reviewed in [12,13]). This loop is the main conduit through which other local signals, such as fibroblast growth factors and bone morphogenetic proteins, exert their function, often impacting on the expression of key transcription factors. A number of systemic and local extrinsic signals (growth hormone, insulin-like growth factors [IGFs]) also play crucial roles in the modulation of chondrocyte activity and bone growth [18,19]. As per the regulation of growth after an insult, it has been proposed that bone catch-up growth is due to a cell-autonomous delay in the normal developmental decline of chondrocyte proliferation, such that when the insult is lifted, the formerly arrested chondrocytes retain a higher proliferative potential correlating with the stage at arrest [9,20]. It was suggested that a similar mechanism applies to other organs [21]. However, the possible contribution of unaffected cells has not been examined, which is important because a cell-autonomous mechanism does not account for cases in which catch-up growth is faster than expected for the observed maturation delay (reviewed in [13,22]).
Here, we developed new mouse models to transiently decrease long-bone growth in mice in order to determine the contributions of cell-autonomous and nonautonomous regulation during catch-up growth. Namely, we blocked proliferation in 50% of the cartilage chondrocytes that drive long-bone elongation, specifically in the left hindlimbs, such that the right limb remains as an internal control. Unexpectedly, left-right symmetry was maintained, revealing the deployment of compensatory mechanisms. Locally, we observed hyperproliferation of wild-type (WT) chondrocytes that mostly compensated for the lack of proliferation of their arrested neighbours. Systemically, a mild growth reduction affected the rest of the body, contributing to maintenance of limb-body proportions. In summary, our results reveal that long-bone catch-up growth shares some similarities with the response of imaginal discs to developmental insults in insects. We found that this response is mostly cell nonautonomous, representing a paradigm shift in the field that opens up new research avenues for basic and translational studies.

An intersectional genetic approach enables inducible p21 misexpression preferentially in left limb chondrocytes
A major roadblock for studies of intra-and inter-organ growth regulation in mouse embryos has been a lack of models in which growth rate can be altered in a specific cell type within an organ, and ideally in only one of two paired organs, leaving the unmanipulated organ as an internal control. To address this deficiency, we devised new mouse models of inducible and transient growth inhibition in the left limb. We generated an Igs7 TRE-LtSL-p21/+ allele, a variant of a double-conditional allele [23], to achieve doxycycline (Dox)-tuneable misexpression of the cell cycle suppressor Cdkn1a (p21 hereafter) [24] in the cells in which activities of the bacteriophage recombinase Cre and of the (reverse) tetracycline transactivator ([r]tTA) intersect ( Fig  1A and 1B). Due to a floxed tdTomato-STOP sequence (LtSL), expression of tdTomato (tdT) takes place in cells expressing (r)tTA but having no history of Cre activity, whereas p21 is expressed in the cell population with a history of Cre and current (r)tTA activity (Fig 1A). We named the general type of allele Dox-controlled and Recombinase Activated Gene Overexpres-sioN (DRAGON). By combining the DRAGON-p21 allele with an asymmetric-Pitx2-enhancer-Cre line expressing Cre in the precursors of the left limb mesenchyme (S1A-S1F Fig) [25] and a cartilage-specific Col2a1-rtTA line containing the reverse tetracycline transactivator under the control of a type II collagen promoter [26] (Fig 1B), Dox-dependent ectopic p21 expression was achieved specifically in non-hypertrophic chondrocytes of the left limb cartilage elements ( Fig 1C and 1C'). Consequently, any growth adjustment detected in the right limb of triple transgenic animals (Pit-Col-p21) when compared to control littermates must be due to activation of a systemic effect or inter-organ communication.

Mosaic local proliferation blockade in the left limb cartilage slightly reduces bone growth but does not alter left-right limb symmetry
We next examined whether mosaic chondrocyte arrest altered left bone growth. As a first test, we compared the lengths of several left forelimb and hindlimb bones of ePit-Col-p21 mice to Pitx2-Cre; Igs7 TRE-LtSL-p21/+ control mice (ePit-p21 hereafter). At E17.5, the left bones were 0.2-0.3 mm (approximately 10%) shorter in animals misexpressing p21 (Fig 2A, n = 7 for ePit-p21; n = 15 for ePit-Col-p21 femora and radii; n = 4 and 11 for humeri and tibiae), indicating that blocking chondrocyte proliferation resulted in decreased bone growth. However, the effect was milder than expected, given that between one-third and two-thirds of chondrocytes were being arrested. This result suggested that compensatory mechanisms that minimized the impact of the p21 insult had been activated in the left limbs. Indeed, at E15.5 or E17.5, no major changes in the length of the proliferative or hypertrophic zones of the growth plate were found (S2F and S2G Fig).
We next took advantage of our unilateral approach that provides an experimental and a control limb within an animal and performed left-right intra-individual comparisons to determine the degree of asymmetry. Unexpectedly, most ePit-Col-p21 bones measured at E17.5 or birth (P0) showed no obvious difference in their left/right length ratio compared to ePit-p21 control littermates. The one exception was a transient small reduction in the size of the left radius compared to the right (Fig 2B and 2C, n ! 4 for ePit-p21; n ! 11 for ePit-Col-p21 at E17.5; n ! 5 and 6 at P0). These results suggested that the right (i.e., control) bones in ePit-Col-p21 mice responded to the impact of p21 expression in the left limbs via a systemic effect or inter-organ communication, such that their growth was reduced similarly to that of the left bones.
first tested for any organ-intrinsic responses and started by examining proliferation of the spared chondrocytes in the hindlimbs. Indeed, the left/right ratio of 5-ethynyl-2'-deoxyuridine (EdU) incorporation by p21 − chondrocytes was higher in experimental animals as compared with controls at E17.5 and P0 but not E15.5 ( Fig 3A and S2H Fig), revealing cell-nonautonomous compensatory proliferation of p21 − cells in the presence of p21 + neighbours. Because p21 − cells did not differ in size from those of control mice (S2I Fig), the hyperproliferation of these cells at E17.5 likely contributes to the lack of a left-specific growth reduction in ePit-Col-p21 embryos. In fact, overall EdU incorporation in left and right ePit-Col-p21 PZs (without distinguishing between p21 + and p21 − cells), while tending to be slightly reduced, was not significantly different, indicating that the compensatory proliferation phenomenon is quite effective  ( Fig 3B). Moreover, the proliferative disadvantage of p21 + versus p21 − chondrocytes in the left limb of ePit-Col-p21 mice resulted in dilution of p21 + chondrocytes from 45%-50% of PZ chondrocytes at E15.5 and E17.5 to approximately 20% at P0 (Fig 3C and 3D; n = 3, 5, and 8, respectively), and this depletion was not due to inactivation of rtTA activity (Fig 3D; n = 3 at E17.5; n = 3 at P0).

Compensatory proliferation involves local cell interactions
As a means to examine whether compensatory proliferation was only dependent on local cellcell interactions, we cultured left and right E15.5 ePit-Col-p21 tibiae (together in the same well) for 2 d with Dox, in the absence of surrounding mesenchyme (Fig 4A). We found that the distal tibia showed chondrocyte proliferation throughout all section levels, with very few or no senescent cells (S3A Fig, n = 3), whereas EdU incorporation was not detected in the inner core of the proximal tibia (bulkier than the distal one), probably because of insufficient nutrient diffusion. We therefore focused our analysis on the distal epiphysis. Similar to our findings in vivo, EdU incorporation in p21 − chondrocytes was significantly higher in the left as compared to the right cultured cartilages (Fig 4B and 4C), suggesting that compensatory proliferation is a bone-intrinsic phenomenon or at least does not require constant interaction with the surrounding mesenchymal tissues. As a control, we tested whether right cartilage proliferation was impaired due to the bone being cultured with the injured left tibia, since this would cause the impression of compensatory proliferation taking place in the left bone. We therefore cultured left and right tibiae from each embryo in different wells (S3B Fig) and found that EdU incorporation in distal right tibiae was not significantly different between bones cultured together (n = 4) or separately (n = 6) from the left bones.

Compensatory proliferation requires a minimum threshold of p21 + chondrocytes
We next addressed whether the proportion of p21 + chondrocytes in the growing cartilage influences the extent of compensatory proliferation. Given that forelimb bones show a lower proportion of p21 + chondrocytes than hindlimb bones within each embryo analysed (S1K Fig;  55.4% ± 11.2% in tibia versus 32.8% ± 7.1% in humerus; n = 3; p = 0.0267 for paired t test), we first tested whether compensatory proliferation was triggered in the proximal humerus. We found that although there was a trend towards increased proliferation in left p21 − chondrocytes, the difference was not significant (Fig 4D; n = 3), suggesting that compensatory proliferation requires a minimum insult threshold to be triggered. Because intrinsic differences between forelimb and hindlimb bones might exist in regards to the compensatory proliferation response, we also tested the threshold hypothesis using only hindlimb bones. In order to induce p21 expression in fewer chondrocytes than in ePit-Col-p21 mice, we made use of a newly generated Col2a1-tTA line (see Materials and methods) in place of the Col2a1-rtTA transgene, such that p21 expression was achieved from approximately E12.5 onwards in the absence of Dox (Pit-tTA-p21 model) (S4A Fig). tTA expression in this line is less extensive than rtTA in the Col2a1-rtTA line, as p21 misexpression was detected in fewer left tibial chondrocytes than in ePit-Col-p21 left tibia (30%-40% at E15.5, 15%-35% at E17.5, 10%-20% at P0; S4A Fig). Consistent with our prediction of a threshold being needed, compensatory proliferation was not detected in the Pit-tTA-p21 model (S4B and S4C Fig). To further investigate whether a minimum insult threshold is required to trigger increased proliferation, we calculated the correlation coefficient between the percentage of p21 + chondrocytes and the extent of proliferation in the PZ from left and right ePit-Col-p21 (in vivo and ex vivo) and Pit-tTA-p21 tibiae, at E17.5 (or E15.5 plus 2 d ex vivo). Segmental linear regression analysis revealed that the extent of EdU incorporation by p21 − chondrocytes did not correlate with the proportion of p21 + neighbours when this proportion was below 35%, but beyond this threshold, there was a linear correlation between both parameters (Fig 4E; n = 26 bones). These results suggest that compensatory proliferation is due to a signal produced in proportion to the number of arrested chondrocytes, that the signal needs to reach a certain threshold to be effective, and that it remains active until at least P0 despite the dilution of p21 + chondrocytes.

Compensatory proliferation is possibly related to epiphyseal cell density
We next asked whether additional cellular changes occur in the left limbs of ePit-Col-p21 mice that could contribute to the growth compensation and potentially correlate with the number of insulted chondrocytes. Because an alteration in cell density can influence organ size, we tested whether cell density was changed in the PZ of ePit-Col-p21 mice. That was indeed the case, and we found a temporal association between the occurrence of compensatory proliferation in the ePit-Col-p21 model (i.e., at E17.5 and P0 but not E15.5) and statistically significant reduction of cell density in the left PZ as compared to the right ( Fig 4F). Notably, left and right PZ cell densities were not significantly different at any stage in ePit-p21 mice ( Fig 4F, n = 12). Moreover, in line with the threshold hypothesis, we found that, at E17.5, there was a certain value of cell density below which EdU incorporation sharply increased in p21 − chondrocytes (

Mosaic local proliferation blockade in chondrocytes of the left limb results in a systemic growth reduction
Given our initial finding that left and right limb bones exhibit reduced growth in ePit-Col-p21 embryos as compared to ePit-p21 littermates (Fig 2), we next investigated whether there was a systemic response to the p21 insult in the left limbs. Because the growth phenotype is quite mild (an approximately 10% reduction), we first confirmed the finding by measuring microcomputerized tomography (μCT)-generated 3D reconstructions instead of flat micrographs (S5A and S5B Fig; n = 7 for ePit-p21 and n = 13 for ePit-Col-p21 embryos). We found a very good correlation between both types of measurements (S5C Fig, n = 80 bones) and therefore used flat micrographs for all measurements in the study. We first tested whether the growth reduction affected the whole body. We found that, in addition to a decrease in right bone length, body weight of E17.5 and P0 ePit-Col-p21 mice-but not E15.5 or E16.5 embryos-was approximately 10% lower than in ePit-p21 littermates (Fig 5A-5C, S5D Fig). Furthermore, the bone-length and weight effects required Dox treatment and therefore p21 expression (Fig 5A-5C). As control experiments, we confirmed that there was no leakiness of the intersectional  5B and 5C). Our results thus revealed a whole-body response to an insult in a specific tissue in mice, similar to what has been described in Drosophila larvae [2][3][4][5].
In order to characterize the cartilage response, we performed an RNA-seq experiment to identify differentially expressed genes (DEGs) between left and right cartilage (PZ plus prehypertrophic region of proximal and distal tibia and femur) of single ePit-Col-p21 embryos at E17.  Fig 5F). Relaxin1, the closest homologue to dilp8, the recently identified [3,27] alarm gene in fly, was not expressed at significant levels in either limb (S6E Fig), suggesting that the mechanism that links the local insult with a systemic response has diverged during evolution. With regards to the relationship between the extent of insult and the induction of a systemic response, Pit-tTA-p21 mice did not trigger a systemic growth defect at E17.5 or P0 (S4D and S4E Fig, summary in Fig 6A), suggesting that the systemic growth reduction, like compensatory chondrocyte proliferation, is only triggered when a certain insult threshold is surpassed in the targeted cartilage.

The systemic growth reduction of ePit-Col-p21 embryos involves impaired placental function and when it is rescued, limb-body proportions are altered
We reasoned that the most likely foetal organ to control systemic growth by responding to a circulating alarm signal is the placenta because in rodents it produces higher IGF levels than any other organ [28] and is considered the main organ controlling foetal growth [29], whereas hepatic IGFs regulate systemic growth mainly after weaning [18]. Placental weight was not diminished in ePit-Col-p21 embryos (n = 19) as compared to ePit-p21 controls (n = 17), such that the placenta/body weight ratio was increased (Fig 6B). This result suggests that placental efficiency is reduced in response to the left-cartilage p21 insult. To determine the status of placental IGF signalling, we tested the expression of several pathway members by qRT-PCR and found that Igf2 levels were increased in the placentas of ePit-Col-p21 embryos as compared to ePit-p21 controls, whereas the levels of Igf1r did not vary significantly (Fig 6C; n = 3 experimental and n = 4 control). Increased expression of IGF2 by the placenta has been seen as part of a placental stress response triggered by prenatal insults such as alcohol exposure, which is also associated with placental functional impairment, increased placental/body weight ratio, and foetal growth restriction [30,31]. Therefore, one possible interpretation of our results is that the left limb cartilage stress response is relayed to the placenta, which then indirectly p21 and Col2a1-rtTA; Igs7 TRE-tdT/+ embryos (eCol-tdT, expressing tdT in all cartilage elements), compared to their controls (ePit-p21 and ePit-tdT) by unpaired t tests. p-Values corrected for multiple comparisons (Holm-Sidak method) are shown. For panel B, n = 22 ePit-p21, n = 26 ePit-Col-p21, n = 25 ePit-tdT, and n = 5 eCol-tdT). For panel C, n = 4, 11, 24, and 5. (D) Model of the systemic growth response after local chondrocyte arrest triggers a stress signal. (E-F) qRT-PCR (panel E) and in situ hybridisation (panel F) for the indicated transcripts in the proliferative plus prehypertrophic zone from ePit-Col-p21 embryos. Panel E shows one of 2 independent experiments with 3 distinct biological replicates each (total n = 6). The -ΔCt (relative to Gapdh) for each stress-related transcript was compared by a paired t test (left versus right). In panel F, n = 2 E15.5, n = 4 E16.5, and n = 6 E17.5 embryos (arrowheads denote Il6 expression). For panel A-C and E, see also S3 Data. Dox, doxycycline; E, embryonic day; qRT-PCR, quantitative realtime polymerase chain reaction; tdT, tdTomato. impacts on foetal growth. Perhaps explaining why increased IGF2 expression does not correlate with enhanced embryo growth, we found an increase in the level of Igf2r (Fig 6C; n = 6 experimental and n = 6 control), which encodes a decoy receptor that can decrease local IGF2 availability [32]. Furthermore, inhibition of IGF2R has been shown to boost placental efficiency [33].
As a means to test whether the systemic growth reduction in ePit-Col-p21 embryos was due to impaired IGF signalling within the placenta, we injected pregnant dams (from E15.25 to E17.25, 3 times per day, Fig 6D) with an IGF2 analogue (Leu 27 -IGF2) that does not cross the placental barrier, can only bind IGF2R (and not IGF1R), and thus was shown to increase placental efficiency [33]. Body and placental weight and femur length of ePit-Col-p21 embryos were compared between litters that were either treated or not treated with Leu 27 -IGF2 to determine the degree to which body/organ size was rescued, and they were also compared with ePit-p21 embryos within treated litters to determine whether Leu 27 -IGF2 differentially affected experimental and control embryo growth. Boosting placental function led to the following results: 1. Femur length of treated ePit-Col-p21 embryos was significantly increased compared to untreated experimental embryos (n = 9 and n = 11, respectively), and within treated litters, femur length was not significantly different between ePit-Col-p21 and ePit-p21 littermates (n = 6), demonstrating preferential rescue of the mutant embryos (Fig 6E and 6F).
2. Whereas placental weight remained unchanged following Leu 27 -IGF2 administration, body weight of treated ePit-Col-p21 embryos was significantly increased compared to untreated experimental embryos (n = 9 and n = 19), and within-litter comparison revealed that body weight was not only rescued but also slightly increased beyond that of ePit-p21 littermates (n = 6), suggesting that ePit-Col-p21 embryos are especially sensitized to the systemic growth factors produced by the placenta (Fig 6G and 6H). Consequently, the placenta/body ratio was notably reduced in treated versus untreated ePit-Col-p21 embryos, to levels even lower than in treated ePit-p21 littermates (Fig 6I and 6J).
3. Because the body size was "over-rescued" as compared to the bones, the femur/body weight ratio of rescued ePit-Col-p21 embryos was diminished compared to ePit-p21 littermates or untreated litters (Fig 6K, n = 5 untreated and n = 6 treated ePit-p21 embryos; n = 6 untreated and n = 8 treated ePit-Col-p21). Taken together, these results suggest that, in the presence of local growth defects, a systemic growth reduction is necessary to maintain limb-body proportions. Unexpectedly, rescue of the systemic effect did not result in leftright asymmetry in ePit-Col-p21 embryos (Fig 6L), indicating that a specific decrease in growth of the unmanipulated limb, which is independent of placental function, contributes to the maintenance of left-right symmetry upon a unilateral insult.

A holistic view of compensatory responses triggered by developmental insults
In summary, our results show that when the embryonic long bones experience mosaic inhibition of chondrocyte proliferation, an adaptive growth response can be triggered that involves cell-nonautonomous local mechanisms and systemic changes during the time frame of the insult, such that body proportions are maintained. We refer to this new type of catch-up growth that happens during an on-going insult as 'adaptive growth' (S7 Fig). Our finding that a local compensatory response occurs during the insult and involves cell-nonautonomous mechanisms is distinct from previous models that proposed that compensation occurs after the insult is lifted and is cell-autonomous [9,11,20]. Therefore, our results introduce a new conceptual framework for interpreting studies of perturbed long-bone growth. Furthermore, the experimental approach we devised for the study of growth regulation in mice makes a strong case for using unilateral perturbation approaches when studying bilateral organs. Although a local response such as compensatory proliferation or reduced cell density could have been unveiled with a mosaic bilateral injury, a subtle body-weight effect would likely be ascribed to the reduced size of all limbs and not to inter-organ communication. Indeed, the hint that prompted us to explore inter-organ communication was the observed reduction in the unmanipulated limb between experimental and control mice. Below, we discuss the potential mechanisms and evolutionary conservation of local and systemic responses to developmental injury.

Compensatory proliferation
We have shown that a few days into mosaic inhibition of proliferation affecting >35% of chondrocytes of the left limb, spared chondrocytes undergo increased proliferation, such that the overall proliferative rate in the left cartilage almost matches that of the right limb. We propose the following order of events, based on correlative data from our study: 1. In response to a reduction in the number of chondrocytes produced, extracellular matrix production is increased (and thus cell density is reduced). As a consequence, the amount of cartilage scaffold being laid down is not reduced, and thus endochondral ossification can proceed at an almost normal rate. This proposed response fits with stereological studies that showed that, in growth plates with low proliferative rates, it is mainly the production of extracellular matrix that contributes to bone growth [34]. In this regard, the small decrease in chondrocyte density seen in Pit-tTA-p21 mice could explain why these mice do not show increased left-right asymmetry despite lacking compensatory proliferation and systemic growth reduction (S4F and S4G Fig). 2. When the insult is extensive enough for cell density to drop below a certain threshold, compensatory proliferation is triggered in spared chondrocytes, to an extent proportional to the number of affected chondrocytes. Because we found that the extent of compensatory proliferation does not linearly correlate with cell density-but it does with the proportion of p21 + chondrocytes-we posit that density plays a permissive rather than an instructive role and that stress signals emanating from p21 + chondrocytes are needed as well. These stress signals could be diffusible molecules or cell-cell interactions, and they could act either in short range, affecting only cells in their proximity, or in long range, via a self-propagated travelling wave [35] and leading to a whole-organ (i.e., community) effect (S7A and S7B Fig).
Moreover, although our ex vivo experiments suggest that compensatory proliferation is a bone-intrinsic process, we cannot rule out that tissues such as the cartilage-bone interface or the perichondrium (also present in the cultures) play a role in modulating compensatory proliferation. To distinguish between all these possibilities, new insult models that allow triggering of focal insult domains of variable size and positions will be required.
3. Once p21 + chondrocytes have been replaced by p21 − cells and the threshold cell density has recovered, compensatory proliferation stops, such that overgrowth is not generated.

Systemic growth reduction
Our results reveal a mild but consistent systemic growth reduction (approximately 10%) in response to a local insult in the cartilage. We propose that the stress response generated in the left cartilage is somehow communicated to the placenta, which in turn systemically reduces growth (S7 Fig). We suggest the following 2 mechanisms that could account for the injured cartilage-placenta communication: 1. Stress signals are produced locally-either by p21 + chondrocytes, p21 − chondrocytes, the perichondrium, or cells at the cartilage-bone interface-are released into circulation, and, when they surpass a threshold, impact on placental efficiency (S7C Fig). It was recently shown that a subpopulation of natural killer (NK) cells that are transiently abundant in the decidual region of the placenta can promote placental function and foetal growth [36]. If the function of these cells was impaired by the circulating stress signals in ePit-Col-p21 embryos, this could negatively impact on placental function and explain the systemic growth reduction.
2. There is a size-monitoring system that detects impaired long-bone growth and leads to a systemic growth reduction that allows the impaired organ to keep up with the rest of the body. Such a mechanism would likely require a central integrator where size-for-age information is stored and compared to actual organ size, but as of yet, there is almost no evidence for such a system (see discussion in [13]).
With regards to potential growth correction treatments, it would be important to determine whether all ePit-Col-p21 organs are equally reduced or whether the musculoskeletal system (which is especially dependent on IGF signalling) is primarily affected. Resolution of the latter question is currently difficult because the embryos are too small for individual organs to be weighed reliably. Volumetric analyses using mesoscopic techniques such as optical projection tomography [37] on embryos expressing fluorescent reporters in the tissue of interest will be necessary to achieve the necessary level of resolution.

Left-right limb crosstalk
An unexpected result of our study is that when placental function is boosted in ePit-Col-p21 concepti through maternal Leu 27 -IGF2 treatment, long-bone growth is not enhanced to the same extent as body weight, resulting in a reduction in the ratio of bone length to body weight (Fig 6). Given that the right cartilage templates are not experiencing the same p21 insult as the left ones, the dampened response of the right skeletal elements to the systemic rescue suggests that the insult in the left cartilage influences growth of the right limb through some sort of left-right crosstalk. A similar crosstalk has been previously proposed in studies of amphibian limb regeneration, in which it was shown that amputation of the contralateral limb at the same rostrocaudal level as the originally amputated limb reduced the regenerative rate of the latter, whereas ipsilateral or contralateral amputation at a different rostrocaudal level did not [38]. Moreover, a study of tibial fracture repair in young rats showed that the healing environment of a fractured bone triggers the release of growthpromoting signals in the growth plate of the fractured bone and that the same signalling is induced in the contralateral growth plate [39]. As previously proposed [38], the most obvious candidate system to mediate crosstalk between the left and right limbs is the nervous system. While the exact mechanism remains to be determined, a recent study showed that peripheral sympathetic nerves might inhibit bone growth in response to sustained social stress [40]. Regardless of the mechanism, these results suggest that the observed systemic growth reduction in ePit-Col-p21 embryos is a combination of 2 effects: reduced growth efficiency of the contralateral bones in response to the left-specific insult, and impairment of placental function that affects the rest of the body.

Evolutionary conservation of compensatory mechanisms
Collectively, our results reveal that the processes leading to coordination of growth within and between organs to achieve normal proportions upon developmental insults are conserved across metazoans. However, the magnitude of the contributions of local and systemic mechanisms likely varies across phyla because the extent of the systemic growth reduction observed in mice seems to be less extreme than in Drosophila, with the caveat that different insults or tissues could elicit distinct responses. The exact underlying mechanisms also vary because we did not observe up-regulation of the dilp8 homologue Relaxin1 in the insulted cartilage. Different molecular mechanisms aside, the compensatory response in vertebrates shares some features with the response in insects, such as our finding that the injured tissue is able to catch up despite being exposed to an environment that stunts growth of the rest of the body. One explanation for this result is that local compensatory proliferation overrides a systemic effect. We further speculate that if the same 'alarm' signal were to trigger both the intrinsic and systemic mechanisms following injury, this would provide an evolutionarily advantageous strategy to achieve robust coordination of organ growth.
While many unknowns remain in the field of organ growth and repair, further exploration of the mechanisms revealed by this study will open exciting new avenues for basic and translational research and lead to an understanding of human growth disorders.

Ethics statement
All animal studies were performed under an approved Institutional Animal Care and Use Committee mouse protocol (#07-01-001) according to MSKCC institutional guidelines.

Study design
To correct for interlitter variability when studying the effect of p21 misexpression on systemic growth, we normalized each measurement from an experimental animal (ePit-Col-p21 or Pit-tTA-p21) to the average measurement for its control littermates (ePit-p21 or Pit-p21). This is important because the absolute measurements vary significantly between litters, in part because they differ in exact developmental stage, number of embryos, and age of the mother [36]. For paired measurements, the use of left/right ratios allowed for intra-individual normalization. For each experiment, the minimum sample size was estimated using an online tool (http://powerandsamplesize.com/Calculators), based on the average SD observed in pilot experiments, to achieve an effect size of 0.03 in the left/right bone length ratio, 0.5 in the left/ right ratio of EdU incorporation, or 10% in normalized systemic measurements, with a power of 0.8 and a 95% CI. In Fig 5B and 5C, 2 embryos (one from the ePit-Col-p21 and one from the eCol-tdT populations) were abnormally small, possibly dead, and were excluded from the analysis. For comparison of qualitative expression, a minimum of 2 specimens per stage and 5 across several stages were used. The investigator measuring bone length was blinded to the treatment/genotype of the specimens. No blinding was done for other measurements. No randomization was used for animal processing.

Statistics
When data were available for control and experimental, a normalized measurement (left/right ratio or percentage of average control mice) was calculated for both. Between different time points, the normalized measurements were compared by multiple unpaired t test with Holm-Sidak correction for multiple comparisons. Within the same time point, comparisons were done by an unpaired Mann-Whitney test (1 variable and 2 conditions), by 1-way ANOVA (1 variable and !3 conditions), or by 2-way ANOVA (2 variables and 2 or more conditions) following a matched (paired) design when possible (indicated when not). When left and right measurements were compared within experimental animals only, paired 2-tailed t test was used. For all ANOVA, alpha = 0.05. All relevant parameters for the statistical tests can be found in S1 Table. When parametric tests were used, data normality was confirmed by Shapiro-Wilk test and equality of variance by F-test. Prism7 software (Graphpad) was used for most analyses. Most graphs show individual values and mean ± SD, unless otherwise indicated.

Animals
To generate the Igs7 TRE-LtSL-p21 mouse line, the NruI-STOP-loxP-tdTomato-SnaBI fragment in the Ai62(TITL-tdT) Flp-in replacement vector [23] was replaced by a custom NruI-tdTomato-STOP-loxP-MluI-HpaI-SnaBI cassette, to generate an empty DRAGON vector. A PCR-amplified Kozak-Cdkn1a cassette was subsequently cloned into the MluI and SnaBI sites to generate the DRAGON-p21 vector. This vector was then used for recombinase-mediated cassette exchange into Igs7-targeted G4 ES cells [23]. Two successfully targeted clones were injected into C2J blastocysts to generate chimeras, obtaining 27 chimeric males (out of 30 born) with 75% to 100% chimerism. Two males from each clone were crossed to BL6 albino mice (Charles River, Wilmington, MA) to assess germline transmission and to establish the new mouse lines. To generate the Col2a1-tTA line, a Kozak-tTA fragment was PCR-amplified from plasmid pEnt L1L3 tTA-3 (Addgene plasmid #27105, gift of Edward Hsiao) and cloned into a vector containing the regulatory region of mouse Col2a1 obtained from plasmid p3000i3020Col2a1 [41]. Backbone-free vector DNA was injected into FVB zygotes to generate transgenic lines. Four out of 11 founders transmitted the Col2a1-tTA allele. The progeny of one of those (founder number 92) expressed the tTA faithfully in the highest percentage of chondrocytes and was bred with Pitx2-Cre animals to generate breeders for the experiments.

Dox treatment
Dox hyclate (Sigma) was added to the drinking water at a final concentration of 1 mg/ml, with 1% sucrose to increase palatability.

Tibial culture
A previously described protocol [43] was slightly adapted to culture embryonic long bones. Briefly, E15.5 tibiae were obtained from the embryos of Dox-treated pregnant females, dissected free of as much soft tissues as possible in ice-cold PBS, and then cultured (at 37˚C, 5% CO 2 ) in 24-well plates with serum-free DMEM (Gibco) containing 0.2% Bovine Serum Albumin (BSA), 0.5 mM L-glutamine, 40 U/ml penicillin/streptomycin (Gibco), 0.05 mg/ml ascorbic acid (Sigma), and 1 mM betaglycerophosphate (Sigma). The medium additionally contained 1 ng/μl Dox to maintain transgene expression. After 2 d, the bones were incubated with 10 μM EdU for 90 min, then fixed in PFA and processed for histological analysis.
Note that after 2 d, we consistently observed growth of 19% to 23% in control limbs as compared to the original length. This is less than in vivo (approximately 87% growth between E15.5 and E17.5), and the main difference seemed to be at the level of the proximal cartilage, which does not proliferate, likely due to insufficient diffusion of nutrients because it is larger than the distal cartilage. We therefore focused our analysis on the distal cartilage, which at these stages is expected to contribute one-third of total growth [44], i.e., approximately 29%, quite similar to the observed growth.

Leu 27 -IGF2 injections
Human Leu 27 -IGF2 (GroPep, Australia) was prepared at 500 ng/μl in sterile 0.01 N HCl solution and kept at 4˚C in between injections. From E15.25 to E17.25, the pregnant dam was subcutaneously injected every 8 h, for a total dose of 1 μg/g of body weight per day.

Skeletal preparations and measurements
Staining of cartilage and bone was performed as described [45]. Bone length was measured on digital micrographs using the Line tool in Adobe Photoshop. Unless otherwise indicated, only the mineralized region was measured.

Micro-CT scans and measurements
Whole femora and tibiae were scanned using a Scanco μCT 35 (Scanco Medical, Brüttisellen, Switzerland) system. Six-μm voxel size, 45 KVp, 0.36-degree rotation step (180-degree angular range), and a 400-ms exposure per view were used for the scans, which were performed in air. Scanco μCT software (HP, DECwindows Motif 1.6) was used for 3D reconstruction and viewing of images. After 3D reconstruction, 'Distance 3D' tool was used for measuring the length of the ossified region. Three measurements were taken and the average derived for each bone. The observer was blinded to the genotype of the mouse.

Sample processing for histology
Mouse embryos were euthanized by hypothermia in cold PBS. Mouse pups were euthanized by decapitation after hypothermia-induced analgesia. Knees (or isolated full tibiae and femora) were dissected out, skinned, and fixed by immersion in 4% paraformaldehyde (PFA; Electron Microscopy Sciences) in PBS for 2 d at 4˚C. After several washes with PBS, the tissue was then cryoprotected, first by brief incubation with a solution of 15% sucrose and then 30% sucrose in PBS for at least 4 h at 4˚C, and then embedded in Cryomatrix (Thermo) using dry-ice-cold isopentane (Sigma). The knees were oriented sagittally and facing each other, with the tibiae on the bottom of the block (i.e., closest to the blade when sectioning). Serial 7-micron sections were collected with a Leica Cryostat on Superfrost slides, allowed to dry for at least 30 min, and stored at −80˚C until used. For all histological techniques, frozen slides were allowed to reach room temperature in a closed box, and Cryomatrix was washed away for 15 min with warm PBS (37˚C).

Immunohistochemistry and TUNEL
Sections were incubated in citrate buffer (10 mM citric acid, 0.05% Tween 20 [pH 6.0]) for 15 min at 90˚C, allowed to cool down, washed with PBSTx (PBS containing 0.1% Triton X-100), blocked with 5% BSA in PBSTx for 30 min at room temperature, and incubated with the primary antibody over night at 4˚C (see list of antibodies below). After PBSTx washes, incubation with Alexa647-and/or Alexa555-conjugated secondary antibodies (Molecular Probes; 1/500 in PBSTx with DAPI) was performed for 1 h at room temperature. After PBSTx washes, the slides were mounted with Fluoro-Gel (Electron Microscopy Sciences). For TUNEL staining, endogenous biotin was blocked after antigen retrieval using the Avidin/Biotin blocking kit (Vector #SP-2001), and TdT enzyme and Biotin-16-dUTP (Sigma #3333566001 and #11093070910) were subsequently used following manufacturer instructions. Biotin-tagged DNA nicks were revealed with Alexa488-or Alexa647-conjugated streptavidin (Molecular Probes, 1/1000) during the incubation with the secondary antibody.

In situ hybridisation
The protocol described in [46] was followed. For embryos and newborns, samples were not decalcified. Except for Col2a1, Col10a1, and Ihh (provided by Dr. Licia Selleri), the templates for most riboprobes were generated by PCR from embryonic cDNA, using primers containing the SP6 or T7 RNA polymerase promoters. Sequence of the primers is available upon request. After purification of the PCR product (Qiagen PCR purification kit), DIG-labelled probes were transcribed following manufacturer instructions (Roche), treated with DNAase for 30 min, and purified by LiCl-mediated precipitation in alcoholic solvent. Probes were kept at −80˚C in 50% formamide (Fluka). For immunohistochemistry after in situ hybridisation, sections were incubated in citrate buffer (10 mM citric acid, 0.05% Tween 20 [pH 6.0]) for 15 min at 90˚C, allowed to cool down, washed with PBSTx, and incubated with 1% H 2 O 2 in PBSTx for 1 h to block endogenous peroxidases. After BSA blocking and primary antibody incubation, endogenous biotin was blocked using Avidin/Biotin Blocking kit (Vector #SP-2001), and then the slides were incubated with a biotinylated secondary antibody. A brown precipitate was obtained using a peroxidase-coupled streptavidin-biotin complex (Vectastain Elite ABC Kit, Vector #PK-6100) and DAB substrate (Vector #SK-4100), following manufacturer instructions.

Imaging
For in vivo experiments, sagittal sections of the knees were imaged, focusing on the region between both menisci and analysing at least 2, and typically 4, sections per limb. For cultured distal tibiae, frontal sections were used because they allow for better identification of the different epiphyseal regions. The transition between round (resting) and flat (columnar) nuclei, roughly describing an arch between the upper point of the wedges formed by the groove of Ranvier, was chosen as the start of the PZ, while the transition towards bigger, more spaced nuclei (pre-hypertrophic) was chosen as the end of the PZ. The point where pericellular matrix is sharply reduced around enlarging chondrocytes was considered as the start of the HZ, while the end of the HZ was marked as the distal end of the last intact chondrocyte. Bright-field and fluorescence images were taken on a Zeiss inverted microscope (Observer.Z1) using Axiovision software (Zeiss). Mosaic pictures were automatically reconstructed from individual 10× (bright-field) or 20× (fluorescence) tiles.

EdU incorporation
Five mg/ml EdU in PBS was injected (50μg/g body weight, s.c for pups, i.p. for adults and pregnant females) 1.5 h before euthanizing the mice. EdU was detected using the Click-iT Alexa488 Imaging Kit (Invitrogen, C-10337) once the immunohistochemistry and/or TUNEL staining were finished on the same slides. The fraction of nuclei that were positive for EdU, p21, or tdT in the PZ of the cartilage was determined using ImageJ or CellProfiler, followed by manual curation.

Cell density analysis
The PZ was cropped from 20×-imaged sections of left and right experimental and control proximal tibial cartilage, stained for DAPI, p21, and EdU. The area of the region of interest (PZ) was measured in pixels using the Histogram tool in Adobe Photoshop and converted into mm 2 using the resolution and scale information. The DAPI channel was segmented and quantified using Cell Profiler. Cell density was calculated as the number of chondrocytes per area unit.

Cell size analysis
The PZ was cropped from 20×-imaged sections of left and right ePit-Col-p21 proximal tibial cartilage. tdT + chondrocytes were segmented and counted, and their individual area was measured using Cell Profiler.

RNA isolation and analysis
The distal left or right femoral and proximal tibial cartilage from E17.5 Pit-Col-p21 embryos was dissected in cold PBS, the condyles and hypertrophic zones removed using a microknife, and the perichondrium removed by a combination of collagenase type II treatment (Worthington, 2 mg/ml in DMEM, 2 min at room temperature) and mechanical dissection. Left and right cartilage fragments from each embryo (number 1, 2, and 3) were kept in separated tubes and flash-frozen in liquid nitrogen. RNA was extracted using Trizol (Invitrogen) and a mechanical tissue disruptor.

RNA-seq
High-quality RNA was deep sequenced (!50 million paired-end reads) by the New York Genome Center. Aligned reads were analysed using DESeq2 tool in R. A paired design was used, such that left and right comparison was performed for each specimen, which minimized the effect of sequencing batch and interspecimen variability. DEGs were obtained using a threshold of adjusted p-value 0.05. NMF library tools were used to generate heatmaps. Enrichment analysis was performed using DAVID [47] and WebGestalt [48].

qRT-PCR
cDNA was synthesized from purified RNA using iScript reverse transcriptase (RT) as described by the manufacturer (Bio-Rad). Each target was amplified in triplicate to obtain an average per sample, using SYBR Green (Applied Biosystems) on a StepOnePlus real-time PCR system (Applied Biosystems). Primer sequences are shown in Table 1. Negative controls (no template) and no-RT cDNA controls were included for each primer/sample combination. Relative expression on each sample was calculated by the 2 −ΔCT method, with Gapdh (for cartilage) or Tbp (for placenta) as a reference.