 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 3% (left/right bone length ratio), or 25% (rest of parameters), with a power of 0.8 and a 95% confidence interval. For comparison of qualitative expression, a minimum of two specimens per stage and five 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. Most analyses were done with Prism 7.0 software.
A previously described protocol (Agoston et al., 2007) was slightly adapted. Briefly, tibiae were obtained from P1 pups, dissected free of soft tissues and allowed to recover from dissection for 6 hr 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). Different quantities of DT in PBS were then added, followed by 24 hr incubation. Tibiae were then fixed in PFA and processed for histological analysis.
We thank the Joyner lab for scientific discussions. We are grateful to Juanma González-Rosa for comments on the manuscript and suggesting the use of Pitx2-Cre (kind gift of Hiroshi Hamada), and Chitra Dahia for insightful discussions, critical reading of the manuscript, providing access to the µCT scanner at the Hospital for Special Surgery and for interpretation of the data. This work was supported by grant R21HD083860 (NIH-NICHD) to ALJ, a National Cancer Institute Cancer Center Support Grant [P30 CA008748] to MSKCC, and by postdoctoral fellowships from HFSP and the Revson Foundation to ARD.
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Thank you for submitting your article "Altered paracrine signaling from the injured knee joint impairs postnatal long bone growth" for consideration by eLife. Your article has been reviewed by two peer reviewers, one of whom, Clifford J Rosen (Reviewer#1) is a member of our Board of Reviewing Editors, and Didier Stainier as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Colin Farquharson (Reviewer #2).
In this manuscript, the authors test the hypothesis that local factors outside the growth plate provide signals to long bones to enhance growth during the critical early phases of postnatal development, particularly when growth hormone is not fully activated. In particular, the authors focus on the paracrine activity of the infrapatellar fat pad as a source of IGF-I and activation of the injury response. They propose that Injury and/or inflammation can impact longitudinal growth via disturbed activation of this tissue and suggest that paracrine regulation of the growth plate is essential for normal early neonatal growth. Indeed, understanding the role of these paracrine factors may contribute to our knowledge base about longitudinal growth and pathologic states of impaired growth due to injury.
1) The authors have not excluded changes in systemic GH/IGF-I axis via the pituitary (particularly since they showed systemic rescue by treating with IGF-I), nor dealt with the possibility in early postnatal development that IGF-II may be altered by inflammation and injury. Delineating changes in these other growth factors is essential for the revision. Furthermore, the discussion of early linear growth is slanted too much toward paracrine factors despite convincing mouse data to the contrary from Arg Efstratiadis and others. Those studies need to be included (see 5 below).
2) Their suggestion that the perichondrium is not the source of altered signaling is not fully supported by the figures and needs clearer delineation and/or further experiments to exclude this source of IGF-I.
3) Further studies examining SOCS3 expression and its role in down regulating IGF-I are necessary as an alternative mechanism.
4) The rescue experiments with ruxo and IGF-I showed lack of a response in the tibia; this needs to be redone or clarified to the extent that it is more demonstrable; we prefer to see a larger N for these experiments.
5) The implications for normal neonatal growth, particularly between days one – twelve is not clear in the discussion and would benefit from further elaboration.
We are glad that the reviewers liked our “provocative model” and we thank them for all their suggestions, as we think the revisions have improved the quality of the manuscript and made the results more compelling.
This is indeed a very important point that was not completely clear in the original manuscript. Since we are performing a unilateral modification, with the right limb as internal control, any left-specific defect cannot be due to changes in a systemic factor(s) (be it GH or IGF1), although it could be due to local changes in the signal transduction machinery that responds to those systemic factors. Importantly, Efstratiadis and others have shown that mutant mice for growth hormone receptor (Ghr) do not show a body weight or a limb length phenotype until P10-P15 (Zhou et al., PNAS 1997, Lupu et al., Dev Biol 2001), while we see a significant effect as early as P4. Therefore, we decided to focus on local IGF signaling. We did not detect major changes of expression for Igf2 or Igf1r (i.e. the other IGF ligand and their common receptor), except in the damaged cells of the prospective articular cartilage, where the Igf2 expression domain is somewhat interrupted (new Figure 4—figure supplement 1) To test the possibility that downstream signaling components were affected, we determined SOCS3 expression as suggested by the reviewer, as it could conceivably be activated by inflammatory cytokines and exert a negative influence on IGF signaling (Ahmed and Farquharson, J. Endocrinology 2010). However, we could not detect differential expression between the left and right GPs in P1-PitDTR animals at P2 or P3 (new Figure 4—figure supplement 1), suggesting that it is the reduced availability of IGF1 from the fat pad that plays a major role in the phenotype. It is also noteworthy that when the same intra-articular (i.e. local) IGF1 injection regimen that we performed in the rescue experiment is reproduced in PitDTR animals not given DT, no overgrowth phenotype is observed (Author response image 1). Actually the injected limb is slightly shorter, perhaps due to the repeated injections.
Left and right bone length in P5 PitDTR animals that were not injected with DT, but that were injected with IGF1 in the left knee from P1 to P4, twice daily. p-values for multiple comparisons test after two-way ANOVA are shown.
We have also expanded the Introduction/Discussion to further clarify the role of IGF signaling in early postnatal development. We explain that deletion of Igf1 in the liver (which diminishes circulating levels by >75%) does not affect limb growth (Yakar et al.,and Sjögren et al., PNAS 1999), whereas chronic overexpression of Igf1 in liver, brain and other organs results in overgrowth of only a subset of organs “without an apparent increase in skeletal growth” (Mathews et al., Endocrinology 1988). Furthermore, one of the most comprehensive studies relating to the trophic role of GH/IGF, by the Efstratiadis lab (Stratikopoulos et al., PNAS 2008), reached the conclusion that “growth hormone-induced, liver-specific IGF1 apparently begins exerting postnatal hormonal action shortly after weaning”. Finally, the fact that deletion of Igf1 in the whole limb mesenchyme greatly diminishes chondrocyte hypertrophy by P7 (