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Nutrient-regulated dynamics of chondroprogenitors in the postnatal murine growth plate

Takeshi Oichi, Joe Kodama, Kimberly Wilson, Hongying Tian, Yuka Imamura, Yu Usami, Yasushi Oshima, Taku Saito, Sakae Tanaka, Masahiro Iwamoto, Satoru Otsuru, Motomi Iwamoto-Enomoto

Posted on: 23 February 2023 , updated on: 27 February 2023

Preprint posted on 21 January 2023

Alumni

Fasted bones grow fast later: chondroprogenitors in the growth plate of murine long bones adapt to dietary restriction, leading to catch-up growth during refeeding.

Selected by Alberto Rosello-Diez, Boya (Hannah) Zhang, Chee Ho H'ng

Categories: developmental biology

Background

One of the most fascinating characteristics of growing animals is that, after a transient developmental perturbation, they show a tendency to regain a normal growth trajectory. This phenomenon is referred to as ‘catch-up growth’, defined by Prader and Tanner (1) as an example of canalization (robustness in the phenotypic traits in response to any perturbations), a concept coined by Waddington (2) and independently proposed by Schmalhausen (3). While some systemic (neuroendocrine) mechanisms were initially proposed to explain catch-up growth (4), the observation that it can independently affect specific organs or tissues shifted the focus towards local mechanisms (5).

 

The long bones within the limbs have been well studied in this regard. Their growth takes place via endochondral ossification, whereby a transient cartilage template is first formed and progressively replaced by bone (6). The primary ossification center in the shaft of the bone soon divides the initial cartilage into two growing units at both bone ends, called growth plates (Figure 1). Cells in the cartilage (chondrocytes) transition, from the ends towards the center, through subsequent differentiation states: chondroprogenitors in the resting zone are progressively recruited into the proliferative pool of flat chondrocytes, arrayed in longitudinal columns, which subsequently enlarge and differentiate towards hypertrophic chondrocytes (Figure 1). These cells either die or transdifferentiate to bone-laying cells (osteoblasts), so that the cartilage matrix is replaced by bone, adding length to both ends of the bone shaft.

 

In order to sustain growth over an extended period of time, this system must find a delicate balance between production of cartilage on one end of the growth plate and destruction on the other. Therefore, chondroprogenitors have been a subject of active research for the last 30 years or so, with a focus on their ability to self-renew and the limit (or lack thereof) of their proliferative potential. On the one hand, a school of thought suggests that chondroprogenitors have a limited proliferative potential that gets progressively exhausted with each cell division (5). In this scenario, catch-up growth is a cell-autonomous process: transient cell-cycle arrest leads to preservation of the proliferative potential in the arrested cells, so that these grow at a faster-than-normal-for-age rate once the insult is lifted. On the other hand, this model does not seem to be complete or even correct, as we have shown that mosaic (i.e. salt-an-pepper) cell-cycle arrest in the growth plate is compensated for by hyperproliferation of spared chondrocytes, which is a cell-nonautonomous response (7). Moreover, recent studies have shown that there is a change in the behavior of chondroprogenitors across development: from not self-renewing during fetal-neonatal stages, so that their pool is depleted as bones grow, to self-renewing and becoming long-lived progenitors or stem cells at juvenile stages, likely due to the influence of extrinsic signals, such as formation of the secondary ossification center (reviewed in (8)).

 

Oichi and colleagues shed light onto this process, by characterizing the fate, stem cell markers and growth potential of chondroprogenitors, as well as bone length and body weight, in mice exposed to three distinct regimes: ad libitum feeding (Normal), dietary restriction (DR), and dietary restriction followed by Refeeding (which causes catch-up growth). The preprint is very revealing, while opening new avenues for future research.

Figure 1: Process of endochondral ossification.

Key findings

Oichi et al. used lineage tracing of Axin2-CreER+ (Axin2+ hereafter) cells to trace the stem cells in the cartilage. They showed that the DR regime was able to stunt bone growth, as expected. Surprisingly, however, this was in part mediated by reduced recruitment (i.e. differentiation) of Axin2+ cells to the proliferative pool in the cartilage, and reciprocal increase of the resting pool (i.e. self-renewal, see Figure 2-middle).

  1. Following refeeding after nutrient restriction, the model demonstrated catch-up growth as evident by the increased growth rate as compared to age- and sex-matched Normal animals, leading to recovery of normal bone length a few weeks later. This correlated with increased differentiation of the Axin2+ cells towards the proliferative pool (Figure 2-right).
  2. Region-specific RNA-seq data and subsequent analysis revealed that IGF-1/PI3K signaling was differentially activated in resting vs. proliferative chondrocytes. Interestingly, IGF-1 signaling was downregulated during the DR regimen, correlating with increased self-renewal capacity and suppressed cell differentiation of the resting cells (Figure 2-middle). Of note, exogenous application of recombinant human (rh) IGF-1 was able to partially reverse this cell behavior.
  3. Conversely, refeeding after DR rescued IGF-1/PI3K signaling in resting chondrocytes, allowing the enhanced recruitment of the previously increased pool of resting cells to the proliferative pool, thus accelerating bone growth.

Figure 2: Summary of findings. Oichi et al. bioRxiv 2023.

What we like about this preprint

This study clearly shows that catch-up growth due to transient nutrient restriction is a two-step process: during DR, the Axin2+ resting cells “save energy” by reducing recruitment into the proliferative pool, preserving their proliferative potential. When the conditions are more favorable, this expanded pool of progenitors with high proliferative potential is the one driving enhanced growth as compared to Normal animals (which have fewer Axin2+ cells by that stage).

Moreover, one of the predictions of the cell-autonomous model of catch-up growth is that, when a bone is smaller than it should, given its chronological age, it will catch-up at a rate similar to that of a younger bone of similar size (which would be its ‘bone age’). Oichi and colleagues provide the exact data needed to test this prediction. In Figure S7, they show that tibial length at P41 in the Refeeding group is similar to tibial length in the P34 Normal group (this is the case for both males and females). A similar growth rate would then be expected for both groups. But this is not always the case. In males, P34 Normal growth rate is ~140 µm/day, but P41 Refeeding growth rate is ~100 µm/day, and the low variability suggests that this difference is significant. In females, however, P34 Normal growth rate is ~110 µm/day, very similar to P41 Refeeding growth rate, as predicted by the cell-autonomous model. The cause of this ‘sexual dimorphism’ remains to be determined, but it suggests that other factors, such as metabolism, hormones or body weight, can modulate the catch-up process.

 

Pending questions

– What about other chondroprogenitors, besides Axin2+ cells? Do they behave similarly?

– Newton et al. (9) showed that mTORC1 activity is required for the stem-cell behavior of self-renewing chondroprogenitors. However, Oichi and colleagues show that replenishment of the reserve pool is promoted by reduced IGF1 signaling, which is typically upstream of mTORC1. It seems that new experiments are required to reconcile these two observations.

– Fig 1e: Total Axin2+ cells were quantified apparently without normalization. What is the proportion of total Axin2+ cells referred to the total number of resting chondrocytes? In other words, is there a change in cell density?

– What happens if prolonged rhIGF-1 treatment is given? Do you expect to see overgrowth (increased differentiation, number and density of column) in the DR + rhIGF-1 chondrocytes?

 

Related research

  1. A. Prader, J. M. Tanner, G. von Harnack, Catch-up growth following illness or starvation. An example of developmental canalization in man. J Pediatr 62, 646-659 (1963).
  2. C. H. Waddington, The strategy of the genes; a discussion of some aspects of theoretical biology. (Allen & Unwin, London,, 1957), pp. ix, 262 p.
  3. I. I. Schmalhausen, Factors of evolution: the theory of stabilizing selection. Factors of evolution: the theory of stabilizing selection. (Blakiston, Oxford, England, 1949), pp. xiv, 327-xiv, 327.
  4. J. M. Tanner, Regulation of Growth in Size in Mammals. Nature 199, 845-850 (1963).
  5. J. Baron et al., Catch-up growth after glucocorticoid excess: a mechanism intrinsic to the growth plate. Endocrinology 135, 1367-1371 (1994).
  6. H. M. Kronenberg, Developmental regulation of the growth plate. Nature 423, 332-336 (2003).
  7. A. Rosello-Diez, L. Madisen, S. Bastide, H. Zeng, A. L. Joyner, Cell-nonautonomous local and systemic responses to cell arrest enable long-bone catch-up growth in developing mice. PLoS Biol 16, e2005086 (2018).
  8. J. C. Lui, Home for a rest: stem cell niche of the postnatal growth plate. J Endocrinol 246, R1-R11 (2020).
  9. P. T. Newton et al., A radical switch in clonality reveals a stem cell niche in the epiphyseal growth plate. Nature 567, 234-238 (2019).

Tags: catch-up growth, growth plate, igf1, mouse

doi: https://doi.org/10.1242/prelights.33860

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Author's response

The author team shared

– What about other chondroprogenitors, besides Axin2+ cells? Do they behave similarly?

Our preliminary single-cell analysis showed that resting chondrocytes consisted of a heterogeneous cell population, with Axin2+ cells overlapping somewhat with PTHrP+ cells. Although we don’t have data to answer this question at present, whether there are differences in the response of these cell populations to external nutrition and what cell populations become active after nutritional resumption is a subject of great interest.

 

– Newton et al. (9) showed that mTORC1 activity is required for the stem-cell behavior of self-renewing chondroprogenitors. However, Oichi and colleagues show that replenishment of the reserve pool is promoted by reduced IGF1 signaling, which is typically upstream of mTORC1. It seems that new experiments are required to reconcile these two observations.

We agree that there is a discrepancy between the results of the Newton et al. paper and the results of our study. We assume that this discrepancy is due to the differences in the mice used and the experimental conditions. To understand the cell-autonomous function of IGF-1 and mTORC1 signaling in chondroprogenitors, we need markers that are specifically expressed in chondroprogenitors of the cartilage growth plate and not expressed in other organs.

 

– Fig 1e: Total Axin2+ cells were quantified apparently without normalization. What is the proportion of total Axin2+ cells referred to the total number of resting chondrocytes? In other words, is there a change in cell density?

Although not quantified and only an impression, the percentage of Axin2-positive cells in the quiescent layer chondrocytes increased after the formation of secondary ossification centers and then decreased.

 

– What happens if prolonged rhIGF-1 treatment is given? Do you expect to see overgrowth (increased differentiation, number and density of column) in the DR + rhIGF-1 chondrocytes?

We do not think that longer administration of rhIGF-1 would improve bone growth phenotype because nutrient deprivation affects/impairs many signaling pathways and factors other than IGF-1 signaling. The importance of IGF-1 signaling in regulation of the growth plate function has been addressed. Our study revealed IGF-1 signaling has an important role in regulating differentiation from resting chondrocytes to proliferative chondrocytes

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