**Background:** Longitudinal bone growth occurs via endochondral ossification at the growth plate, where chondroprogenitors in the resting zone maintain chondrocyte turnover. Malnutrition is a leading cause of childhood growth retardation, and catch-up growth (accelerated growth after nutritional recovery) is well-described but its cellular mechanism is poorly understood. Wnt/β-catenin signaling, read out by Axin2 expression, marks stem cells in multiple adult tissues. This study used Axin2CreERT2 lineage tracing to investigate how nutrient availability regulates chondroprogenitor dynamics.
**Methods:** Axin2CreERT2 mice were crossed with R26RZsGreen, R26RTdTomato, or R26RConfetti reporters. Tamoxifen (120 μg/g body weight) was injected for 3 consecutive days from P23 (or P0, P42, P84) to label Axin2+ cells. A mouse catch-up growth model was established by 50% dietary restriction (DR) from P27 for 7 days followed by ad libitum refeeding. Fate-mapping quantified ZsGreen+ cells in the top 50 μm of the resting zone and ZsGreen+ columns. Proliferation was assessed by EdU labeling, Ki67 in situ hybridization, and phospho-histone H3 immunostaining. Resting chondrocytes were identified by Clu in situ hybridization. Transcriptome analysis used laser microdissection (LMD) of resting vs. proliferative chondrocytes from P30 growth plates followed by RNA-seq (GSE192840). IGF-1/PI3K signaling was evaluated by serum IGF-1 ELISA, Igf-1 in situ hybridization, and p-Akt immunohistochemistry. Pharmacological interventions included the IGF-1 receptor inhibitor picropodophyllin (PPP, 20 μg/g) and recombinant human IGF-1 (rhIGF-1, 1 μg/g).
**Key Results:** Axin2+ cells appeared in the resting zone only after secondary ossification center formation. At P26, 96.0% ± 0.71% of Axin2+ cells were EdU-negative, 26.5% ± 4.4% expressed Ki67, 75.8% ± 14.2% expressed Cd73, and 33.3% ± 7.34% expressed Pthrp. Lineage tracing showed that labeled cells formed short columns (<10 cells) by P30 and long columns (≥10 cells) by P33, with contribution persisting for at least 6 months. Clonal analysis with Confetti reporter showed 23.3% ± 6.35% of columns were monoclonal. Seven-day DR increased ZsGreen+ cells in the top 50 μm by 30% (P=0.0125) and Clu+ resting chondrocytes by 56% (P=0.0008) while reducing ZsGreen+ columns (P<0.0001) and column density (P=0.0245). DR reduced EdU incorporation in the growth plate to 0.9% ± 0.5% vs. 10.0% ± 2.9% in controls (P<0.0001), but increased Ki67+ (55.8% ± 9.9% vs. 40.8% ± 11.1%, P=0.0045) and p-H3+ (26.8% ± 12.0% vs. 12.6% ± 6.9%, P=0.0035) cells among ZsGreen+ resting chondrocytes. After refeeding, ZsGreen+ columns increased significantly vs. continued DR at P36 (P=0.0052) and P41 (P<0.0001), and by P48 the catch-up group had more columns than ad libitum controls (P=0.0461). The increased ratio of column formation was significantly higher in catch-up vs. control at P41 (P=0.0043) and P48 (P<0.0001). RNA-seq identified 1,442 differentially expressed genes between resting and proliferative chondrocytes (fold change >±2, FDR<0.01). PI3K-Akt signaling was enriched in resting chondrocytes (KEGG:04151). p-Akt localized specifically to the resting and hypertrophic zones. Igf-1 expression was 4-fold higher in resting vs. proliferative chondrocytes by CPM (P=0.0041). PPP reduced p-Akt+ cells in the resting zone by ~50% (P=0.0435). DR reduced serum IGF-1 (P<0.0001), local Igf-1 expression (P<0.0001), and p-Akt+ cells among ZsGreen+ resting chondrocytes (P<0.0001); all recovered with refeeding. One-day fasting reduced serum IGF-1 (P<0.0001) and Igf-1 expression (P=0.0008), and rhIGF-1 restored p-Akt levels (P=0.0008). In DR mice, 7-day rhIGF-1 treatment increased ZsGreen+ columns (P<0.0001), reduced ZsGreen+ cells in the top 50 μm by 17% (P=0.0002), reduced Clu+ cells by 26% (P=0.0002), and increased column density (P=0.0084), without affecting Ki67 expression in resting chondrocytes (P=0.6669).
**Clinical Implications:** This study reveals a novel cellular mechanism for catch-up growth: nutrient deprivation causes chondroprogenitors to accumulate in the resting zone by self-replication while blocking their differentiation, creating a pool of cells ready to rapidly form columns upon nutritional recovery. IGF-1 signaling is a key mediator of this differentiation block, and exogenous IGF-1 can partially reverse it. These findings advance understanding of bone growth regulation and may inform noninvasive therapies for human growth disorders, particularly those related to malnutrition. The Axin2CreERT2 model provides a tool to study chondroprogenitor dynamics under various growth-inhibiting conditions.