ORF47-01 - Spatial Transcriptomic Analysis of the Effects of Retinoic Acid Excess on Epiphyseal Growth Plate

Introduction: High-dose retinoids are used to treat childhood neuroblastoma. However, this treatment has adverse effects on growth plate cartilage, resulting in an advanced bone age and severe short stature.

Objective: This study investigates the molecular mechanisms by which high-dose retinoic acid (RA) affects growth plate chondrocytes in postnatal mice.

Methods: C57BL/6 mice received a single dose of DMSO (vehicle) or all-trans RA at 20 mg/kg body on postnatal day 11. After 24 hours, proximal tibial growth plates were harvested for histology and molecular analysis using VisiumHD spatial transcriptomics profiling.

Results: 24 hours after RA treatment, histological analysis revealed shorter proliferative zone (p=0.03) length in RA treated mice than the controls group. Spatial transcriptomic analysis profiling revealed that RA altered gene expression in all three zones of the growth plate: 75 upregulated and 123 downregulated genes in the resting zone, 57 upregulated and 89 downregulated genes in the proliferative zone, and 116 upregulated and 44 downregulated genes in the hypertrophic zone. Ingenuity pathway analysis (IPA) and Enricher pathway analysis identified multiple affected pathways which are important for bone development and endochondral ossification in all three zones. Interestingly, in the resting zone, RA treatment induced downregulation of growth hormone receptor (Ghr), which is known to crosstalk with RAR/RXR receptor signaling. In the proliferative zone, we found a dramatic downregulation of Tcf7, which is important for activation of WNT signaling and chondrocyte proliferation. In the hypertrophic zone, we found that RA induced upregulation of Crebbp, as well as Runx1 and Runx3, suggesting accelerated hypertrophic differentiation.

Conclusions: Our findings suggest that high-dose retinoic acid treatment has widespread effects on gene expression in all three zones of the growth plate, potentially affecting stem cell maintenance, chondrocyte proliferation, and hypertrophic differentiation. Further analysis of these data has the potential to provide insights into the mechanisms by which high-dose retinoids impair linear growth in children. Similar spatial transcriptomic approach using Visium HD will likely be useful for study of other disease models in the growth plate to provide mechanistic insights that were not possible in the past.