Location
Philadelphia, PA
Start Date
1-5-2024 1:00 PM
End Date
1-5-2024 4:00 PM
Description
INTRODUCTION: The Parathyroid hormone-related protein (PTHrP, the product of the Pth-like hormone, Pthlh, gene)/Indian hedgehog (Ihh) feedback loop is a fundamental regulator of skeletal growth plates. The chondrocytes within the growth plate undergo the sequential resting, proliferative, and hypertrophic stages before being replaced by bone. Just prior to undergoing hypertrophy, pre-hypertrophic chondrocytes express Ihh, which signals back to resting cells to promote the expression of Pthlh. PTHrP then signals proliferating chondrocytes to limit their differentiation to hypertrophy and maintain a pool of mitotic cells in the growth plates of typical long bones. However, the metatarsals (MT) do not grow like typical long bones and instead form a growth plate at only one end. The opposite end undergoes direct ossification, with the primary center of ossification invading the cartilaginous epiphysis. The MT is a useful model for comparing the expression of the genes involved in the PTHrP/Ihh feedback loop in chondrocytes that do and do not form growth plates.
OBJECTIVE: We quantify the expression of Pthlh and Ihh in the two ends of the mouse MT to test the hypothesis that the PTHrP/Ihh feedback loop is specific to growth plate formation.
METHODS: We performed qRT-PCR on neonatal (P0), 4-day (P4), and 9-day-old (P9) mouse distal and proximal MT1 and MT3. Tissues were collected in RNA Protect Tissue Reagent (Qiagen), and RNA was extracted and purified using PureLink RNA Mini Kit with PureLink DNase (Invitrogen). cDNA was synthesized using the ProScript II kit (NEB). qPCR was performed on a StepOnePlus RT-PCR system using the SYBR Green Master reagent (Applied Biosystems). Lung served as a tissue control, and Axin1 was an internal control. Three technical replicates were used for each RT-PCR run. Cellular expression of Pthlh and Pth1r was assessed by in situ hybridization using the RNAscope™ (ACD Bio) standard protocol with a custom bone unmasking reagent.
RESULTS: qPCR shows that Pthlh is consistently expressed at higher levels in the distal growth plate-forming end of the MT3 at all three ages. Results are more varied for Ihh in the MT3 and for both genes in the relatively developmentally delayed MT1. In situ hybridization shows that Pthlh is located in the perichondrium and articular chondrocytes at both ends of the MT3, but a strong band of expression occurs only in reserve chondrocytes in the distal growth plate. Strong Ihh expression is observed in pre-hypertrophic chondrocytes at both ends of the P0 and P4 MT3. At P9, Ihh was expressed in pre-hypertrophic chondrocytes of the distal growth plate and throughout the epiphysis of the proximal end.
CONCLUSION: These data indicate there is a quantifiable difference in Pthlh expression between growth plate forming and non-forming ends of the mouse metatarsal. The reduction of Pthlh expression in the non-growth plate-forming end results from the loss of PTHrP+ cells in the resting zone. This population has been hypothesized to act as a progenitor pool of growth plate chondrocytes, and its loss appears to underlie the differential growth of the mammalian metatarsal.
Embargo Period
7-2-2025
Quantifying Pthlh and Ihh gene expression in the growth plate forming and non-forming ends of the mouse metatarsal
Philadelphia, PA
INTRODUCTION: The Parathyroid hormone-related protein (PTHrP, the product of the Pth-like hormone, Pthlh, gene)/Indian hedgehog (Ihh) feedback loop is a fundamental regulator of skeletal growth plates. The chondrocytes within the growth plate undergo the sequential resting, proliferative, and hypertrophic stages before being replaced by bone. Just prior to undergoing hypertrophy, pre-hypertrophic chondrocytes express Ihh, which signals back to resting cells to promote the expression of Pthlh. PTHrP then signals proliferating chondrocytes to limit their differentiation to hypertrophy and maintain a pool of mitotic cells in the growth plates of typical long bones. However, the metatarsals (MT) do not grow like typical long bones and instead form a growth plate at only one end. The opposite end undergoes direct ossification, with the primary center of ossification invading the cartilaginous epiphysis. The MT is a useful model for comparing the expression of the genes involved in the PTHrP/Ihh feedback loop in chondrocytes that do and do not form growth plates.
OBJECTIVE: We quantify the expression of Pthlh and Ihh in the two ends of the mouse MT to test the hypothesis that the PTHrP/Ihh feedback loop is specific to growth plate formation.
METHODS: We performed qRT-PCR on neonatal (P0), 4-day (P4), and 9-day-old (P9) mouse distal and proximal MT1 and MT3. Tissues were collected in RNA Protect Tissue Reagent (Qiagen), and RNA was extracted and purified using PureLink RNA Mini Kit with PureLink DNase (Invitrogen). cDNA was synthesized using the ProScript II kit (NEB). qPCR was performed on a StepOnePlus RT-PCR system using the SYBR Green Master reagent (Applied Biosystems). Lung served as a tissue control, and Axin1 was an internal control. Three technical replicates were used for each RT-PCR run. Cellular expression of Pthlh and Pth1r was assessed by in situ hybridization using the RNAscope™ (ACD Bio) standard protocol with a custom bone unmasking reagent.
RESULTS: qPCR shows that Pthlh is consistently expressed at higher levels in the distal growth plate-forming end of the MT3 at all three ages. Results are more varied for Ihh in the MT3 and for both genes in the relatively developmentally delayed MT1. In situ hybridization shows that Pthlh is located in the perichondrium and articular chondrocytes at both ends of the MT3, but a strong band of expression occurs only in reserve chondrocytes in the distal growth plate. Strong Ihh expression is observed in pre-hypertrophic chondrocytes at both ends of the P0 and P4 MT3. At P9, Ihh was expressed in pre-hypertrophic chondrocytes of the distal growth plate and throughout the epiphysis of the proximal end.
CONCLUSION: These data indicate there is a quantifiable difference in Pthlh expression between growth plate forming and non-forming ends of the mouse metatarsal. The reduction of Pthlh expression in the non-growth plate-forming end results from the loss of PTHrP+ cells in the resting zone. This population has been hypothesized to act as a progenitor pool of growth plate chondrocytes, and its loss appears to underlie the differential growth of the mammalian metatarsal.