Expression of Wnt7b and Smoc2 in correlation with role of growth plate formation
Location
Philadelphia, PA
Start Date
30-4-2025 1:00 PM
End Date
30-4-2025 4:00 PM
Description
Introduction: Growth plate chondrocytes originate from PTHrP-expressing progenitor cells in the reserve zone and then differentiate through the columnar and hypertrophic zone. This process is controlled by signaling between chondrocyte zones and the surrounding perichondrium. Previously, we used RNA sequencing to compare the growth plate forming and non-forming ends of neonatal mouse metatarsals to identify differentially expressed genes (DEGs) specific to growth plate formation. To further identify genes that may be associated with progenitor cells, we cross-referenced our dataset with a study identifying DEGs between PTHrP-expressing and non-expressing growth plate chondrocytes. Two of the most DEGs are Wnt7b and Smoc2. Wnt7b is pivotal in bone development, osteoblast differentiation, and bone healing. Smoc2, a negative regulator of osteogenic differentiation and extracellular matrix (ECM) mineralization, plays a crucial role in bone growth.
Objective: To further understand the roles of Wnt7b and Smoc2 in the growth plate we perform in situ hybridization to identify their spatial expression patterns in neonatal mouse metatarsals.
Methods: Histological specimens consisted of hind limbs collected at P0, P4, and P9 from FVB/NJ mice. Samples were prepared using fixation in an RNase-free preparation of 4% paraformaldehyde, decalcified in an RNase-free preparation of Morse’s solution (22.5% formic acid and 10% sodium citrate) for 24 hours. They were then embedded in paraffin following standard procedures. We performed in situ hybridization (ISH) on paraffin-embedded sections using RNAscope™ 2.5 HD Assay-RED (ACD Bio) for Wnt7b and Smoc2 following RNAscope™ standard protocol, except replacing standard antigen retrieval processes with the use of an RNAscope™ proprietary antigen retrieval enzyme for bone and cartilage tissue.
Results: Wnt7b shows little expression in the chondrocytes within either end of the metatarsal at all three ages. At P0, Wnt7b is strongly expressed in the perichondrium around the growth plate at the distal end, while expression is weaker and more diffuse proximally. At later ages, expression remains strong in the distal perichondrium but becomes progressively weaker proximally until it is lost by P9.
Smoc2 is strongly expressed throughout the distal metatarsals including the epiphyseal, columnar, and hypertrophic zones as well as the perichondrium at all three ages. Proximally, the expression domain is similar but noticeably weaker than in the distal end. At P9, expression is reduced in hypertrophic chondrocytes in the distal epiphysis and largely lost in the proximal end.
Conclusion: We observe a striking difference in the intensity of expression of Wnt7b and Smoc2 between the distal and proximal ends of metatarsal. Their expression in the reserve and columnar zones and the perichondrium supports a role in regulating growth plate function through modulation of reserve chondrocyte progenitor cells.
Embargo Period
5-20-2025
Expression of Wnt7b and Smoc2 in correlation with role of growth plate formation
Philadelphia, PA
Introduction: Growth plate chondrocytes originate from PTHrP-expressing progenitor cells in the reserve zone and then differentiate through the columnar and hypertrophic zone. This process is controlled by signaling between chondrocyte zones and the surrounding perichondrium. Previously, we used RNA sequencing to compare the growth plate forming and non-forming ends of neonatal mouse metatarsals to identify differentially expressed genes (DEGs) specific to growth plate formation. To further identify genes that may be associated with progenitor cells, we cross-referenced our dataset with a study identifying DEGs between PTHrP-expressing and non-expressing growth plate chondrocytes. Two of the most DEGs are Wnt7b and Smoc2. Wnt7b is pivotal in bone development, osteoblast differentiation, and bone healing. Smoc2, a negative regulator of osteogenic differentiation and extracellular matrix (ECM) mineralization, plays a crucial role in bone growth.
Objective: To further understand the roles of Wnt7b and Smoc2 in the growth plate we perform in situ hybridization to identify their spatial expression patterns in neonatal mouse metatarsals.
Methods: Histological specimens consisted of hind limbs collected at P0, P4, and P9 from FVB/NJ mice. Samples were prepared using fixation in an RNase-free preparation of 4% paraformaldehyde, decalcified in an RNase-free preparation of Morse’s solution (22.5% formic acid and 10% sodium citrate) for 24 hours. They were then embedded in paraffin following standard procedures. We performed in situ hybridization (ISH) on paraffin-embedded sections using RNAscope™ 2.5 HD Assay-RED (ACD Bio) for Wnt7b and Smoc2 following RNAscope™ standard protocol, except replacing standard antigen retrieval processes with the use of an RNAscope™ proprietary antigen retrieval enzyme for bone and cartilage tissue.
Results: Wnt7b shows little expression in the chondrocytes within either end of the metatarsal at all three ages. At P0, Wnt7b is strongly expressed in the perichondrium around the growth plate at the distal end, while expression is weaker and more diffuse proximally. At later ages, expression remains strong in the distal perichondrium but becomes progressively weaker proximally until it is lost by P9.
Smoc2 is strongly expressed throughout the distal metatarsals including the epiphyseal, columnar, and hypertrophic zones as well as the perichondrium at all three ages. Proximally, the expression domain is similar but noticeably weaker than in the distal end. At P9, expression is reduced in hypertrophic chondrocytes in the distal epiphysis and largely lost in the proximal end.
Conclusion: We observe a striking difference in the intensity of expression of Wnt7b and Smoc2 between the distal and proximal ends of metatarsal. Their expression in the reserve and columnar zones and the perichondrium supports a role in regulating growth plate function through modulation of reserve chondrocyte progenitor cells.