Differential expression of ApoD and Nppc in the mouse metatarsal indicates a role in growth plate formation
Location
Philadelphia, PA
Start Date
1-5-2024 1:00 PM
End Date
1-5-2024 4:00 PM
Description
Introduction: Apolipoprotein D (ApoD) and natriuretic peptide precursor C (NPPC) have numerous physiological and regulatory functions. ApoD lowers the risk of cardiovascular disease by facilitating the transport and metabolism of cholesterol, is an antioxidant with anti-inflammatory roles in areas of tissue damage and injury, and regulates myelination within the nervous system to protect the integrity of nervous signal transduction pathways. Nppc is a precursor molecule for atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). ANP and BNP are regulatory proteins responsible for maintaining euvolemia in the body. Additionally, CNP is released by endothelial cells of the vasculature to promote vasodilation and is released by cardiac myocytes to prevent remodeling in the presence of injury.
While these genes are not normally suspected to be associated with the growth plate, a recent survey of gene expression suggests otherwise. In a survey of comparative gene expression in the growth plate-forming and non-forming end of 4-day-old (P4) and 9-day-old (P9) mouse metatarsals, ApoD and NPPC were significantly differentially expressed in the growth plate.
We investigate the roles of ApoD and NPPC in bone development using in situ hybridization to identify the spatial and cellular expression of these genes in the growth plate of neonatal mouse metatarsals.
Objective: The objective of this research is to investigate the degree of ApoD and NPPC gene expression at the level of the growth plate, and their potential implications in bone metabolism.
Methods: Histological specimens consisted of hind limbs collected at postnatal days 0, 4, and 9 (P0, P4, 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 ApoD and Nppc 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: Our results showed that Nppc expression increased in intensity from P0 to P9 in the perichondrium surrounding the growth plate region of the metatarsals and distal phalanges. P0 and P4 showed diffuse expression in the perichondrium, but P9 had a more intense expression adjacent to the proliferative zone of the growth plate specifically. Similarly, ApoD expression was also observed in the perichondrium surrounding the growth plate regions of the metatarsals and phalanges. In the P9 samples, expression was strong within the proliferative zone of the growth plate itself.
Conclusion: We observe a striking difference in the expression of Nppc and ApoD between the distal and proximal perichondrium of the metatarsal. These results support a role for the genes in the development of growth plate formation and suggest differential regulation of gene expression between the two ends of the metatarsal.
Embargo Period
5-23-2024
Differential expression of ApoD and Nppc in the mouse metatarsal indicates a role in growth plate formation
Philadelphia, PA
Introduction: Apolipoprotein D (ApoD) and natriuretic peptide precursor C (NPPC) have numerous physiological and regulatory functions. ApoD lowers the risk of cardiovascular disease by facilitating the transport and metabolism of cholesterol, is an antioxidant with anti-inflammatory roles in areas of tissue damage and injury, and regulates myelination within the nervous system to protect the integrity of nervous signal transduction pathways. Nppc is a precursor molecule for atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). ANP and BNP are regulatory proteins responsible for maintaining euvolemia in the body. Additionally, CNP is released by endothelial cells of the vasculature to promote vasodilation and is released by cardiac myocytes to prevent remodeling in the presence of injury.
While these genes are not normally suspected to be associated with the growth plate, a recent survey of gene expression suggests otherwise. In a survey of comparative gene expression in the growth plate-forming and non-forming end of 4-day-old (P4) and 9-day-old (P9) mouse metatarsals, ApoD and NPPC were significantly differentially expressed in the growth plate.
We investigate the roles of ApoD and NPPC in bone development using in situ hybridization to identify the spatial and cellular expression of these genes in the growth plate of neonatal mouse metatarsals.
Objective: The objective of this research is to investigate the degree of ApoD and NPPC gene expression at the level of the growth plate, and their potential implications in bone metabolism.
Methods: Histological specimens consisted of hind limbs collected at postnatal days 0, 4, and 9 (P0, P4, 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 ApoD and Nppc 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: Our results showed that Nppc expression increased in intensity from P0 to P9 in the perichondrium surrounding the growth plate region of the metatarsals and distal phalanges. P0 and P4 showed diffuse expression in the perichondrium, but P9 had a more intense expression adjacent to the proliferative zone of the growth plate specifically. Similarly, ApoD expression was also observed in the perichondrium surrounding the growth plate regions of the metatarsals and phalanges. In the P9 samples, expression was strong within the proliferative zone of the growth plate itself.
Conclusion: We observe a striking difference in the expression of Nppc and ApoD between the distal and proximal perichondrium of the metatarsal. These results support a role for the genes in the development of growth plate formation and suggest differential regulation of gene expression between the two ends of the metatarsal.