Strong differential gene expression of Sall1 and Pax9 between the proximal and distal ends of the developing mouse metatarsal.
Location
Philadelphia, PA
Start Date
1-5-2024 1:00 PM
End Date
1-5-2024 4:00 PM
Description
Introduction:
Spalt-like transcription factor 1 (Sall1) and Paired box gene 9 (Pax9) both play important roles in limb development. Sall1 is expressed in distal limb mesenchyme, and it plays a role in anterior-posterior limb axis patterning and hand and thumb development. Pax9 plays a critical role in the formation of the axial skeleton, sclerotome formation, and chondrocyte differentiation in early development. In terms of distal limb development, the loss of Pax9 has been shown to result in polydactyly.
The cartilaginous growth plates are crucial for long bone growth and Pax9 has been shown to play a role in chondrocyte differentiation. Most long bones form growth plates at both ends, yet in the metacarpals and metatarsals, [delete comma] the growth plate forms at only one end. The opposite end undergoes direct ossification invading the cartilaginous epiphysis and replacing it with bone. In a survey of relative gene expression between the growth plate forming and non-forming ends of the mouse metatarsal, Sall1 and Pax9 had increased expression in the non-forming end of the bone. Therefore, we used in situ hybridization to determine the spatial and cellular expression of these genes in neonatal metatarsals to better understand the role of these genes in skeletal development.
Objective: The primary objective of this study is to investigate the level of gene expression of Pax9 and Sall1 within growth plates and their role in limb development.
Methods: Histological specimens consisted of mouse hind limbs collected at postnatal days 0, 4, and 9 (P0, P4, P9). 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 Sall1 and Pax9 following RNAscope™ standard protocol, except [for] replacing standard antigen retrieval processes with the use of an RNAscope™ proprietary antigen retrieval enzyme for bone and cartilage tissue.
Results: Both Sall1 and Pax9 have generally similar patterns of expression. At both, P0 and P4, these genes are observed throughout in the perichondrium and the columnar and epiphyseal chondrocytes on the proximal end of the metatarsal. Distally, expression is much reduced with only sporadic expression in the perichondrium and epiphyseal and columnar chondrocytes. Interestingly, Sall1 and Pax9 are expressed in the chondrocytes within the tarsals and in tissues surrounding the distal phalanges. At P9, Sall1 and Pax9 are expressed strongest in the proximal periosteum/perichondrium and in the adjoining ligamentous structures of the proximal metatarsals and tarsals.
Conclusion/Discussion: This analysis confirms that Sall1 and Pax9 are more heavily expressed in the proximal, non-growth plate forming regions of metatarsals. Their expression in the chondrocytes of the proximal epiphysis may indicate a role in repressing growth plate formation. The expression in perichondrium and surrounding connective tissues suggest a role in the development of the ligaments that stabilize the tarsal-metatarsal region.
Embargo Period
5-23-2024
Strong differential gene expression of Sall1 and Pax9 between the proximal and distal ends of the developing mouse metatarsal.
Philadelphia, PA
Introduction:
Spalt-like transcription factor 1 (Sall1) and Paired box gene 9 (Pax9) both play important roles in limb development. Sall1 is expressed in distal limb mesenchyme, and it plays a role in anterior-posterior limb axis patterning and hand and thumb development. Pax9 plays a critical role in the formation of the axial skeleton, sclerotome formation, and chondrocyte differentiation in early development. In terms of distal limb development, the loss of Pax9 has been shown to result in polydactyly.
The cartilaginous growth plates are crucial for long bone growth and Pax9 has been shown to play a role in chondrocyte differentiation. Most long bones form growth plates at both ends, yet in the metacarpals and metatarsals, [delete comma] the growth plate forms at only one end. The opposite end undergoes direct ossification invading the cartilaginous epiphysis and replacing it with bone. In a survey of relative gene expression between the growth plate forming and non-forming ends of the mouse metatarsal, Sall1 and Pax9 had increased expression in the non-forming end of the bone. Therefore, we used in situ hybridization to determine the spatial and cellular expression of these genes in neonatal metatarsals to better understand the role of these genes in skeletal development.
Objective: The primary objective of this study is to investigate the level of gene expression of Pax9 and Sall1 within growth plates and their role in limb development.
Methods: Histological specimens consisted of mouse hind limbs collected at postnatal days 0, 4, and 9 (P0, P4, P9). 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 Sall1 and Pax9 following RNAscope™ standard protocol, except [for] replacing standard antigen retrieval processes with the use of an RNAscope™ proprietary antigen retrieval enzyme for bone and cartilage tissue.
Results: Both Sall1 and Pax9 have generally similar patterns of expression. At both, P0 and P4, these genes are observed throughout in the perichondrium and the columnar and epiphyseal chondrocytes on the proximal end of the metatarsal. Distally, expression is much reduced with only sporadic expression in the perichondrium and epiphyseal and columnar chondrocytes. Interestingly, Sall1 and Pax9 are expressed in the chondrocytes within the tarsals and in tissues surrounding the distal phalanges. At P9, Sall1 and Pax9 are expressed strongest in the proximal periosteum/perichondrium and in the adjoining ligamentous structures of the proximal metatarsals and tarsals.
Conclusion/Discussion: This analysis confirms that Sall1 and Pax9 are more heavily expressed in the proximal, non-growth plate forming regions of metatarsals. Their expression in the chondrocytes of the proximal epiphysis may indicate a role in repressing growth plate formation. The expression in perichondrium and surrounding connective tissues suggest a role in the development of the ligaments that stabilize the tarsal-metatarsal region.