Comparison of Hoxa11 and Hoxd11 expression in the mouse metatarsal
Location
Philadelphia, PA
Start Date
30-4-2025 1:00 PM
End Date
30-4-2025 4:00 PM
Description
Introduction: Hox genes are a highly conserved group of regulatory genes that code for transcription factors involved in embryological limb development and skeletal patterning. They occur in homologous clusters A, B, C, and D on separate chromosomes. There has been extensive research in hox genes' roles in embryological limb development, however, their function in postnatal bone growth is less well known. In this study, we specifically focus on the postnatal role of the Hoxa11 and Hoxd11 genes on metatarsal bone growth. The metatarsal forms a growth plate on one end and undergoes direct ossification on the other which makes it ideal for observations concerning gene expression specific to bone growth. Target deletions of Hoxd11 and Hoxa11 result in mispatterning of the forelimb and hindlimb, along with disruption of growth plate formation. Comparison of Hoxa11 and Hoxd11 can also determine if there are any differences in expression between homologous genes from different clusters.
Objective: Identify the spatial distribution of Hoxa11 and Hoxd11 expression during metatarsal ossification to determine their respective roles in growth plate function and skeletal growth.
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 Hoxa11 and Hoxd11 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: Both Hoxa11 and Hoxd11 are expressed in the cellular nuclei throughout the metatarsal as all ages. However, there are intriguing differences between the genes. In general, Hoxa11 is expressed diffusely in the epiphysis, with a higher proportion of expression in columnar chondrocytes. In contrast, highest Hoxd11 expression occurs in epiphyseal and reserve zones, with the distal epiphysis tending to show a higher proportion of expression than the proximal end. Both genes are expressed throughout the perichondrium/periosteum. However, Hoxa11 is expressed at a much greater intensity than Hoxd11.
Conclusions: The continued expression of Hoxa11 and Hoxd11 in the postnatal metatarsal indicates that both genes have a continuing role in skeletal growth. In particular, Hoxd11 may have a more primary function in chondrocyte differentiation in the growth plate, given its relative strength and location of expression to both Hoxa11 and the proximal non-growth plate forming end of the metatarsal. The intense expression of Hoxa11 in the perichondrium/periosteum may indicate a role in patterning skeletal growth.
Embargo Period
5-19-2025
Comparison of Hoxa11 and Hoxd11 expression in the mouse metatarsal
Philadelphia, PA
Introduction: Hox genes are a highly conserved group of regulatory genes that code for transcription factors involved in embryological limb development and skeletal patterning. They occur in homologous clusters A, B, C, and D on separate chromosomes. There has been extensive research in hox genes' roles in embryological limb development, however, their function in postnatal bone growth is less well known. In this study, we specifically focus on the postnatal role of the Hoxa11 and Hoxd11 genes on metatarsal bone growth. The metatarsal forms a growth plate on one end and undergoes direct ossification on the other which makes it ideal for observations concerning gene expression specific to bone growth. Target deletions of Hoxd11 and Hoxa11 result in mispatterning of the forelimb and hindlimb, along with disruption of growth plate formation. Comparison of Hoxa11 and Hoxd11 can also determine if there are any differences in expression between homologous genes from different clusters.
Objective: Identify the spatial distribution of Hoxa11 and Hoxd11 expression during metatarsal ossification to determine their respective roles in growth plate function and skeletal growth.
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 Hoxa11 and Hoxd11 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: Both Hoxa11 and Hoxd11 are expressed in the cellular nuclei throughout the metatarsal as all ages. However, there are intriguing differences between the genes. In general, Hoxa11 is expressed diffusely in the epiphysis, with a higher proportion of expression in columnar chondrocytes. In contrast, highest Hoxd11 expression occurs in epiphyseal and reserve zones, with the distal epiphysis tending to show a higher proportion of expression than the proximal end. Both genes are expressed throughout the perichondrium/periosteum. However, Hoxa11 is expressed at a much greater intensity than Hoxd11.
Conclusions: The continued expression of Hoxa11 and Hoxd11 in the postnatal metatarsal indicates that both genes have a continuing role in skeletal growth. In particular, Hoxd11 may have a more primary function in chondrocyte differentiation in the growth plate, given its relative strength and location of expression to both Hoxa11 and the proximal non-growth plate forming end of the metatarsal. The intense expression of Hoxa11 in the perichondrium/periosteum may indicate a role in patterning skeletal growth.