PTHrP & Ihh expression in Hoxd11 knockout metatarsal growth plate formation
Location
Philadelphia, PA
Start Date
17-4-2026 1:30 PM
End Date
17-4-2026 2:30 PM
Description
NTRODUCTION: Hox genes play a fundamental role in patterning the growth and structure of the limb skeleton. Hoxd11 is expressed in two phases, an early phase corresponding to the limb and proximal carpals and distal phase corresponding to digits 2-5. Hoxd11 is known to affect skeletal growth. Loss of function of Hoxa11 and Hoxd11 results in a shortened tibia and fibula and minute radius and ulna that lack growth plates. Increased dosage of Hoxd11 results in longer metacarpals. Metacarpals and metatarsals (MT) are unusual in that they form only a single growth plate. Previously, we used the MT as a natural model to understand gene expression specific to growth plate formation. PTHrP (the product of the Pthlh gene)/IHH feedback loop is a key regulator of growth plates. Reserve chondrocytes express Pthlh which limits differentiation of proliferative chondrocytes in the columnar zone. Just prior to undergoing hypertrophy, pre-hypertrophic chondrocytes express Ihh which signals back to resting cells to promote the expression of Pthlh. We have observed that Pthlh expression in the reserve zone is absent prior to the loss of one of the MT growth plates.
OBJECTIVE: We aim to investigate the relationship between Hoxd11 loss-of-function and Pthlh and Ihh expression in MTs. This will form a baseline by which we can compare to future studies of Hox loss-of-function and Pthlh and Ihh expression throughout the limb skeleton.
METHODS: Histological specimens consisted of hind limbs collected at P0 from Svlmj/B6 mice from Hoxd11+/- breeding pairs. Samples were fixed in an RNase-free preparation of 4% paraformaldehyde, decalcified in RNase-free Morse’s solution (22.5% formic acid and 10% sodium citrate) for 24 hours, and embedded in paraffin following standard procedures. Tailsnips were collected for genotyping. In situ hybridization (ISH) was performed on paraffin-embedded sections using RNAscope™ 2.5 HD Assay-RED (ACD Bio) probes for Pthlh and Ihh, following the standard RNAscope™ protocol with modification using a proprietary antigen retrieval enzyme optimized for bone and cartilage tissue.
RESULTS: Our results indicate that homozygous Hoxd11 loss-of-function mouse limbs may display a slightly stronger expression of Ihh in the pre-hypertrophic zone of metatarsal growth plates compared to wild type specimens. We observe no significant difference in expression of Pthlh in the perichondrium and periarticular chondrocytes of homozygous Hoxd11 loss-of-function specimens compared to wild type.
CONCLUSION: Pthlh and Ihh expression in loss-of-function and wild type Hoxd11 mice indicate similar expression patterns within the growth plate regions at the developmental stage examined. Loss of Hoxd11 function does not appear to alter the expression of Pthlh in reserve chondrocyte. Future work will be done in the forelimb to compare to bones such as the metacarpals and pisiform which are likely more susceptible to reductions in Hoxd11 loss-of-function.
Embargo Period
5-20-2026
PTHrP & Ihh expression in Hoxd11 knockout metatarsal growth plate formation
Philadelphia, PA
NTRODUCTION: Hox genes play a fundamental role in patterning the growth and structure of the limb skeleton. Hoxd11 is expressed in two phases, an early phase corresponding to the limb and proximal carpals and distal phase corresponding to digits 2-5. Hoxd11 is known to affect skeletal growth. Loss of function of Hoxa11 and Hoxd11 results in a shortened tibia and fibula and minute radius and ulna that lack growth plates. Increased dosage of Hoxd11 results in longer metacarpals. Metacarpals and metatarsals (MT) are unusual in that they form only a single growth plate. Previously, we used the MT as a natural model to understand gene expression specific to growth plate formation. PTHrP (the product of the Pthlh gene)/IHH feedback loop is a key regulator of growth plates. Reserve chondrocytes express Pthlh which limits differentiation of proliferative chondrocytes in the columnar zone. Just prior to undergoing hypertrophy, pre-hypertrophic chondrocytes express Ihh which signals back to resting cells to promote the expression of Pthlh. We have observed that Pthlh expression in the reserve zone is absent prior to the loss of one of the MT growth plates.
OBJECTIVE: We aim to investigate the relationship between Hoxd11 loss-of-function and Pthlh and Ihh expression in MTs. This will form a baseline by which we can compare to future studies of Hox loss-of-function and Pthlh and Ihh expression throughout the limb skeleton.
METHODS: Histological specimens consisted of hind limbs collected at P0 from Svlmj/B6 mice from Hoxd11+/- breeding pairs. Samples were fixed in an RNase-free preparation of 4% paraformaldehyde, decalcified in RNase-free Morse’s solution (22.5% formic acid and 10% sodium citrate) for 24 hours, and embedded in paraffin following standard procedures. Tailsnips were collected for genotyping. In situ hybridization (ISH) was performed on paraffin-embedded sections using RNAscope™ 2.5 HD Assay-RED (ACD Bio) probes for Pthlh and Ihh, following the standard RNAscope™ protocol with modification using a proprietary antigen retrieval enzyme optimized for bone and cartilage tissue.
RESULTS: Our results indicate that homozygous Hoxd11 loss-of-function mouse limbs may display a slightly stronger expression of Ihh in the pre-hypertrophic zone of metatarsal growth plates compared to wild type specimens. We observe no significant difference in expression of Pthlh in the perichondrium and periarticular chondrocytes of homozygous Hoxd11 loss-of-function specimens compared to wild type.
CONCLUSION: Pthlh and Ihh expression in loss-of-function and wild type Hoxd11 mice indicate similar expression patterns within the growth plate regions at the developmental stage examined. Loss of Hoxd11 function does not appear to alter the expression of Pthlh in reserve chondrocyte. Future work will be done in the forelimb to compare to bones such as the metacarpals and pisiform which are likely more susceptible to reductions in Hoxd11 loss-of-function.