Loss of Foxc1 and Foxc2 function in chondroprogenitor cells disrupts endochondral ossification

Abstract

Endochondral ossification initiates the growth of the majority of the mammalian skeleton and is tightly controlled through gene regulatory networks. The forkhead box transcription factors Foxc1 and Foxc2 regulate aspects of osteoblast function in the formation of the skeleton, but their roles in chondrocytes to control endochondral ossification are less clear. Here, we demonstrate that Foxc1 expression is directly regulated by the activity of SRY (sex-determining region Y)-box 9, one of the earliest transcription factors to specify the chondrocyte lineage. Moreover, we demonstrate that elevated expression of Foxc1 promotes chondrocyte differentiation in mouse embryonic stem cells and loss of Foxc1 function inhibits chondrogenesis in vitro . Using chondrocyte-targeted deletion of Foxc1 and Foxc2 in mice, we reveal a role for these factors in chondrocyte differentiation in vivo . Loss of both Foxc1 and Foxc2 caused a general skeletal dysplasia predominantly affecting the vertebral column. The long bones of the limbs were smaller, mineralization was reduced, and organization of the growth plate was disrupted; in particular, the stacked columnar organization of the proliferative chondrocyte layer was reduced in size and cell proliferation was decreased. Differential gene expression analysis indicated disrupted expression patterns of chondrogenesis and ossification genes throughout the entire process of endochondral ossification in chondrocyte-specific Foxc1/Foxc2 KO embryos. Our results suggest that Foxc1 and Foxc2 are required for normal chondrocyte differentiation and function, as loss of both genes results in disorganization of the growth plate, reduced chondrocyte proliferation, and delays in chondrocyte hypertrophy that prevents ossification of the skeleton.

Foxc1andFoxc2function in osteochondral progenitors for the progression through chondrocyte hypertrophy and mineralization of the primary ossification center

“…Impaired formation of cartilaginous elements in early deletion of Foxc1 and Foxc2 in Sox9-cre expressing cells Foxc1 and Foxc2 genes are expressed in condensing mesenchyme cells at the onset of chondrogenesis in the limb skeleton (Almubarak et al, 2021;Hiemisch et al, 1998). We examined chondrocyte differentiation (by Alcian blue staining) in embryos engineered to contain floxed alleles of both these transcription factors plus a Cre recombinase that had been knocked in to the 3' UTR of the Sox9 locus (Sox9 ires-Cre ; Akiyama et al, 2005;Sasman et al, 2012).…”

“…The copyright holder for this preprint (which this version posted April 27, 2023. ; https://doi.org/10.1101/2023.04.26.538325 doi: bioRxiv preprint and Foxc2, we created a conditional mouse model that deleted both genes in the chondrocyte lineage. These Col2-cre;Foxc1 Δ/Δ ;Foxc2 Δ/Δ mutants displayed impaired chondrocyte differentiation and led to a general skeletal hypoplasia without affecting vital organ systems (Almubarak et al, 2021). The rib cage and vertebral column was more affected in these compound homozygous mice compared to the bones in the limb.…”

“…The transcription factors Forkhead Box C1 and C2 (FOXC1 and FOXC2) are important regulators of skeletal development (Almubarak et al, 2021;Kume et al, 1998;Winnier et al, 1997;Yoshida et al, 2015). These factors display overlapping expression in skeletal progenitors of the limb and the vertebrae (Almubarak et al, 2021;Hiemisch et al, 1998). Mice with homozygous null mutations in either Foxc1 and Foxc2 display malformations in the axial skeleton (the skull, vertebral column, and rib cage).…”

“…The rib cage and vertebral column was more affected in these compound homozygous mice compared to the bones in the limb. Given that Foxc1 and Foxc2 are abundantly expressed in the condensing limb bud mesenchyme (Almubarak et al, 2021;Hiemisch et al, 1998), such phenotypic differences between the axial and appendicular skeleton were surprising. The mesenchyme that forms the axial skeleton is derived from the sclerotome.…”

Myelodysplastic syndromes disable support of human hematopoietic stem and progenitor cells by CD271, VCAM- 1 and CD146 expressing marrow niche cells

“…2A, B). NMVL cells are also likely enriched for human CXCL12 Abundant Reticular (CAR) cells, previously shown to be a critical HSC-supportive population, since NVML have increased expression not only of CXCL12 but also of the characteristic CAR cell genes FOXC1 and EBF3 [27][28][29] (Fig. 2A,B).…”