Understanding the role of Foxc1 and Foxc2 in embryonic bone development
Creators: Almubarak, Asra
Publisher: name: University of Alberta Library
Year: 2022

<p>Endochondral ossification is a skeletal development process where mesenchymal progenitors differentiate into chondrocytes that will be eventually replaced by bone. Disrupting this process causes skeletal dysplasia. FOXC1 and FOXC2 are two transcription factors of the forkhead box family that express at early stages of endochondral ossification. The similar expression pattern and similar endochondral ossification phenotype observed in Foxc1 or Foxc2 mutant mice, indicate a possible compensation between the two genes. Compound Foxc1 and Foxc2 mutant mice die in utero before any skeletal elements are formed. In order to study the molecular and biological aspects of Fox1 and Foxc2 function in endochondral ossification, we employed both in vitro and in vivo systems. ATDC5 prechondrocytic cells, mouse embryonic stem cells (mESCs) and U2OS osteosarcoma cells were used to investigate molecular function of FOXC1.  In addition, we generated two conditional mutant mouse models to study of the role for Foxc1 and Foxc2 in skeletal development. First, both Foxc genes were deleted in  chondrocytes (Col2-cre;Foxc1Δ/Δ;Foxc2Δ/Δ ). Second Foxc1 and Foxc2 were deleted in condensing limb bud mesenchyme ( Prx1-cre;Foxc1Δ/Δ;Foxc2Δ/Δ). Our analysis showed that SRY (sex-determining region Y)-box 9 (SOX9), a master regulatory transcription factor of chondrogenesis directly regulates Foxc1 expression. We also demonstrated that Foxc1 overexpression enhances chondrocyte differentiation in mouse embryonic stem cells, while loss of Foxc1 function inhibits chondrogenesis in ATDC5 cells. Conditional KO of Foxc1 and Foxc2 in chondrocytes in mice led to general skeletal dysplasia with preferential abnormality in the axial skeleton including the vertebral column compared to the appendicular skeleton. The long bones were smaller due to a disorganized growth plate, reduced columnar chondrocyte proliferation and impaired mineralization. Moreover, Col2-cre;Foxc1Δ/Δ;Foxc2Δ/Δ mice displayed complete lack of chondrogenesis in the cervical vertebrae and delayed endochondral development of the thoracic vertebrae. In the intervertebral discs, the anulus fibrosus was not formed and the nucleus pulposus was irregularly shaped.<br>
Prx1-Cre conditional deletion of Foxc1 and Foxc2 in early limb bud mesenchyme resulted in mice with shorter, bowed limbs that exhibited reduced mineralization, thinner digits, and smaller bone eminences. In the growth plate, absence of both Foxc genes impaired the formation of Ihh expressing cells which compromised proliferation and lead to a delayed entry into hypertrophy at E14.5. At E16.5, COLX hypertrophic chondrocyte domains were strikingly mineralized and expanded into the primary ossification center of the mutant limb, with disrupted mineralization of the primary ossification center. Foxc1 and Foxc2 KO compromised phosphate-regulating gene with homologies to endopeptidases on the X chromosome (Phex) expression in the primary ossification center leading to Osteopontin (OPN) stabilization and decreased mineralization. </p>

<p>Previously reported stabilization of B-CATENIN in hypertrophic chondrocytes led to similar growth plate abnormalities to those in Prx1-cre;Foxc1Δ/Δ;Foxc2Δ/Δ  mice limbs. In vitro analysis addressed the functional association between FOXC1 and B-CATENIN and demonstrated that overexpression of Foxc1 inhibited b-CATENIN activity in U2OS cells. Moreover, we demonstrate that FOXC1 DNA-binding is required in order to completely inhibit B-CATENIN activity, and not through FOXC1 physical binding with B-CATENIN protein. Our findings suggest that Foxc1 and Foxc2 are essential for regulating different aspects of endochondral ossification, and that loss of Foxc1 and Foxc2 function impaired the progression of various stages of endochondral ossification in axial and appendicular skeleton.</p>