An increasing body of evidence strongly suggests that low oxygen tension is not only a component of many human disorders including cancer, heart attack and stroke, but it is also important in normal fetal development and cell differentiation. At the AAOS sponsored Developmental Biology in Orthopaedics symposium, I had the opportunity to present my research on how the hypoxia inducible factor -1a (HIF-1a) regulates differentiation of the limb bud mesenchyme and joint development.
HIF-1a is a crucial mediator of the adaptive response of cells to hypoxia. Previous research has shown that this transcription factor is critical for survival of hypoxic chondrocytes and negatively regulates chondrocyte proliferation in later stages of endochondral bone development. The goal of my study was to investigate whether HIF-1a has a role in the formation of mesenchyme condensations, in commitment of mesenchymal cells towards chondrocytes, and in early stages of chondrocyte differentiation.
Our lab provided evidence that mesenchymal condensations are hypoxic; then, by whole mount in situ hybridization and immunohistochemistry analysis, we demonstrated that HIF-1a is expressed in limb bud mesenchyme. Lastly, by using a novel reporter mouse, we showed that HIF-1a is transcriptionally active in limb bud mesenchyme as early as day 10.5 of embryonic development.
To investigate the role of HIF-1a in mesenchymal condensation and early chondrogenesis, we thus conditionally inactivated HIF-1a in limb bud mesenchyme. Mutant mice were viable but presented an extreme shortening of both forelimbs and hindlimbs. Whole mount skeletal preparations, standard histology, in situ hybridization analysis and Tunel assay were performed at different times of fetal and postnatal development.
Conditional knockout of HIF-1a in limb bud mesenchyme did not impair mesenchyme condensation but did delay the formation of the cartilaginous primordia. Late hypertrophic differentiation was also delayed as a result, very likely, of the delay in early chondrogenesis. Furthermore, mutant mice showed a striking impairment of joint development, which was secondary to a delay of joint specification. This study revealed a crucial, and previously unrecognized, role of HIF-1a in differentiation during embryonic development, which occurred in absence of significant loss of cell viability and was not secondary to impairment of angiogenesis.