Exploring the impact of energy metabolism in periosteal cells transcriptional profile.

Glycolytic metabolism, once considered merely a consequence of cellular function, has emerged as a crucial regulator of cellular differentiation. Both glycolysis and oxidative phosphorylation (OXPhos) are vital for osteoblastogenesis. Osteoblasts are the cells that make bone. To deepen our understanding of OXPhos's role in bone mass accrual, we engineered a mutant mouse model deficient in Mitochondrial Transcription Factor A (TFAM) specifically in mesenchymal progenitors within the limb bud and their descendants, using the PRX1-Cre driver system. TFAM is essential for transcription of the mitochondrial genes that encode critical components of the electron transport chain, thereby governing OXPhos. Our TFAM mutant mice exhibit marked shortening of long bones, evident at birth and progressing with age, accompanied by growth plate abnormalities and bone deformities. Critically, these mice develop spontaneous fractures in long bone mid-shafts by three weeks of age, indicating significant compromise in bone integrity. Comparative analyses using MicroCT and histomorphometry reveal drastic cortical bone thinning in mutants due to severely impaired osteoid deposition and bone formation, especially in the diaphyseal periosteum. Furthermore, an increased number of osteoclasts at this site suggests that an elevated bone resorption contributes to the phenotype. Acknowledging the active role of energy metabolism in cell differentiation and function, we measured ATP production in PRX1 lineage periosteal cells. TFAM deletion led to a substantial decrease in steady-state intracellular ATP levels, highlighting a critical metabolic deficit. To rectify this metabolic defect and potentially correct the resultant phenotype, we bred TFAM mice with another mouse line expressing mutated, oxygen-independent Hypoxia Inducible Factor 1alpha (HIF1dPA) within PRX1 lineage cells (yielding TFAM/HIF1dPA mice). These double mutants did not experience spontaneous fractures, and their cortical bone thickness and the ability of periosteal cells to accumulate osteoid was fully restored. Therefore, by examining the transcriptional profile of periosteal cells, our aim is to determine whether TFAM deficiency alters their genetic landscape and if HIF1 activation can reverse these changes. Our goal is to provide new insights into the role of energy metabolism in shaping the transcriptome landscape, thereby influencing cell differentiation and activity. To investigate the impact of energy metabolism on the transcriptional profile of periosteal cells, we established two mouse lines: one with a mutant phenotype characterized by a lack of mitochondrial transcription factor A (TFAM) in PRX1 lineage cells. We conditionally knocked out TFAM in uncommitted mesenchymal progenitors and their descendants using PRX1-Cre drivers and a mouse line homozygous for a floxed Tfam allele (TFAMfl/fl). PRX1-Cre mice express Cre recombinase in mesenchymal cells that give rise to chondrocytes and osteoblasts in long bones and calvaria. Subsequently, we generated double mutants lacking TFAM and expressing a constitutively stabilized form of HIF1α protein in mesenchymal progenitors and their descendants (referred to as PRX1;TFAM;HIF1dPA or TFAM;HIF1dPA). scRNA-seq was performed on periosteal cells isolated from the long bones hindlimbs of three-week-old mice. The resulting data were utilized to identify distinct cell clusters within the periosteum and to conduct differential gene expression analysis between control mice and their mutant counterparts for each line.