Fetal rat calvaria cells plated at very low density generate discrete colonies, some of which are bone colonies (nodules) from individual osteoprogenitors that divide and differentiate. We have analyzed the relationship between cell proliferation and acquisition of tissue-specific differentiation markers in bone colonies followed individually from the original single cell to the fully mineralized state. The size distribution of fully formed nodules is unimodal, suggesting that the coupling between proliferation and differentiation of osteoprogenitor cells is governed by a stochastic element, but distributed around an optimum, corresponding to the peak colony size/division potential. Kinetic analysis of colony growth showed that osteoprogenitors undergo 9-10 population doublings before the appearance of the first morphologically differentiated osteoblasts in the developing colony. Double immunolabeling showed that these proliferating cells express a gradient of bone markers, from proliferative alkaline phosphatase-negative cells at the periphery of colonies, to postmitotic, osteocalcin-producing osteoblasts at the centers. An inverse relationship exists between cell division and expression of osteocalcin, the latter being restricted to late-stage, BrdU-negative osteoblasts, while the expression of all other markers is acquired before the cessation of proliferation, but not concomitantly. Bone sialoprotein expression is biphasic, detectable in some of the early, alkaline phosphatase-negative cells, and again later in both late preosteoblast (BrdU-positive) and osteoblast (BrdU-negative, osteocalcin-positive) cells. In late-stage, heavily mineralized nodules, staining for osteocalcin and bone sialoprotein is not detectable in the oldest/most mature cells. Our observations support the view that the bone nodule "tissue-like" structure, originating from a single osteoprogenitor and finally encompassing mineralized matrix production, recapitulates successive stages of the osteoblast differentiation pathway, in a proliferation/maturation sequence. Understanding the complexity of the proliferation/differentiation kinetics that occurs within bone nodules will aid in the qualitative and/or quantitative interpretation of tissue-specific marker expression during osteoblastic differentiation.

BackgroundResident fibroblasts synthesize the cardiac extracellular matrix, and can undergo phenotype conversion to myofibroblasts to augment matrix production, impairing function and contributing to organ failure. A significant gap in our understanding of the transcriptional regulation of these processes exists. Given the key role of this phenotype conversion in fibrotic disease, the identification of such novel transcriptional regulators may yield new targets for therapies for fibrosis.ResultsUsing explanted primary cardiac fibroblasts in gain- and loss-of-function studies, we found that scleraxis critically controls cardiac fibroblast/myofibroblast phenotype by direct transcriptional regulation of myriad genes that effectively define these cells, including extracellular matrix components and α-smooth muscle actin. Scleraxis furthermore potentiated the TGFβ/Smad3 signaling pathway, a key regulator of myofibroblast conversion, by facilitating transcription complex formation. While scleraxis promoted fibroblast to myofibroblast conversion, loss of scleraxis attenuated myofibroblast function and gene expression. These results were confirmed in scleraxis knockout mice, which were cardiac matrix-deficient and lost ~50 % of their complement of cardiac fibroblasts, with evidence of impaired epithelial-to-mesenchymal transition (EMT). Scleraxis directly transactivated several EMT marker genes, and was sufficient to induce mesenchymal/fibroblast phenotype conversion of A549 epithelial cells. Conversely, loss of scleraxis attenuated TGFβ-induced EMT marker expression.ConclusionsOur results demonstrate that scleraxis is a novel and potent regulator of cellular progression along the continuum culminating in the cardiac myofibroblast phenotype. Scleraxis was both sufficient to drive conversion, and required for full conversion to occur. Scleraxis fulfills this role by direct transcriptional regulation of key target genes, and by facilitating TGFβ/Smad signaling. Given the key role of fibroblast to myofibroblast conversion in fibrotic diseases in the heart and other tissue types, scleraxis may be an important target for therapeutic development.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0243-8) contains supplementary material, which is available to authorized users.

Fetal rat calvaria cells plated at very low density generate discrete colonies, some of which are bone colonies (nodules) from individual osteoprogenitors that divide and differentiate. We have analyzed the relationship between cell proliferation and acquisition of tissue-specific differentiation markers in bone colonies followed individually from the original single cell to the fully mineralized state. The size distribution of fully formed nodules is unimodal, suggesting that the coupling between proliferation and differentiation of osteoprogenitor cells is governed by a stochastic element, but distributed around an optimum, corresponding to the peak colony size/division potential. Kinetic analysis of colony growth showed that osteoprogenitors undergo 9-10 population doublings before the appearance of the first morphologically differentiated osteoblasts in the developing colony. Double immunolabeling showed that these proliferating cells express a gradient of bone markers, from proliferative alkaline phosphatase-negative cells at the periphery of colonies, to postmitotic, osteocalcin-producing osteoblasts at the centers. An inverse relationship exists between cell division and expression of osteocalcin, the latter being restricted to late-stage, BrdU-negative osteoblasts, while the expression of all other markers is acquired before the cessation of proliferation, but not concomitantly. Bone sialoprotein expression is biphasic, detectable in some of the early, alkaline phosphatase-negative cells, and again later in both late preosteoblast (BrdU-positive) and osteoblast (BrdU-negative, osteocalcin-positive) cells. In late-stage, heavily mineralized nodules, staining for osteocalcin and bone sialoprotein is not detectable in the oldest/most mature cells. Our observations support the view that the bone nodule "tissue-like" structure, originating from a single osteoprogenitor and finally encompassing mineralized matrix production, recapitulates successive stages of the osteoblast differentiation pathway, in a proliferation/maturation sequence. Understanding the complexity of the proliferation/differentiation kinetics that occurs within bone nodules will aid in the qualitative and/or quantitative interpretation of tissue-specific marker expression during osteoblastic differentiation.