Abstract
The primordium of the limb contains a number of progenitors far superior to those necessary to form the skeletal components of this appendage. During the course of development, precursors that do not follow the skeletogenic program are removed by cell senescence and apoptosis. The formation of the digits provides the most representative example of embryonic remodeling via cell degeneration. In the hand/foot regions of the embryonic vertebrate limb (autopod), the interdigital tissue and the zones of interphalangeal joint formation undergo massive degeneration that accounts for jointed and free digit morphology. Developmental senescence and caspase-dependent apoptosis are considered responsible for these remodeling processes. Our study uncovers a new upstream level of regulation of remodeling by the epigenetic regulators Uhrf1 and Uhrf2 genes. These genes are spatially and temporally expressed in the pre-apoptotic regions. UHRF1 and UHRF2 showed a nuclear localization associated with foci of methylated cytosine. Interestingly, nuclear labeling increased in cells progressing through the stages of degeneration prior to TUNEL positivity. Functional analysis in cultured limb skeletal progenitors via the overexpression of either UHRF1 or UHRF2 inhibited chondrogenesis and induced cell senescence and apoptosis accompanied with changes in global and regional DNA methylation. Uhrfs modulated canonical cell differentiation factors, such as Sox9 and Scleraxis, promoted apoptosis via up-regulation of Bak1, and induced cell senescence, by arresting progenitors at the S phase and upregulating the expression of p21. Expression of Uhrf genes in vivo was positively modulated by FGF signaling. In the micromass culture assay Uhrf1 was down-regulated as the progenitors lost stemness and differentiated into cartilage. Together, our findings emphasize the importance of tuning the balance between cell differentiation and cell stemness as a central step in the initiation of the so-called “embryonic programmed cell death” and suggest that the structural organization of the chromatin, via epigenetic modifications, may be a precocious and critical factor in these regulatory events.
Highlights
The autopod is a paddle shaped structure consisting of a core of skeletal progenitors covered by the ectoderm
While the interdigital application of exogenous BMPs in the embryonic limb induces cell death and DNA damage, the same BMPs stimulate cell proliferation and differentiation in the tip of the fingers, that are constituted by the same skeletal progenitors[6]
We show that Uhrf[1] and Uhrf[2] genes are expressed in the interdigital mesoderm and interphalangeal joints where undifferentiated cells undergo senescence and apoptosis
Summary
The autopod is a paddle shaped structure consisting of a core of skeletal progenitors covered by the ectoderm. Sanchez-Fernandez et al Cell Death and Disease (2019)10:347 associated with lysosomal activation[2,3], and cell senescence[4] All these degenerative routes are preceded by intense DNA damage[5], which is suggestive of common upstream regulation. While the interdigital application of exogenous BMPs in the embryonic limb induces cell death and DNA damage, the same BMPs stimulate cell proliferation and differentiation in the tip of the fingers, that are constituted by the same skeletal progenitors[6]. This finding suggests that complementary signals cooperate with BMPs to trigger the degenerative events. DNA damage, cell senescence, and apoptosis are associated with epigenetic and chromatin architecture alterations of cancerous cells[7] that may determine tumorigenesis and sensitivity to chemotherapy and irradiation[8,9]
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