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.
