Tassy, O. | bioRxiv Science

bioRxiv2017

Multiple inputs ensure yeast cell size homeostasis during cell cycle progression

Coordination of cell growth and division is essential for proper cell function. In budding yeast, although some molecular mechanisms responsible for cell size control during G1 have been elucidated, the mechanism by which cell size homeostasis is established and maintained throughout the cell cycle remains to be discovered. Here, we developed a new technique based on quantification of histone levels to monitor cell cycle progression in individual yeast cells with unprecedented accuracy. Our analysis establishes the existence of a strong mechanism controlling bud size in G2/M that prevents premature entry into mitosis, and contributes significantly to the overall control of size variability during the cell cycle. While most G1/S regulation mutants do not display any strongly impaired size homeostasis, mutants in which B-type cyclin regulation is altered display large cell-to-cell size variability. Our study thus demonstrates that size homeostasis is not controlled by a G1-specific mechanism but is likely to be an emergent property resulting from the integration of several mechanisms, including the control of cyclin B-Cdk activity, that coordinate cell and bud growth with division.

cell biology↗

bioRxiv2017

Recapitulating Early Development Of Mouse Musculoskeletal Precursors Of The Paraxial Mesoderm In Vitro

In vertebrates, body skeletal muscles and axial skeleton derive from the paraxial mesoderm which flanks the neural tube and notochord. The paraxial mesoderm forms in the posterior region of the embryo as presomitic mesoderm (PSM), which generates the embryonic segments called somites. Here, we characterized gene signatures identified using microarray series from the mouse PSM and compared the PSM transcriptome dynamics to that of the developing neural tube. In contrast to the PSM where an abrupt transcriptome reorganisation occurs at the level of the determination front, we show that transcriptome changes are progressive during parallel stages of neural tube differentiation. We show that these early differentiation stages of the paraxial mesoderm can be efficiently recapitulated in monolayer culture in vitro using murine Embryonic Stem (ES) cells. We describe a serum-containing protocol which parallels in vivo tissue maturation allowing differentiation of ES cells towards a paraxial mesoderm fate. We show that R-spondin treatment or Wnt activation alone can induce posterior PSM markers in both mouse and human ES/iPS cells but acquisition of a committed posterior PSM fate requires BMP inhibition to prevent induced cells to drift to a lateral plate mesoderm identity. We show that posterior PSM-like cells induced from mouse ES cells can be further differentiated in vitro to acquire an anterior PSM Pax3-positive identity. When grafted into injured adult muscle, these induced PSM-like precursors generated large numbers of immature muscle fibers. We further show that exposing ES-derived PSM-like cells to a brief FGF inhibition step followed by culture in horse serum-containing medium allows efficient recapitulation of the myogenic program. Differentiating ES cells first produce mononucleated embryonic myocytes and subsequently multinucleated myotubes, as well as Pax7-positive cells. The protocol described here results in improved differentiation and maturation of mouse muscle fibers differentiated in vitro over serum-free protocols. It provides an efficient system for the study of myogenic processes otherwise difficult to study in vivo such as fusion or satellite cell differentiation.

developmental biology↗