Epithelial cell division in the Xenopus laevis embryo during gastrulation

Abstract

How vertebrate epithelial cells divide in vivo and how the cellular environment influences cell division is currently poorly understood. A sine qua non condition to study cell division in situ is the ease of observation of cell division. This is fulfilled in the Xenopus embryo at the gastrula stage where polarized epithelial cells divide with a high frequency at the surface of the organism. Recently, using this model system, we have shown that epithelial cells divide by asymmetric furrowing and that the mode of cell division is regulated during development. Here, we further characterize epithelial cell division in situ. To this end, we used confocal microscopy to study epithelial cell division in the ectoderm of the Xenopus laevis gastrula. Cell division was followed either by indirect immunofluorescence in fixed embryos or by live imaging of embryos transiently expressing diverse fluorescent proteins. Here, we show that during cytokinesis, the plasma membranes of the two daughter cells are usually separated by a gap. For most divisions, daughter cells make contacts basally at a distance from the furrow tip which creates an inverted teardrop-like shaped volume tightly associated with the furrow. At the end of cytokinesis, the inverted teardrop is resorbed; thus it is a transient structure. Several proteins involved in cytokinesis are localized at the tip of the inverted teardrop suggesting that the formation of the gap could be an active process. We also show that intercalation of neighboring cells between daughter cells occasionally occurs during cytokinesis. Our results reveal an additional level of complexity in the relationship between dividing cells and also with their neighboring cells during cytokinesis in the Xenopus embryo epithelium.

Maintenance of the Epithelial Barrier and Remodeling of Cell-Cell Junctions during Cytokinesis

“…This difference could be due to a number of factors including the distinct structure and molecular components of the junctions, differences in cortical or junctional tension, or differing requirements for barrier function. Previous studies in vertebrates have demonstrated that cells neighboring the cleavage furrow can intercalate between the recently divided cells [27, 63]. This is consistent with our observations that cells initially form a short interface between neighbors, and even one hour after cytokinesis, many daughter cells are still separated by neighbor cells.…”

Actomyosin-generated tension on cadherin is similar between dividing and non-dividing epithelial cells in early Xenopus laevis embryos

“…For indirect immunofluorescence, embryos were fixed with 2% trichloroacetic acid (TCA) in F1 1X medium for 2 hours at room temperature, devitelinated, permeabilized in PBS plus 1% triton X-100 (PBST 1%) for 10 minutes and saturated one hour in PBST 0.1% plus BSA 1% as previously described23. They were then incubated with the following antibodies: anti-GFP (I-16, Santa Cruz, clone 7.1 and 13.1 11 914 460 001, Roche), anti-Xenopus C-cadherin (clone 6B6, DSHB), anti-α-catenin (PA1-25081 Thermo Scientific), anti β-catenin (H102, Sc-7199) or anti-p120 catenin (a kind gift of Dr P McCrea).…”

“…To elucidate these questions, we have investigated tension applied on cadherin during cell division in Xenopus embryo epithelium. At blastula stage, in situ observation of cell division is possible since polarized epithelial cells divide with a high frequency at the surface of the organism23. We have employed the approach of E-cadherin FRET tension sensor14 in developing Xenopus leavis embryos.…”

Tight junctions negatively regulate mechanical forces applied to adherens junctions in vertebrate epithelial tissue

“…All animal experiments were performed in accordance with the approved protocols and guidelines at Rennes 1 University by the Comité Rennais d'Ethique en Matiere d'Expérimentation Animale (C2EA-07) and the French Ministry for Education and Research (3523813). Eggs were fertilised in vitro and embryos were collected as described previously (Hatte et al, 2014). After de-jelling, embryos were placed in 5% Ficoll in F1 buffer (10 mM HEPES pH 7.6, 31.2 mM NaCl, 1.75 mM KCl, 59 μM MgCl 2 , 2 mM NaHCO 3 , and 0.25 mM CaCl 2 ) for microinjection.…”