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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 embryoepithelium.
Supplementary Fig. S1. The gap is specific to dividing cells in living cell embryos. A wide-field of GFP-GPI expressing epithelial cells in gastrula
is shown. White arrows point on the gap between the two daughter cells surrounded by interphase cells. Such a gap is observed only in cytokinetic
cells, it is not detected between interphase cells.
Supplementary Fig. S2. At the clone periphery, some cells are isolated from the main cell group. A wide-field of GFP-GPI expressing gastrula
showing labelled green cells separated from the main GFP-GPI expressing cell group by unlabeled cells (black).
Supplementary Fig. S3. Neighboring cells intercalate between the two daughter cells during cytokinesis Live-imaging of GFP-GPI and RFP-GPI
(merge of green and red are shown) expressing embryo showing neighboring cells which intercalate between the two daughter cells during cytokinesis.
Two confocal planes are shown. Orthogonal projections along the daughter cell axis (d, white dashed lines) and the neighboring cells axis (n,
grey dashed line) are shown. The white arrow on the orthogonal projection (d) points on the two neighboring cells which intercalate between the two
daughter cells. Time is given in minutes. 0 is defined as the time before any sign of membrane contraction can be detected. Scale bar: 10 mm
Fig. 1. During epithelial cell cytokinesis in the
Xenopus gastrula, a gap transiently forms between
the plasma membranes of the two daughters cells.
(A,B) Albino embryos were fixed at the gastrula stage
and indirect immunofluorescence was performed with
anti-xMELK antibodies (red and stained for DNA with
TO-PRO-3 (blue). (A) Three optical sections are shown.
An orthogonal projection along the daughter cells axis
perpendicular to the cytokinetic furrow (white dashed
line) is shown (orthog.). High magnifications of the
insets in orthogonal projections (a’ and a”) are shown.
On the left column, the gap between daughter cells is
visible over the entire height of daughter cells wheras
on the right daughter cells are in contacts basally to
the gap thus closing the gap and creating an inverted
teardrop-like shape volume. (B) A wide-field of the
epithelium presented in A showing two cytokinetic
cells surrounded by interphase cells. White arrows
point on the gap between the two daughter cells. Such
a gap is not detected between interphase cells. (C)
Time-lapse imaging of a dividing cell expressing GFPGPI.
Embryos were microinjected in one blastomere
at the two cells stage with mRNA encoding GFP-GPI
and observed when they reached the gastrula stage
11. A high magnification of the gap is shown in the
insert. Time is given in minutes. 0 is defined as the
time before any sign of membrane contraction can be
detected. Scale bars: 10 mm.
Fig. 2. Three types of epithelial
cell cytokinesis in
Xenopus laevis gastrula.
Live-imaging of GFP-GPI,
green, (A,B) and GFPGPI+RFP-GPI,
merge of
green and red, (C) expressing
embryos showing a dividing
cell with the inverted teardrop
between the daughter
cells and which progresses
with the cleavage furrow
(A), a cell which divides
without formation of a gap
(B), and neighboring cells
which intercalate between
the two daughter cells during
cytokinesis (C). Two (A,C)
or three (B) confocal planes
are shown. Orthogonal projections
along the daughter
cell axis (white dashed
lines) are shown in (A,B).
In the orthogonal projection
in (A), a high magnification
of the teardrop is shown in
the insert. Arrowhead over
the orthogonal projection in
(B) indicates that the furrow
ingresses asymmetrically
from the apical membrane
towards the basal region. In (C) two orthogonal projections respectively along the daughter cells axis (d) and the neighboring cells axis (n, grey dashed
line) are shown. The white arrow on the orthogonal projection (d) points on the two neighboring cells which intercalate between the two daughter cells.
Time is given in minutes. 0 is defined as the time before any sign of membrane contraction can be detected. Scale bars: 10 mm.
Fig. 3. Anillin, actin and myosin II heavy chain (MHC) localize at the leading edge of the inverted teardrop during epithelial cell cytokinesis in gastrula. Localizations of GFP-anillin + RFP-GPI (respectively green and red, a merge is presented)
(A), actin detected with the mcherry-Utr-CH probe (B), and myosin II heavy chain
(MHC) using the SF9-GFP intrabody (C). For each, 3 confocal planes and orthogonal projections along the daughter cells (d) and neighboring cells (n) axes are shown. The arrows point on the gap between the two daughter cells. Scale bars, 10 mm.
Fig. 4. Dynamics of the cleavage furrow during epithelial cell cytokinesis.
(A) Graphs plotting the distance between the cytokinetic furrow and
the apical membrane over time (n=4) showing that cytokinesis is initially
rapid and becomes slower when the furrow is 2.5-4.5 mm of the apical
membrane. (B) Quantification of the gap width during the fast and the
slow phases progression of the cytokinetic furrow over time. The same
cells as in (A) were analyzed
