DOI: 10.5061/dryad.ghx3ffbmx
title: Physically asymmetric division of the C. elegans zygote ensures invariably successful embryogenesis

description: Asymmetric divisions that yield daughter cells of different sizes are
 frequent during early embryogenesis, but the importance of such a physical
 difference for successful development remains poorly understood. Here, we
 investigated this question using the first division of C. elegans embryos,
 which yields a large AB cell and a small P1 cell. We equalized AB and P1
 sizes using acute genetic inactivation or optogenetic manipulation of the
 spindle positioning protein LIN-5. We uncovered that only some embryos
 tolerated equalization, and that there was a size asymmetry threshold for
 viability. Cell lineage analysis of equalized embryos revealed an array of
 defects, including faster cell cycle progression in P1 descendants, as
 well as defects in cell positioning, division orientation and cell fate.
 Moreover, equalized embryos were more susceptible to external compression.
 Overall, we conclude that unequal first cleavage is essential for
 invariably successful embryonic development of C. elegans.
description: 3D time-lapse
 recordings of embryos expressing mCherry∷H2B in either wild-type
 or <i>lin‑5(ev571)</i> background were first pre-processed to
 enhance the nuclear signal and remove noise with the Noise2Void/CARE
 machine learning pipeline (Krull et al., 2019). Thereafter, a custom Fiji
 macro was used to correct the drift using the first polar body as a bright
 fiducial marker. The lineage was then traced using a level-set image
 segmentation and model evolution implemented in MATLAB (MathWorks, USA) as
 described previously (Dzyubachyk et al., 2010; Krüger et al., 2015), and
 corrected in the WormDeLux Java-based lineage editor (Jelier et al.,
 2016). Results were exported in the StarryNite format (Bao and Murray,
 2011). Cells were then automatically named in the lineage editor according
 to the canonical lineage (Sulston et al., 1983), and manually checked
 afterward for possible errors, especially in mutant embryos that often
 substantially deviated in division orientation and cell positioning from
 the wild-type model used for naming.  As mentioned in
 the manuscript, the source code used for the analysis of deposited data is
 available at <b>https://github.com/UPGON/worm-rules-eLife</b>.  R_00_import_Lineages.R was used to load and align data in time and space. R_01_Analysis_pipeline.R contains code and links to an additional R files in the repo used for statistical analysis of the data. .tb_button {padding:1px;cursor:pointer;border-right: 1px solid #8b8b8b;border-left: 1px solid #FFF;border-bottom: 1px solid #fff;}.tb_button.hover {borer:2px outset #def; background-color: #f8f8f8 !important;}.ws_toolbar {z-index:100000} .ws_toolbar .ws_tb_btn {cursor:pointer;border:1px solid #555;padding:3px} .tb_highlight{background-color:yellow} .tb_hide {visibility:hidden} .ws_toolbar img {padding:2px;margin:0px}
description: See 200723_all_lineaged_embryos.xlsx for annotations
 of embryos. Raw lineage files in StarryNite format
 are in zip_lineages.zip Processed and aligned lineage
 data, as well as embryo annotation, are to be found
 in 200723_embryos.RData RData file in the
 contains following objects: Embs, Nuclei, Timing and functions
 getCellFates and SaveNuclei  <b>Embs </b> -
 it is the table with all samples and ID + additional important information
 about embryos: "ID" - unique ID
 "AB_rel" - relative size of the larger cell AB with
 respect to the whole embryo "Outcome" -
 whether embryo hatched or died "Genotype" -
 either wild-type or temperature-sensitive background
 (<i>ev571</i> is the allele of <i>lin-5 </i>that
 we use to make embryos "equalized")
 "Experiment" with following values:
 meta - this means that embryo was upshifted during metaphase and
 should be equalized.<br> ctrls - experiments of two
 kinds: <i>ev571 </i>embryos upshifted during 2-cell stage
 (will be normally unequal and nearly fully viable) 2. second control are
 WT embryos (non-ts) that were imaged at exactly the same conditions
 as <i>ev571</i> (17°C) - ids GZ01-GZ10
  "AB_size"  - absolute size (2D cross-section) of AB
 cell in um2        "P1_size"         
    "pixel" - pixel size in um             
   "Z_slicing" - the distance between slices
 in 3D in um (this is important because distances in the coordinate system
 are expressed in relative units Z-slicing/pixelSize)
 "length", "height" - the incomplete manual
 measurement of the length and width of embryo
 "interval", "TimeExpantionFactor" - the
 interval between individual frames of the time-lapse movie in seconds and
 expansion factor used to align embryogenesis of all my embryos
 <b>Nuclei</b> 3D coordinates for
 each cell at every time point for every embryo. It
 contains lists named according to Embryo ID that contains data.frames
 named "t001" - "tLastTimeFrame" <br> each
 data.frame you find cells present at a given time... for
 example: Nuclei$EM01$t001 ID ALIVE
 PRED SUCC1 SUCC2   X    Y     Z     SIZE  IDENTITY<br> 1     1   -1 
            1           -1 .      175  78 16.0    66         
 ABp <br> 2     1   -1             2           -1        324 213
 18.5   59          EMS<br> 3     1   -1             4           -1 
       432 129 18.5   58          P2<br> 4     1   -1             3 
          -1        148 203 21.5   60          ABa ALIVE
 is irrelevant because all cells in my embryos at the end of lineaging were
 alive, PRED - i don't who what that means.<br> If a cell
 divides it will have some ID value in SUCC2 
 <b>Timing</b> It is large
 data.frame containing extracted and scaled timing for individual cells,
 time when they have appeared (StartTime), and when they divided (EndTime).
 "Frame" refers to the time-point in the Nuclei. It also contains
 some movement statistics, which are not normalized for the embryo
 size. Function
 <strong>getCellFates()</strong> accepts vector of Cell names
 and returns a named vector with all corresponding fates of down the road
 for each cell.  Function
 <strong>SaveNuclei()</strong> takes list of time-points (e.g.
 SaveNuclei(Nuclei$GZ01, "Projects/GZ01")) and saves them as an
 folder with StaryNite formated files, which can be opened it with the 3D
 viewer.   .tb_button
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 outset #def; background-color: #f8f8f8 !important;}.ws_toolbar
 {z-index:100000} .ws_toolbar .ws_tb_btn {cursor:pointer;border:1px solid
 #555;padding:3px} .tb_highlight{background-color:yellow} .tb_hide
 {visibility:hidden} .ws_toolbar img {padding:2px;margin:0px}