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.

3D time-lapse recordings of embryos expressing mCherry∷H2B in either wild-type or lin‑5(ev571) 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 https://github.com/UPGON/worm-rules-eLife. 

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.

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• 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 

Embs  - 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 (ev571 is the allele of lin-5 that we use to make embryos "equalized")

"Experiment" with following values:

meta - this means that embryo was upshifted during metaphase and should be equalized.
ctrls - experiments of two kinds: ev571 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 ev571 (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

Nuclei

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" 
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
1     1   -1             1           -1 .      175  78 16.0    66          ABp 
2     1   -1             2           -1        324 213 18.5   59          EMS
3     1   -1             4           -1        432 129 18.5   58          P2
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.
If a cell divides it will have some ID value in SUCC2 

Timing

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 getCellFates() accepts vector of Cell names and returns a named vector with all corresponding fates of down the road for each cell. 

Function SaveNuclei() 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.

 

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