Biology Reference
In-Depth Information
allows lineages to be generated in a straightforward manner through most of embryo-
genesis. The image degradation in deeper DIC focal planes limits complete linea-
ging of single embryos from DIC. Although computer image analysis can identify
nuclei in DIC images (
Hamahashi et al., 2005
) the low contrast of DIC images makes
this computationally challenging.
2. Semiautomated Lineage Analysis
With the advent of histone-GFP reporters marking all embryonic nuclei
(
Ooi et al., 2006
), imaging and lineage analysis of embryo development could
now be studied using the laser confocal microscope. The high fluorescence contrast
of nuclear-localized GFP allows identification of nuclei by computer image seg-
mentation in a more reliable fashion than from DIC data. Although photobleaching
and phototoxicity limit the image quality, it is possible to image histone-GFP
throughout embryonic development. The histone-GFP itself is presumably synthe-
sized anew during each cell cycle, partly compensating for photobleaching effects.
The Waterston lab implemented the first histone::GFP imaging platform for follow-
ing embryo development through gastrulation and morphogenesis (
Murray et al.,
2006
). To track moving and dividing nuclei the Starrynite software was developed
(
Bao et al., 2006
). Additional visualization software allows curation of the tracking
data and extraction of lineage information (
Boyle et al., 2006
).
The accuracy of the original Starrynite software declined in embryos with
>
350 nuclei. To allow nuclear tracking in later embryos several approaches are
being tested. One approach is to optimize the segmentation algorithm for
images of optically sectioned nuclei (
Santella et al., 2010
). Another approach,
currently implemented in our laboratory (see below), is to curate nuclear iden-
tification at each time point, such that the automated tracking at the next time
point t+1 starts with corrected information. A second modification to the search
algorithm is to constrain the search for a particular nucleus (or its daughters) at
t+1 only in the local neighborhood of its previous position at t. Rather than
performing de novo segmentation, in this approach the maximum amount of
information available at
t
is used for performing the segmentation and tracking
for
t+1 (
Table III
).
3. Extent of Variation in the Wild-Type Lineage
The ability to completely track all nuclei in individual embryos has prompted
further examination of the degree of variability in wild-type development.
Automated lineage tracing has confirmed the high degree of invariance in the
assignment of cell fates, with the known exceptions of the midline cell pairs men-
tioned above. Some cell-division axes in the C lineage display variability. Cell
division times can vary by a factor of 10% between embryos (
Sulston et al.,
1983
), but within individual embryos the relative timing of cell divisions is highly
consistent. The high degree of correlation of cell cycles within an individual embryo
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