of 11
Supplemental m
ethods
Plasmid construction and injection
We have designed NNT as follows: NLS sequence (PK
KKRKV)
(Kalderon et al., 1984)
was linked together 3 times with aspartic acid to create 3xNLS [as described before by
(Chertkova et al., 2020)
]. T he 3xNLS was fused with a 6D or 6G linker to codon optimized
GFP nanobody
(Bothma et al., 2018). The 3xNLS-
6D-GFP nanobody (NNT) sequence
was ordered from IDT as gBlocks and inserted into pUASt-
attB (5xUAS) vector, a gift from
Konrad Basler (Bischof et al., 2007), using EcoRI and Xbal cutting sites. NNT constructs
were injected by Rainbow Transgenic Flies, Inc. using site specific injection into the
second (6D version, R8622) and third (6D and 6G versions, attp40w) chromosomes.
Image processing and data analysis
Intensity over time curve was calculated in the following way. Images were analyzed using
ImageJ. Z projections of the 20 Z-stacks of the green channel were created using the sum
slices function. Images were rotated by marking the midline and rotating into a horizontal
position with the head pointing to the left. Background was subtracted using the rolling
ball function with 50 μm radius. A rectangle was selected and placed close to the midline
while avoiding yolk signal (120x60 μm for 2xPE-
Gal4, and 40x140 μm for Kr
-Gal4) to
measure and plot the mean intensity value versus time curve using the Plot Z-axis profile
command. For Kr-
Gal4, we pla
ced the rectangle closer to the posterior side where the
signal was still detectable at the end of the movie. The resulting intensity/time curves were
shifted in time, such that the 0 time point corresponds to furrow closure (determined based
on the bright
field and green channel images) and 0 intensity corresponds to minimal
intensity detected. We averaged data from three embryos. To determine the difference in
timing of signal appearance we defined a 150 A.U. as minimum intensity (a threshold
Development: doi:10.1242/dev.199822: Supplementary information
Development • Supplementary information
when by eye we could see signal) and calculated intersection of the average intensity
curves and the 150 A.U. line. Signal was starting at time (average ± SD, n=3 embryos)
relative to furrow closure (t=0), for 2xPE-Gal4 driver, 2xPE_NNT = -
18 ± 2.2 min,
2xPE_NLS_GFP =
14 ± 5.5 min and for Kr
-Gal4 driver, Kr_NNT= -
11 ± 2.3 min,
Kr_NLS_GFP = 4 ± 3.9 min). Similarly we have calculated the intersection point of the
two average curves (NNT and NLS_GFP) to determine the time when they were reaching
the same intensity level (average curves were intersect at 42 min and at 48 min after
furrow closure (t=0) respectively for 2xPE and Kr-Gal4). Intersections were calculated
using Python and data were plotted in Gnuplot.
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Development • Supplementary information
Fig. S1. Individual intensity curves for figure: NaNuTrap driven fluorescent
signal precedes NLS-G
FP signal (Fig. 2D,
G)
. (
A, B
) 2xPE-Gal4 (A) and Kr (B) drivers
were used to label mesoderm and ectodermal
cells respectively by driving the
expression of the NNT construct (blue) or an NLS-GFP construct (green), see also Fig.
2. Individual intensity curves for embryos (open marks) and mean intensity of GFP
signal shown (closed marks) over time. NNT-driven signal intensity shown in blue, NLS-
GFP shown in green (n=3 embryos, error bars s.e.m.). All curves were shifted in time
such that t=0 time point corresponds to furrow closure.
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Development • Supplementary information
Fig. S2. Labelling
cell nuclei
using
NaNuTrap
with Gal4
drivers
(A,B,C
)
Snapshots from live movie
s of embryo
s expressing NNT using the PrD-
Gal4 (
A),
Hairy-
Gal4
(B) and En -Gal4 driver
s (C).
Image
s show representative time point
s of the
movie
s starting when the signal was first detected. EGFP was deposited maternally.
Embryo
s were imaged from the ventral (A) or lateral (B,C) side, with anterior to the left.
Note that the autofluorescent signal from the yol
k is yellow (B,C) and can be separated
from the GFP signal shown in green (see Methods). Arrow
s indicate cell nuclei in frames
associated with the initial signal present only in a few cells. Time i
s shown in hh:mm
format.
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Development • Supplementary information
Movie 1. Early cell nuclei labelling using
NNT construct and 2xPE
-Gal4 driver.
2xPE-
Gal4 is driving the expression of the NNT construct with EGFP deposited maternally (left), or
the NLS-GFP construct (right) to label the mesoderm. Time-lapse movies start during
cellularization at stage 5 (25 min before furrow closure). In this and subsequent movies, the
embryo was imaged from the ventral side, with anterior to the left.
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Movie 2.
Early cell nuclei labelling using NNT construct and Kr
-Gal4 driver.
Kr- Gal4
is driving the expression of the NNT construct with EGFP deposited maternally (left), or
the NLS
-GFP construct (right). Time-lapse movies start during cellularization at stage 5
(i.e. 40 min before furrow closure).
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Movie 3. Early expression with the help of NNT using the existing TTG balancer
line.
Twi -Gal4 of the TTG (Twi
-Gal4, 2xEGFP) balancer line is driving the expression of
the NNT construct with EGFP deposited maternally (left). Right image shows a time-
lapse
movie of the TTG balancer line. Time-lapse movies start during cellularization at sta
ge 5
(i.e. 40 min before furrow closure).
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Movie 4.
Cell nuclei labelling using NNT construct and En
-Gal4 driver
. En - Gal4 is
driving the expression of the NNT construct with EGFP deposited maternally. Movie starts
at stage 8/9.
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Movie 5.
Early cell nuclei
labelling (using maternal expressed GFP) combined with
late cell nuclei labelling (using zygotically expressed NLS-
GFP).
2xPE-Gal4 is driving
the expression of the NNT construct (with EGFP deposited maternally) together with NLS-
GFP construct to label the mesoderm (using the 5xUAS::NNT, UAS::NLS-GFP fly line).
Time -lapse movie starts during cellularization at stage 5 (i.e. 40 min before furrow
closure).
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Development • Supplementary information
Movie 6. Automatic tracking of cells in the early
Drosophila
embryo in which nuclei
are labelled with NaNuTrap method.
Cells in Movie 2 (i.e., Kr-Gal4 driving expression
of NNT in presence of EGFP deposited maternally) tracked automatically without manual
correction using the Imaris software tracking function. Spot detection and tracking can be
initiated ea
rlier compared to conventional cell labelling methods as cell nuclei are labelled
more effectively using NNT (e.g. see Movie 2). Color bar indicates average speed of the
tracks (0-
4.5 μm/min).
Development: doi:10.1242/dev.199822: Supplementary information
Development • Supplementary information
Movie 7. Loss of NNT driven signal is an indicator of cell div
ision.
Movie shows
dividing mesoderm cells labelled with NNT (green) while histone is labelled in all cells
(red, His2Av-mRFP1). To label the mesoderm, we collected embryos from the cross of
His2Av
-
mRFP1; NNT, Vasa::EGFP x Twi-
Gal4.
Development: doi:10.1242/dev.199822: Supplementary information
Development • Supplementary information