Current Biology, Volume 23
Variation in the Dorsal Gradient
Distribution Is a Source for Modified
Scaling of Germ Layers in Drosophila
Juan Sebastian Chahda, Rui Sousa-Neves, and Claudia Mieko Mizutani
J.S.C., R.S.-N., and C.M.M. designed the research, analyzed data, and wrote the manuscript.
J.S.C. and C.M.M. performed the experiments. R.S.-N. by isolated and established the Santa
Maria isogenic line for the hybrid experiments.
Figure S1 (related to Figures 1 and 4). Graphs of Dl quantification obtained from individual
embryos and normalization method employed.
Figure S2 (related to Figure 2). Activation of twi expression in hybrid embryos and conservation
in the expression domains of columnar neural identity genes in the Drosophila species.
Figure S3 (related to Figure 3). Quantification of nuclear packing density.
Figure S4 (related to Experimental Procedures). Method used for scoring triploid embryos.
Supplemental Experimental Procedures. Explanation about scoring of genotypes,
quantification methods and transformation of Dl graphs.
Normalization of Dl Gradient Allows Cross-Species Comparison
Antibodies raised against several D. melanogaster proteins have been widely used with success
in other species to visualize expression patterns as well as to quantify morphogenetic gradients
(e.g. Bcd gradient in ). Our quantification method accurately extracts species-specific Dl
gradient shapes by normalizing raw fluorescent data obtained from antibody stainings (see
methods) to compensate for experimental variation and possible differences in antibody affinities
across species. It is important to emphasize that even if different affinities for the antibody exist
in other species (which formally it is not known), because the kinetics of antibody-protein
binding relies on constants of association (Ka) and dissociation (Kd), the readings along the
entire curve would increase or decrease after normalization is made (see figure S1). To
normalize Dl levels in the 30 most ventral nuclei, the data point (nuclei; i) with the lowest
fluorescent intensity was subtracted from each data point. Next, each data point was divided by
the sum of all the data points (
percentage all fluorescent values obtained. Therefore, even if differences in antibody kinetics
exist across species, the shape of the Dl gradient can be assessed, because raw fluorescent values
will be equally increased or reduced at every data point, but an individual data point’s value in
terms percentage of all fluorescence obtained is unchanged (Figure S1A). Figure S1B shows the
average shape of the Dl gradient in different species (Figures 1F-I) and in D. melanogaster
mutants (Figures 4E-F) obtained from individual embryos.
To test if sna and twi are activated by same Dl threshold levels across species, we
detected nascent transcripts at the border between the mesoderm and neuroectoderm of D.
melanogaster/D. simulans hybrids. The in situ displayed in Figure 2A-F shows that the lowest Dl
levels required to activate sna expression in both D. melanogaster and D. simulans are identical.
Figures S2A and B show that the threshold level of Dl needed to activate twi expression is also
the same across these species.
With the shift of the mesodermal boundary in these species (as well as in D. busckii),
there is a corresponding shit in the dorsal most border of the neuroectoderm, since the
neuroectodermal domain contains similar numbers of sog expressing cells in the species D.
melanogaster, D. busckii, D. simulans, D. sechellia (. Figures S2C-F show that the columnar
neuroectodermal genes maintain same domain and borders within the neuroectoderm.
In order to investigate a possible cause of Dl gradient modification within species, we
analyzed the size and packing density of blastoderm nuclei using anti-Laminin staining. Figure 3
shows that the diameter of blastoderm nuclei is different among D. busckii, D. melanogaster, D.
simulans and D. sechellia. To quantify the apparent differences in nuclear packing density, we
used image analysis tools to extract the areas between nuclei and calculated the number of pixels
of these areas (Figure S4).
??? ). After normalization, each data point represents a
Figure S1, Related to Figures 1 and 4.
(A) Hypothetical normalization from two species with exact Dorsal gradient shapes but different
(B) Normalized intensity levels of nuclear Dl protein (y-axis) per individual nucleus (x-axis)
obtained from individual embryos. Graphs are centered on the ventral midline (x=15) based on
sna expression domain, and extend dorsally from the center to the left (x=0) and right (x=30).
D. busckii embryos (n=5), D. melanogaster (n=12), D. simulans (n=10), D. sechellia (n=12), D.
melanogaster ssm haploids (n=9) and gyn-2; gyn-3 triploids (n=8).
Figure S2, Related to Figure 2.
(A and B) Sensitivity of twi activation is identical between D. melanogaster and D. simulans.
Ventral view of whole mount blastoderm embryos stained for twi mRNA (red) and DAPI nuclear
(A) Hybrid embryo from D. melanogaster mother and D. simulans father.
(B) Hybrid embryo from D. simulans mother and D. melanogaster father. Note that there are two
nuclear transcription dots present in all cells along the border of twi expression, indicating that
both copies of the twi gene from each species are activated at same Dl levels. The abutting cells
outside the mesoderm have both twi copies turned off.
(C–F) Domains of columnar neural identity genes are conserved in D. busckii, D. simulans, D.
melanogaster, D. sechellia, with similar numbers of cells within the vnd (green), ind (red) and
msh (white) domains. For D. busckii (C) we were able to determine the size of vnd and ind
domains by doing a double staining with an antibody anti-Ind (red), and an in situ for sog
(green). At the stage when ind expression is observed, sog mRNA levels demarcates the ventral
border of the neuroectoderm, but it is decreased from the dorsal most part of the neuroectoderm
Figure S3, Related to Figure 3. Quantification of Differences in Nuclear Packing Density
(A) Confocal images of anti-Laminin staining from a single plane at the center of nuclei in
different Drosophila species, as indicated.
(B) Image segmentation from (A) with colored space between nuclei shown in black, and space
occupied by nuclei shown in white.
(C) Histogram of pixel numbers from white areas corresponding to the nuclei for each species.
The higher the pixel value (y-axis), the greater is the nuclear packing density.
Figure S4, Related to Experimental Procedures. Genotyping of gyn Triploids by Counting
the Number of sna Nuclear Transcripts
The identification of gyn triploids was verified by the presence of three nuclear dots in embryos
stained for the autosomal gene sna mRNA, instead of two copies in wild-type.
(A) Cross-section from a diploid gyn. Arrow indicates a nucleus with two nuclear dots on the
(B) Maximum confocal projection showing surface view of embryo from which (A) was cut
from; note the presence of two nuclear dots in all nuclei shown.
(C) Cross-section from a triploid gyn. Arrow indicates a nucleus with three nuclear dots.
(D) Embryo from which (C) was cut from; note the presence of three nuclear dots in a maximum
Supplemental Experimental Procedures
Scoring of Hybrid Crosses and Embryos with Altered Ploidy
The hybrid progeny was confirmed by scoring classical unisexual progeny. When D.
melanogaster are used as mothers, only females hatch in the F1 and those have defects in
dorsoventral bristles and are sterile. The F1 of the reverse cross is composed exclusively by
sterile males, which have genital arcs of intermediate size between D. melanogaster and D.
simulans or D. sechellia. Over 200 adult hybrids were recovered, sexed and confirmed to be
infertile (atrophied testis and ovary).
Haploid embryos were generated by mating homozygous females w, ssm (a gift from
James Erickson) [4,5] to wild-type males. 100% of the progeny of this cross develop as haploids.
Triploid embryos were generated by mating gynogenetic-2; gynogenetic-3 (gyn) double
homozygous females to wild-type males (stock obtained from Bloomington Stock Center).
About 12% of the embryos derived from this cross develop as triploids. To maintain the 12%
triploidy rate, gyn2; gyn3 females were mated to sterile males (ms(3)K81) and F1 females that
developed parthenogetically  were used to generate triploid embryos, as described above.
Haploid and triploid embryos were identified by their nuclear density and size at the onset of
cellularization, and the number of nuclear nascent transcripts for autosomal genes (e.g. sna) as
one copy in haploids, two copies in diploids and three copies in triploids (See Figure S6, below).
Measurements of Egg Size, Nuclear Numbers, Size, and Packing Densities
For measuring the anteroposterior axis, eggs with chorion were mounted in 70% glycerol/PBS
with a small amount of glass beads (150-210 µm size, Polysciences) to prevent flattening caused
by coverslip. Eggs were imaged on a Confocal microscope (Zeiss LSM700) at an approximate
mid-section focal plane. Zeiss AxioVision 4.8 software was used to obtain distances from
anterior to posterior poles of eggs. For DV measurement of D. simulans and D. melanogaster,
additional intact embryos of were analyzed using upright imaging in glycerin jelly  after a
treatment in bleach to remove the chorion, 15 min fixation, de-vitellinization in
heptane/methanol, post-fixation for 15 min and staining with DAPI. Confocal images were
obtained for 10 D. melanogaster and 15 D. simulans embryos at the focal plane with largest DV
perimeter, and analyzed using Image J. These additional measurements confirmed that these two
species have statistically identical DV diameters.
Method for Dl Gradient Graph Transformation Based on Data from Hybrids
After normalization of Dl levels was performed as described above, we were able to transform
graphs representing Dl gradient shape to directly compare relative Dl levels. This transformation
was possible after determining that the threshold levels of Dl required to activate transcription of
snail or twist in D. melanogaster, D. simulans and D. sechellia is identical (Figure 2 in paper).
We arbitrarily set a value of “one” to the data point representing the last nucleus to express
snail/twist. These Dl threshold levels correspond to the dorsal mesodermal border, or the last
cells with lowest amount of Dl that still express snail or twist. Next, we corrected the gradient of
each species by multiplying every point on the curve by a factor X, where X=1/Y, and Y equals
to the normalized fluorescent value of the last nucleus, on average, expressing snail/twist. For
example, if in the D. melanogaster gradient the fluorescent intensity of the most ventral nucleus
is 3x more intense than the sixth nucleus located on the dorsal mesodermal border, then after the
transformation, the sixth nucleus will have a value of 1 and the most ventral nucleus will have a
value of 3.
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