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).