Transcript Document

A morphogenic framework for analyzing gene expression in Drosophila melanogaster blastoderms.
Soile V.E. Keränen1, Cris L. Luengo Hendriks1, Charless Fowlkes2, Gunther H. Weber3, Oliver Rübel4, Min-Yu Huang3, Clara N. Henriquez1,
Hanchuna Peng, Lisa Simirenko1, Damir Sudar1, Bernd Hamann3, Jitendra Malik2, Michael Eisen1, Mark D. Biggin1, David W. Knowles1, Berkeley
Drosophila Transcription Network Project.
1) Lawrence Berkeley National Laboratory, Berkeley, CA. 2) Department of Electrical Engineering and Computer Science, UC Berkeley, Berkeley, CA.
3) Institute for Data Analysis and Visualization (IDAV), UC Davis, Davis, CA 4) University of Kaiserslautern, Kaiserslautern, Germany
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ABSTRACT
To fully understand and be able to computationally model
the spatial complexity of developmental regulatory
networks, it is critical to measure gene expression patterns
at the resolution of individual cells. To this end, we have
developed image analysis techniques for extracting 3D
embryo morphology and quantifying gene expression at
cellular resolution in Drosophila melanogaster blastoderm
embryos (see also Luengo et al., Fowlkes et al., Knowles et
al.). Using these methods, we have discovered that well
before gastrulation there are complex a/p and d/v nuclear
density changes around the blastoderm. These changing
densities correspond to nuclear movements whose
directions and magnitudes we have estimated using both
time-lapse images of live, histone2A-GFP embryos and
pointcloud data from fixed embryos. Because these nuclear
movements are larger that the diameter of a cell or the x,y,z
separation between different genes’ expression patterns in
the embryo, our data indicate that blastoderm pattern
formation needs to be analyzed in a morphodynamic, rather
than a morphostatic environment. As an example, we
describe the movement of pair-rule and gap gene stripes
during stage 5 and compare these to the predicted nuclear
flow.
Live cell data shows that the local density differences
are real and result from pregastrula nuclear movements
The xyz-positions of the blastoderm nuclei and the expression intensity of two genes around each
nucleus are extracted from a confocal image (A) using an algorithm (B) and converted into computer
readable data table (C). This data can be transformed into other forms, like an unrolled view of ~6000
blastoderm nuclei showing normalized gene expression levels (D).
Sytox image
D
V
V
D
D
40
60
80
% egg length
cells
Corresponding unrolled
view showing eve and
sna expression
eve (green), sna (red), isodensity (white)
DNA
cytoplasm
atten. corr.
A
Cy3 image
B
C
id,
1,
2,
3,
4,
5,
6,
x,
102.36,
264.63,
225.91,
318.42,
110.18,
340.48,
y,
142.14,
172.01,
174.99,
48.34,
34.40,
73.79,
z,
Nx,
Ny,
Nz,
112.00,-0.396, 0.851, 0.344,
79.36, 0.103, 0.972,-0.208,
88.65,-0.030, 0.999,-0.015,
138.91, 0.095,-0.744, 0.660,
109.65,-0.186,-0.913, 0.362,
37.548, 0.205,-0.299,-0.931,
. . .
density map
pointcloud
file
Cou image
Vn,
207.96,
281.73,
185.79,
182.46,
127.81,
208.26,
Vc,
605.36,
599.90,
418.35,
464.19,
432.01,
607.49,
Sytox,
52.18,
82.12,
85.32,
37.61,
55.78,
80.23,
Cy3_n,
23.55,
31.67,
35.63,
19.31,
24.12,
33.04,
Cy3_a,
18.76,
34.97,
31.27,
15.15,
23.53,
26.75,
Cy3_b,
22.55,
15.95,
14.77,
12.47,
12.19,
21.24,
Cy3_g,
22.10,
31.93,
34.00,
17.55,
19.71,
28.91,
Cou_n,
11.95,
21.06,
19.59,
21.01,
13.81,
31.48,
Cou_a,
8.13,
12.56,
20.53,
13.78,
7.57,
20.69,
Cou_b,
28.01,
41.40,
38.80,
26.87,
28.16,
50.45,
Density maps
Cou_ g
12.04
19.12
21.35
17.53
12.40
26.96
Unrolling the embryo
Density changes through time
D
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Explaining the complex expression movement in blastoderm embryos requires models that include morphogenic interactions
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As known, the expression borders of gap genes gt, hb and kr shift anteriorly (1). Also the expression borders of the posterior-middle stripes of pair-rule genes shift anteriorly,
anterior stripes posteriorly. However, dorsal parts of the stripes often move differently from ventral parts of the stripes. Computing the actual difference of this shift requires both
accurate 3D shapes of embryos, and further modeling that includes the cell movements and density changes to determine what fraction of observed pattern movement is caused
by differential induction and what fraction follows from the cell flow. Below are some initial predictions on cell movement and its role in shift of selecte eve, ftz, gt, hb and kr pattern
elements (indicated by while arrows on black embryos).
late
D
20
nuclei
profiles of each individual cell, these changes need to
be included in the spatio-temporal analysis of
expression patterns and gene regulation. When we
divided the embryos in different age cohorts to measure
the expression shifts, we found that also the local
nuclear densities change.
3
normals
Live embryo density maps
0
markers
Computational analysis shows nuclear density changes
in stage 5 Drosophila melanogaster blastoderm through
time
If we want to accurately map the temporal expression
surface shell
The maps below are composites of 23 time-lapse images of GFP-H2A embryos at
different dorso-ventral orientations.
early
2
From confocal images we can get computer analyzable, cellular
resolution spatial expression data
early border position:
late border position:
100
0
Dorsal
20
40
60
80
% egg length
border position projected
from cell movement:
Pattern shift
100
Dorso-lateral
Lateral
Nuclear flow field maps
Arrows indicate direction and distance
of the projected movement.
Ventral
Ventro-lateral
a/p and d/v patterning mutants have altered nuclear density patterns
Changes in a/p and d/v patterning
wild
system can cause changes in
density patterns obserbved in an type
average wild type embryo.
In gd7 mutants the denser dorsal
patch in wild type embryos
extends ventrally and laterally.
a/p density is also disturbed.
In Toll10B mutants, the
development of denser patches in
general is disturbed.
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The d/v differences in ftz stripe positions is likely to depend
on d/v axial patterning of morphogenesis
gd7 has been classified as a fully
dorsalizing mutation. In gd7-mutants,
the dorsal sides of the stripes
resemble the wild type stripes, but the
ventral sides of the ftz-stripes draw
together like dorsal wild type ftzstripes (A).
A
Toll10B has been classified as a fully
ventralized phenotype. In Toll10B
mutant embryos, however, the late ftz
stripes similar to wild type ventrally but
not as close to each other in dorsal
side as in the late wild type embryos
(B).
Although the effects in Toll10B might result from disturbances in cell adhesion (2),
gd7 and bcd9 results suggest that correct 3D density patterns depend on cooperation between a/p and d/v signals.
Toll10B
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3D nuclear density patterns change rapidly during stage 5
in Drosophila blastoderm. This involves pre-gastrula
morphogenic movements of blastoderm nuclei.
Because a/p expression borders of different elements and
different genes shift differently in d/v axis, 3D analyses
of expression patterns are essential for modeling
regulatory interactions.
Toll10B
In bcd9 a/p patterning mutants the
posterior denser patch is
duplicated, but also dorsal denser
bcd9
patch is less developed.
Conclusions:
Cellular resolution expression analysis of Drosphila
blastoderm requires multiple morphological maps and
positional correspondence tables for each nucleus
between the maps of different stages.
gd7
It has long been known that dorsal
sides of ftz stripes are closer to each
other than ventral sides and that this
pattern is disturbed in d/v mutants (3).
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B
gd7
Because
and
mutants also
show aberrant density patterns (left),
the abnormal stripe movement is likely
to result, at least in part, from the d/v
regulation of morphogenetic
movements.
Comparison between mutant and wild type embryos shows
that d/v and a/p density patterns are connected, as are
d/v elements of a/p pattern changes.
Novel models are required to analyse:
1) which part of the expression change is caused by direct
regulatory interactions between cells
(induction/inhibition) and which part of the change is
due to the shifting cell positions
2) how the a/p and d/v patterning axes interact with each
other
References:
Any mutant analyses for disturbances in blastoderm pattern formation
need to also account for disturbances in morphogenic movement.
1)
2)
3)
Jaeger et al. (2004) Nature 40(6997):368-371.
Keith and Gay (1990) EMBO Journal 9(13):4299-4306
Carroll et al. (1987) Development 99(3):327-332.