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Extraction and comparison of
gene expression patterns from
2D RNA in situ hybridization
images
BIOINFORMATICS
Gene expression
Vol. 26, no. 6, 2010, pages 761-769
Outline
•
•
•
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In situ hybridization
Introduction
Berkeley Drosophila in situ database
Approach
– Image registration
– Representation of expression patterns
– Correspondence of expression similarities to expert
annotation
– Expression similarity of known co-regulated genes
In situ hybridization (ISH)
• a type of hybridization that uses a labeled
complementary DNA or RNA strand (i.e.
probe) to localize a specific DNA or RNA
sequence in a portion or section of tissue,
if the tissue is small enough (e.g. plant
seeds, Drosophila embryos)
In situ hybridization
• In situ hybridization of wild type Drosophila embryos at
different developmental stages for the RNA from a gene
called hunchback
Introduction
• Recent advancements in high-throughput
imaging.
• Methods for capturing these spatial and/or
temporal expression patterns
– In situ hybridization
– Fluorescent reporter constructs or tags
• Are still frequently assessed by subjective
qualitative comparisons
Introduction
• A particular interesting and fundamental
problem - to compare two samples on the
level of their expression profiles
– For instance, the same gene under different
conditions or across different species
– different genes with the goal to cluster them
akin to approaches developed for microarray
data.
Introduction
• Several problems arise when comparing image
expression images:
– To process the raw input images
• to eliminate noise under a typical large range of imaging
condition (e.g. different viewpoints, different locations,
multiple specimens per image)
• to perform normalizations
– Represent the expression patterns and to specify
appropriate similarity metrics capable of assessing
similarity
– Assess the significance of observed similarities
Introduction
• Use a dataset of Drosophila embryonic
expression patterns
• There main contribution
– Robust and fully automated image analysis
techniques
– Comprehensively compare different similarity
metrics
– A new significance testing framework for
spatial similarity scores through constrained
realization Monte Carlo simulations
Berkeley Drosophila in situ
database
• 78621 images of 3724 genes expressed across
six time windows (covering the developmental
stages 1-3, 4-6, 7-8, 9-10, 11-12, 13-15).
• Focus on the subset of 27157 images covering
3127 genes acquired during the 4-6 stages
– Crucial information is not yet provided for later stages
– Are not subject to the additional complexity that
expression in later stages
Image registration
• Prior to any quantitative analysis, necessary to normalize and
register the images to a common frame
Image registration
• statistical shape models
– Signed distance maps to describe object
contours
• Negative distance - inside the object
• Positive distances – outside
• Magnitude - actual distance
Image registration
– Drosophila shape model was automatically
created from a manually curated set of 120
embryo images
• The contours of the embryo were manually
segmented and transformed into signed distance
map
• Automatically normalized in size
– Minimizing the distance of each individual signed
distance map to the mean signed distance map
• The resulting normalized maps were analyzed
using hierarchical principal component analysis
(PCA) decomposition
Image registration
• Model the filtered intensity values around
the contour the embryos
– Provide characteristic priors
– Bin the intensities observed in distances from
-25 to 25, while remaining bins are not
included.
Image registration
(A) Example image showing the creation of the training set
(B) A subset of 120 images used for the training of the shape model
(C) The training set is normalized in size
(D) Four of the principal shapes of the embryo, These images depict 2
standard deviations of the principal component from the mean of the signed
distance map.
Image registration
• The task of image registration – to find the
optimal set of parameters 
– Two categories:
• Rigid transformation parameters of the image r
• The principle shape components of our shape prior s
– These tow sets of parameters are simultaneously
optimized using an in-house implementation of a
particle swarm optimizer.
• This approach estimates both the average
shape of an embryo, as well as the main
components of variation of embryo shape
Representation of expression
patterns
• We are interested in comparing the global
2D expression pattern.
• After registration, the transformed image T
and shape mode S are used to calculate
column and row vectors of expression
data
Correspondence of expression
similarities to expert annotations
• To determine how the similarity values
computed by each metric corresponded to
manually annotated expression terms
• metrics
– Spatial metrics
• Haar wavelets (HWs)
• Spatial mutual information (SMI)
– Non-spatial metrics
• Mean squared error (MSE)
• Mutua information (MI)
Correspondence of expression
similarities to expert annotations
• For each scoring metric, we calculated an
enrichment significance for each
annotation term.
• Describe how often genes annotated with
a particular ontology term show the
strongest similarity to genes annotated
with the same term.
Correspondence of expression
similarities to expert annotations
P-value cutoff of 0.05, SMI
performed the best, with 22
of the 29 annotation terms
being significantly enriched.
Expression similarity of known coregulated genes
• We use SMI and significance tests to
validate known biological interactions,
suggesting their usefulness for inference
on biological data.
• Gene regulation and spatial patterning are
a tightly coupled process
– Transcription factor acting as activators for a
gene are often co-expressed in similar spatial
regions
Expression similarity of known coregulated genes
A, C => Nubbin and dichaete
bottom of (A,C) => The extracted expression vector
B,D => a set of random realizations with constraints on the correlation
between spatially adjacent expression values
E => distribution of similarity values
Expression similarity of known coregulated genes
Significance of the pairwise score :
Blue : >0.1
Green : (0.1, 0.05]
Yellow : (0.05, 0.01]
Red :<0.01
Expression similarity of known coregulated genes
• Example
– pdm2 and nubbin (also known as pdm1) share
function roles
– Ocelliless (also known as orthodenticle) is positively
regulated by bicoid
– Ubx indirectly regulated dichaete through the
intermediate activation of dpp
– Hunchback represses the expression of nubbin, pdm2
and Ubx
– Giant and Krueppel mutually repress each other
Expression similarity of known coregulated genes
• Not all known interactions are detected as
significant
– A spatial expression pattern of a gene is the
result of complex interaction of many genes
across several time stages