Jclub 2007

Aleksandr Tkachenko

The article:

http://dx.doi.org/10.1371/journal.pgen.0030178

In Situ Hybridization (ISH)

● Expression of genes is manifested by the production of RNA transcripts within cells

● ISH uses a labeled complementary RNA strand to localize a specific RNA sequence in a portion of tissue

● As few as five mRNA molecules per cell can be detected

In Situ Hybridization (ISH)

● validation of regulatory networks● discovery of network components

Hence, we need:● automatic, high-throughput technology● tools to manipulate expression data for

individual genes (atlases)

Possible applications:

Genepaint.orgis a digital atlas of gene expression patterns

in the mouse

Features:

● Enables to retrieve expression patterns by gene ids, anatomical locations or sequence homology

● Provides high-resolution visualization

Genepaint.org

● Hierarchical controlled vocabulary of majority of anatomical structures (organs, tissues)

● Attributes:

– Type of expression (regional, scattered)

– Intensity (not detected, low, medium, strong)

Annotation of expression patterns

Genepaint.orgAnnotation of expression patterns

Genepaint.orgHierarchical Clustering

● Genes● Anatomical regions

Genepaint.orgHierarchical Clustering

Distance metric

● depends on both an expression intensity and a type of pattern

● satisfies criteria:

– genes that do not express in any of the same anatomical regions are maximally distant

– the distance metric is adaptive to the range of expression strengths for a gene.

– pattern is secondary to intensity

Genepaint.orgHierarchical Clustering

Results:

tissues that have similar embryonic origin and/or similar physiological functions group together

there are groups of genes with similar expression patterns – can form a part of a common signaling pathway.

Experiment

Clustering results:

7 meaningful groups of anatomical sections12 groups of genes

Organism: midgestation mouse embryo

Anatomical sections: ≈ 90

Genes: ≈1000

Experiment

Focus on group containing Pax6.

Hypothesis: if genes demonstrate similar behavior in this cluster, they could form a component of regulatory network. We'll try to identify genes regulated by Pax6.

Experiment

Verification:

1.Knockout of Pax6 caused a change in expression of 17 genes in neocortex

2.Electromobility shift assays identified 12 genes not previously known as targets of Pax6 regulation.

Conclusion

In situ hybridization when combined with appropriate data mining tools is an effective method for discovery and validation of regulatory network components