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Anatomical Localization
BeeSpace 5th Annual Workshop
Institute for Genomic Biology
University of Illinois at Urbana-Champaign
Urbana, IL| MAY 22, 2009
Localization of gene expression
• behavior (including social behavior) is the result of
activation or inhibition of neural circuits (circuits =
channels for information flow)
• gene expression regulates both short and long term
tendencies for circuits to be active
• a strong prediction in behavioral genomics is that
genes that influence specific behaviors will be
expressed by specific subsets of neurons
• we “layer” new information about patterns of gene
expression on top of pre-existing neural circuit
diagrams
In situ hybridization
DNA → RNA → protein
in situ hybridization is a method for localization of
specific mRNA sequences in preserved tissue
(sections, whole mounts, or dispersed cells) by
hybridizing a complementary nucleotide probe to the
RNA of interest
Requirements for in situ hybridization
• sequence of the target gene
• design of a probe specific for the target gene
• selection of a probe label and synthesis of the
labeled probe (microgram quantities)
• tissue in which the target gene is known to be
expressed
• pretreatment, hybridization, post-hybridization,
preparation of tissue for microscopy
• interpretation of images with reference to controls
(no probe, sense probe, multiple probes, colocalization with protein, qRT-PCR, common sense)
BeeSpace in situ project
• use sequence information from the Honey Bee
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Genome Project to create “whole genome” DNA
microarrays
use DNA microarrays to examine gene expression in
the brains of bees in different behavioral states
generate list of “behaviorally relevant” genes
use in situ hybridization to link gene expression to
neural circuits
minor delays in preparing high quality microarrays
led to adoption of a pre-existing list of genes
encoding the entire set of bee peptide precursors
(maintains focus on “from genome to neural circuit”)
What is a peptide?
DNA
RNA → protein
DNA → RNA
→→
pre-propeptide
→ peptide
• peptide: short (20 – 30) sequence of
amino acids
• pre-propeptide: peptides are encoded
in the genome as part of larger peptide
precursors; enzyme-mediated
cleavage creates functional
neuropeptides
• neuropeptide: peptide synthesized and
secreted by a neuron, acts as a
neurohormone or neuromodulator
Peptide processing
Identifying bee neuropeptides
• Hummon et al. (2006) From the genome to the
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proteome: uncovering peptides in the Apis brain.
Science 314: 647-649.
36 genes in the bee genome encode peptide
precursors
200 peptides predicted, existence of 100 confirmed
“right size” set of behaviorally relevant genes
why important? chemical analysis provides a
“snapshot” of what is present at one point in time;
localization of precursors reveals ALL possibilities
From sequence to circuit
• Honey Bee Genome Project publishes sequence, ID
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assignments based on gene prediction algorithms
MALDI-TOF MS, qRT-PCR confirmation of gene
expression in Hummon et al.
manual curation of peptide precursor sequence
design of primers to confirm gene identity
design of primers with promoters attached to
generate anti-sense and sense probes
choice of label for visualization
synthesis of labeled probe (in vitro transcription)
optimization of hybridization conditions
Manual curation is labor-intensive
confirm gene
predictions by
aligning predicted
proteins with
known
orthologues
checking RNA for
phylogenetic
start, stop,
analysis to
intron/exon
identify true
boundaries, splice
orthologues
sites
confirming that
primers generate
sequencing PCR
product of
products
expected size
Alignment
Example: bee gene for pigment-dispersing factor (PDF) peptide
all species:
known peptides
& algorithms
predicted or experimentally
determined amino acid
sequences
Work flow
probes
tested,
ready-togo
curation completed
preparation of sections
Current project status
• manual curation of honey bee peptide precursor genes
• primer design for honey bee peptide precursor genes
• primers tested for specificity (RNA gels) and efficacy
(qRT-PCR)
• optimization of protocol for non-fluorescent probe label
(digoxigenin, DIG)
• test of fluorescein (FITC) as a hapten for fluorescent
labeling with secondary antibodies
• Initiated
localization studies with non-fluorescent probes
Pending
• studies with multiple
labels using fluorescent probes
• literature review
In Progress
Examples of in situ results
Localization of PDF precursor
in the bee brain using a DIGlabeled probe and enzymatic
color reaction
Localization of PDF precursor
in the bee brain using a FITClabeled probe and enzymatic
color reaction
Examples of in situ results
mRNA for
sNPF
precursor
(red)
brain
Alexa 546
DIG probe for
vitellogenin protein in
fat body (purple)
FITC probe for
vitellogenin
protein in fat
body (green)
In situ products of BeeSpace
• complete manual curation of all peptide precursor
genes in the honey bee genome
• database of primers specific for each peptide
precursor
• maps of location of neuropeptide precursor gene
expression in the honey bee brain
• detailed protocols for production of in situ probes
• hapten-labeled for high efficiency
• fluorescent-labeled for co-localization of multiple
probes in a single tissue section using confocal
microscopy
Acknowledgements
NSF
WFU
UIUC
Deacons beat the Tar Heels 92-89
January 11, 2009