Anatomical Localization

1
Anatomical Localization

• BeeSpace 5th Annual Workshop
• Institute for Genomic Biology
• University of Illinois at Urbana-Champaign

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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

3
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

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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,
  co-localization with protein, qRT-PCR, common
  sense)

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BeeSpace in situ project

• use sequence information from the Honey Bee
  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)

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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

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Peptide processing
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Identifying bee neuropeptides

• Hummon et al. (2006) From the genome to the
  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

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From sequence to circuit

• Honey Bee Genome Project publishes sequence, ID
  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

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Manual curation is labor-intensive
confirm gene predictions by
aligning predicted proteins with known orthologues
checking RNA for start, stop, intron/exon
boundaries, splice sites
phylogenetic analysis to identify true orthologues
confirming that primers generate product of
expected size
sequencing PCR products
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Alignment
Example bee gene for pigment-dispersing factor
(PDF) peptide
all species known peptides algorithms
predicted or experimentally determined amino acid
sequences
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Work flow
curation completed
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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
• localization studies with non-fluorescent probes
• studies with multiple labels using fluorescent
  probes
• literature review

Initiated
Pending
In Progress
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Examples of in situ results
Localization of PDF precursor in the bee brain
using a FITC-labeled probe and enzymatic color
reaction
Localization of PDF precursor in the bee brain
using a DIG-labeled probe and enzymatic color
reaction
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Examples of in situ results
mRNA for sNPF precursor (red)
Alexa 546
brain
FITC probe for vitellogenin protein in fat body
(green)
DIG probe for vitellogenin protein in fat body
(purple)
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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

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Acknowledgements

• NSF
• WFU
• UIUC

Deacons beat the Tar Heels 92-89 January 11, 2009