Urbana, IL| MAY 22, 2009

Anatomical Localization

BeeSpace 5th Annual Workshop

Institute for Genomic Biology

University of Illinois at Urbana-Champaign

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

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”)

What is a peptide?

DNA → RNA → proteinDNA → 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 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 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 start, stop, intron/exon

boundaries, splice sites

phylogenetic analysis to

identify true orthologues

confirming that primers generate

product of expected size

sequencing PCR products

Alignment

Example: bee gene for pigment-dispersing factor (PDF) peptide

all species: known peptides

& algorithms

predicted or experimentally determined amino acid

sequences

Work flow

preparation of sections

curation completedprobes tested,

ready-to-go

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

In ProgressPending

Examples of in situ results

Localization of PDF precursor in the bee brain using a DIG-labeled probe and enzymatic

color reaction

Localization of PDF precursor in the bee brain using a FITC-labeled probe and enzymatic

color reaction

Examples of in situ results

mRNA for sNPF

precursor (red)

Alexa 546

FITC probe for vitellogenin protein in fat body (green)

DIG probe for vitellogenin protein in fat body (purple)

brain

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

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