1. Urbana, IL| MAY 22, 2009 Anatomical Localization BeeSpace 5 th Annual Workshop Institute for Genomic Biology University of Illinois at Urbana-Champaign

2. 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

4. 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)

5. 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”)

6. 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

7. Peptide processing

8. 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

9. 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

10. 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

11. Alignment Example: bee gene for pigment-dispersing factor (PDF) peptide all species: known peptides & algorithms predicted or experimentally determined amino acid sequences

12. Work flow preparation of sections curation completed probes tested, ready-to- go

13. 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 Progress Pending

14. 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

15. 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

16. 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

17. Acknowledgements NSF WFU UIUC Deacons beat the Tar Heels 92-89 January 11, 2009