Transcript No Slide Title

BeeSpace
Biology Overview
BeeSpace Workshop
IGB, UIUC • 21 May 2006
BeeSpace Goals
Analyze the relative contributions of
Nature and Nurture in
Societal Roles in Honey Bees
Experimentally measure brain gene expression for
important societal roles during normal behavior
varying heredity (nature) and environment (nurture)
Interactively annotate gene functions for important gene
clusters using concept navigation across biological
literature representing community knowledge
Overview
Understanding Social Behavior
• Honey bees have only 1 million neurons
Yet…
• Worker bee exhibits social behavior
• She forages when she is not hungry
but the Hive is
• She fights when she is not threatened
but the Hive is
Biology: The Model Organism
Western Honey Bee, Apis mellifera has become a
primary model for social behavior
Complex social behavior in controllable “urban”
environment
• Normal Behavior – honey bees live in the wild
• Controllable Environment – hives can be modified
Small size manageable with current genomic
technology
• Capture bees on-the-fly during normal behavior
• Record gene expression for whole-brain or brainregion
Biological Foundations of BeeSpace
• There is a robust relationship between brain
gene expression & social behavior
• Gene expression is the “first phenotype” and
can be used to understand nature/nurture
Nature and Nurture
both act on the genome
Heredity
Environment
(Whitfield et al, 2002)
Principal Societal Roles
in the Honey Bee Colony
Home
Comb build, Remove corpses, Hygienic behavior
(remove diseased brood)
Offspring
Brood care, Attend queen, Personal reproduction
(worker)
Defense
Guard, Soldier
Food
Forage for nectar, Forage for pollen, Forage for water
Forage for resin, Scout, Process food (nectar to honey)
Dance communication: sender, Dance communication:
receiver
Nature/Nurture Dissection I
Defense
Roles: Guard and Soldier (Hunt, Hoffmann, Alaux)
Nature: Types of Bees (European, African)
Nurture: Level of Threat (Alarm pheromone; Alaux)
Nature/Nurture Dissection II
Role: Forager (onset age of foraging)
Hereditary factors:
European and Africanized Bees (Hunt, Hoffmann)
ligustica and mellifera (Leconte, )
High/Low Pollen Hoarding Lines (Page, )
Nature/Nurture Dissection III
Role: forager (onset age of foraging)
Social factors
Precocious vs Normal Forager
(Newman, Zhang, Ament)
Normal vs Overage Nurse
Typical vs Single-cohort colony
Males (?)
Queens (Corona, Hughes)
Nature/Nurture Dissection IV
Role: Forager (onset age of foraging)
Physiological Manipulations affecting:
cGMP, Manganese, Insulin, Vitellogenin
Juvenile Hormone
TOFA (Fatty Acid inhibitor)
(Maleszka, Ament)
Brood Pheromone, Queen Pheromone (Alaux)
Nature/Nurture Dissection V
Dissection of Dance Communication
Species differences, Pts. 1 & 2 (Sarma)
“Distance genes” (Sarma)
“Direction genes” (Brockmann)
Time training (Moore and Naeger)
Scouts (Liang)
Experimental Status
• Genome Complete and Microarray Fabricated
(one + year late)
• Bees collected for Experiments
– 3/4 last summer; 1/4 this summer
• Experiments complete with EST array
• Experiments in progress with full array
• Initial Use of Interactive Annotation on-going
• Planning Meta-level Functional Analysis
Goals of Functional Analysis
• Identify genes regulated by heredity and
environment… so what?
• Discover candidate genes (gene clusters,
molecular pathways) for behavioral
regulation… set up for causal experiments
– Kr-h1 (Grozinger lab)
– In situ analysis project (Fahrbach lab)
Examplar: Regulation of Brain Kr-h1
Expression by Queen pheromone
Christina Grozinger,
NC State
Regulates worker behavior:
Retinue response
Delay nurse to forager transition**
Inhibit queen rearing
Inhibit ovary development
Worker responses to queen
pheromone
In young bees:
Retinue response (Slessor et al, Science 1988)
Alter brain gene expression patterns (Grozinger et al PNAS 2003)
In forager bees:
No retinue/attraction
Still have antennal responses (Pham-Delegue et al 1993)
How is response modulated??
Do foragers respond to QP at all?
• Look at brain gene expression
– Kr-h1: downregulated by QP in the brains of
young bees
• Place 30 foragers and 30 day-olds in a cage
with a queen for 3 days
• JH levels much higher in
foragers than young bees
• Treat young bees with
methoprene (JH analog)
• Methoprene reduces QP’s
effects on gene
expression
Kr-h1 relative expression levels
Does JH modulate response?
Grozinger and Robinson JCPA, 2007
• But Met-treated bees still
attracted to QP in retinue
Grozinger et al,
Naturwissenschaften,
2007
What does Kr-h1 do?
• Downregulated by QP (2x) (Grozinger et al 2003)
• High in foragers compared to nurses (4x)
(Grozinger and Robinson 2007)
• QP regulates transition to foraging… so Kr-h1 involved
in foraging?
• What aspect of foraging? (Fussnecker and Grozinger in prep)
– Preparation for flight
– Neuroanatomical changes
– Phototaxis
Function of Kr-h1?
But what does Kr-h1 actually DO???
Study effects on neuron structure in Drosophila
(Tzumin Lee, UMass)
Use MARCM to study single neurons
WT
LOF
GOF
Shi, Lin, Grinberg, Grozinger, Robinson, Lee. Dev Neurobio. in press
Associated with permanent brain
changes?
• MB expand in foragers
• When foragers revert to
nursing behavior, MB stay the
same size (Fahrbach et al
2003)
• Is Kr-h1 associated with flight
behavior, or “permanent”
brain changes?
• Kr-h1 expression correlates
with permanent change in
brain
(Fussnecker and Grozinger, in prep)
Effect of cGMP on Kr-h1
expression?
Background: cGMP treatment causes premature onset of positive
phototaxis (Ben-Shahar et al 2003)
(Fussnecker and Grozinger, in prep)
Regulated directly/indirectly by
cGMP?
• cGMP response element
(Hum et al 2004)
Consensus
Apis
Drosophila
Aedes
Tribolium
AaAtRKaNTTCaAcAKTY
AAATAGTCTTCCAAAGTA
AAACATTCTTCAAAATTC
AAATGTTTTTCCAAATTG
AAATATTTTTCTAAATTT
N = ATCG; R = A, G; K = G/T; Y = T/C
(Fussnecker and Grozinger, in prep)
Comparative studies… Bumble bees
Honey bee
• Kr-h1 associated with foraging…
– QP regulates transition from nursing to
foraging
– Foragers less responsive to QP
• Kr-h1 & foraging or Kr-h1 & QP?
Bumble bee
• Nurse vs. forager
– determined by size
• Queen presence regulates
dominance status (reproduction)
• In collaboration with Guy Bloch…
Z. Huang
Comparative studies… Bumble bees
• Partially sequenced
Bt_Kr-h1 and Bt_PKG
– the foraging gene;
increased expression
in honey bee forager
brain
(Fan, Patch, Bloch and Grozinger,
prelim results)
Associated with flight?
Z. Huang, MSU
• Drones become
competent to take mating
flights when they are
approx 5-7 days old
• Compare drones of
different ages, with and
without flight experience
• Kr-h1 expression matches
likelihood of flight; not
affected by experience
(Fussnecker and Grozinger, in prep)
Cellular localization of gene expression
using in situ hybridization
•
•
•
•
information in the brain flows
through chains of connected
neurons (circuits)
different populations of neurons
express distinctive
complements of proteins (gene
products)
in situ hybridization localizes
mRNAs encoding specific
proteins to individual neurons
this allows gene expression to
be studied in the context of the
neural circuits that produce
behavior
BeeSpace in situ hybridization projects
Fahrbach Lab
• Goal: create “brain maps” for genes identified in microarray
studies as associated with bee behavior using in situ
hybridization
– leverages more than a century of neuroanatomical studies BUT
– requires develop of efficient approaches
– current bottlenecks: frozen sections (10 μm), color reaction to
reveal digoxigenin-labeled probes
– proposed solutions: Vibratome sections (100 μm), fluorescent
probes
• Test set: 36 neuropeptide-encoding genes identified in the bee
genome by a UIUC team using bioinformatics and proteomics
(Hummon et al. 2006 Science paper)
– set is “right size” for a test of new methods
– peptides are expressed abundantly in very small populations of
neurons (often fewer than 10, out of approximately 1 million total
neurons in the bee brain), so identification is unambiguous
– data produced will be of interest to insect neurobiologists
independent of methods advances
Progress report
• Rodrigo Velarde (Ph.D.,
Entomology, UIUC, May
2007) has recently joined
Fahrbach laboratory at Wake
Forest University
• Velarde’s previous
experience in the Robinson
laboratory included use of in
situ hybridization to map the
feeding-related neuropeptide
NPF
• as a postdoctoral researcher,
Velarde will coordinate all
BeeSpace in situ
hybridization projects
Goals of Functional Analysis
• Array studies as papers?
• Identify genes regulated by heredity and
environment… so what?
Ghostbusters Paradigm (descriptive, then
analytical)
MAPK (Whitfield lab, Jason Ebaugh)
• Discover candidate genes (gene clusters,
gene pathways) for behavioral regulation…
set up for causal experiments
Kr-h1 (Grozinger lab)