Genetic Analysis of Behavior. Goals and Assumptions Goal: Begin to dissect circuitry that controls...
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Transcript of Genetic Analysis of Behavior. Goals and Assumptions Goal: Begin to dissect circuitry that controls...
Genetic Analysis of Behavior
Goals and Assumptions
Goal: Begin to dissect circuitry that controls larval (and possibly) behavior
Assumptions: Larval neurons derived from single NB share
functional properties Can generate Gal4 lines expressed in a single (or
several) brain NB and progeny Can effectively eliminate neural function in single-
neuroblast neuronal progeny
Adult Brain
Adult brain Brain regions
Protocerebrum Deutocerebrum Tritocerebrum Optic lobes
Larval Brain and Development
Larval brain is derived from embryonic procephalic NBs 106 NBs/side Form at s8-11 in
stereotyped pattern Brain regions
Protocerebrum (A, C, P) Deutocerebrum Tritocerebrum
Stereotypic Formation of pNBs
pNB addition is continuous; no obvious waves
Stereotypic Formation of pNBs
Mapping (A) Dpn protein (blue) (B-H) svp-lacZ (brown)
and en protein (blue)
Proneural Gene Expression
Proneural genes expressed during NB formation similar to vnc NBs 78 pNB (74%) express proneural gene 28 pNBs (26%) don’t
Proneural expression L’sc: 64 pNBs Ac: 19 pNBs Sc: 18 pNBs Ato: 7 pNBs
Overlap Ac and Sc overlap in some pNBs but not
others (most don’t) Ac and Sc can also overlap with L’sc Ato overlaps with Sc in only 1 pNB
Molecular Map of pNBs
Mapped 34 genes onto pNB map Proneural Gap Pair-rule Segment polarity D/V Homeotic Early eye Glia Others
Each pNB has unique molecular identity
Assumption: some of these genes activate proneural gene expression in cell-type specific way
Larval Brain Organization
Neurons cortex Axons neuropile Compartments separated
by glia? Neuropile compartments
synaptic connections
NB neuron cluster axons with similar synaptic targets
Larval Brain Neuron Clusters
pNB neurons axon bundle
Larval Brain Neuron Cluster
NB GMCs Neurons
Larval Brain Axon Compartments
Microcircuit (neuron cluster) axon bundle Macrocircuit (multiple neuronal clusters) join together
via projection neurons to form a macrocircuit
Summary
Each pNB is unique Most pNBs express proneural genes Each pNB gives rise to a discrete cluster of brain cells that send axons
to similar synaptic targets Confirmation by single cell MARCM?
Do Neuronal Clusters Control Similar Behavioral Functions
Don’t really know Can study with Gal4 lines
Block neurotransmission Behaviors
Locomotion: can break down into multiple components Straight ahead speed; turning ability
Touch and pain Olfaction and gustation Digestion Feeding Hypoxia response Social behavior
UAS Lines for Analysis of Larval Behavior
UAS-TeTxLC Tetanus toxin light chain: blocks neurotransmission
Cleaves synaptobrevin and blocks evoked transmitter release Weak (TNT-E) and strong (TNT-G) forms
UAS-shibirets
Dominant-negative form of dynamin that blocks synaptic vesicle recycling and neurotransmission
4C-Gal4 Causes Larvae to Circle
Screened 150 Gal4 lines for Larval Locomotion Defects 4C-Gal4 UAS-TeTxLC
Larvae circle 4 other Gal4 lines affect turning and straight moves Expression of toxin in small numbers of vnc motorneurons or
interneurons or in some brain regions do not affect behavior
Summary: can study larval behavior with Gal4 lines
4C-Gal4 Expression
Expressed of 4C-Gal4 is in 200 neurons, possibly including Sim+ CX cells
Generate Single pNB Gal4 Lines: Atonal Gene Regulation
Generate large number of Gal4 lines that are expressed in one or a few pNBs
Use proneural gene CRMs to generate single pNB Gal4 lines Why proneural genes?
Expressed in many pNBs Proneural genes are the direct targets of positional information
cues and have individual pNB-specific enhancers Good assumption, but not much data
Ato is modular regarding cell type (ch, eye, antenna, embryo) but was not further subdivided to find CRM for specific precursors
Generate Single pNB Gal4 Lines: AS-C Gene Regulation
AS-C genes Deletion and transgenic analysis
indicate NB and SOP-specific enhancers
Labeling Lineages Not Just Precursors
pNB enhancer-Gal4 is only transiently expressed Include UAS-Gal4 to maintain expression (not well tested)
pNB enh-Gal4 UAS-Gal4 UAS-TeTxLC should express TeTxLC in lineage throughout development
Maybe need enhanced version UAS-Gal4-VP16 Another more-complicated option
pNB enh-Gal4 UAS-FLP actin-[Flp-out]-Gal4 UAS-TeTxLC
Proneural Genomic Organization
Regulatory regions overlap since AS-C genes are linked ac: 5’ flank: 8.8 kb; 3’ flank is 25.1 kb sc: 5’ flank: 25.1 kb; 3’ flank: 12.2 kb l’sc: 5’ flank: 12.2 kb; 3’ flank: 17.7 kb Overall region between y and pcl: 67.2 kb
ato: 5’ flank: 7.9 kb; 3’ flank: 10.1 kb Overall region between CG9630 and CG11671: 18.1 kb
Proneural Gene Transgenic Analysis
Initially PCR all 2 kb fragments with 100 bp overlap into shuttle vector with Gateway sites (pENTR/D-TOPO)
Use Gateway cloning to move fragments into C31 Gal4 vector with Gateway sites
Inject into C31 recipient line with endogenous integrase (50% efficiency into genomic site
Screen for expression in specific pNBs with appropriate proneural and other pNB markers
Gateway Cloning
Uses in vitro reaction (no fragment purification)
Avoids having to clone into large vectors
Can use same Entry Clone to introduce insert into multiple vectors
Uses phage att sites (L, R) for in vitro recombination
C31 Integration
Single host genomic site with recipient cassette Avoids position effects that can affect gene regulation
Uses phage C31 integration sites (P and B) Host site has w+ gene (already exists) between P sites Donor plasmid can have y+ gene in replacement cassette but
unnecessary Between Donor plasmid P sites, need Gateway att sites adjacent to
promoter-Gal4 Inject plasmid into host with integrase present (~50% integration)
Further Regulatory Region Dissection
Assay 2 kb fragments even if expressed in multiple pNBs for larval behavioral defects if no behavioral defect, then no further dissection is required
If behavioral defects are observed, then 2 kb fragments will be further subdivided into 500 bp (or smaller) fragments and screened to obtain more specific enhancers
Also can mutate specific transcription factor binding sites to acquire more specific enhancers E.g. 500 bp fragment drives expression in 6 pNBs, two are En+,
two are Eagle+, and one is Vnd+ mutate En, Eag, and Vnd sites to acquire fragment that is expressed in a single pNB
Conclusions
Main goal is behavioral analysis Other goals:
Could generate additional Gal4 lines using genes besides proneural genes that are expressed in precursors or discrete cell types (e.g. sim or a number of early patterning genes)
However, early patterning genes (e.g. engrailed) may not have enhancers that can be completely subdivided
Analysis could be useful for dissection of adult behaviors, etc. Also analyze VNC for specific lateral CNS NBs and midline cell
expression Drivers also useful for mapping axonal pathways, neural cell
lineages, and misexpression of genes including DNs for genetic studies on axonogenesis, neural function, and behavior
Will provide enormous information and detail regarding NB formation and regulation of proneural genes important evolutionary consequences
Similar strategy can be employed to study midline cells and other cell types