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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

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goals and assumptions
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
  • Adult brain
    • Brain regions
      • Protocerebrum
      • Deutocerebrum
      • Tritocerebrum
      • Optic lobes
larval brain and development
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
Stereotypic Formation of pNBs
  • pNB addition is continuous; no obvious waves
stereotypic formation of pnbs1
Stereotypic Formation of pNBs
  • Mapping
    • (A) Dpn protein (blue)
    • (B-H) svp-lacZ (brown) and en protein (blue)
proneural gene expression
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
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
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
Larval Brain Neuron Clusters

pNB  neurons  axon bundle

larval brain axon compartments
Larval Brain Axon Compartments
  • Microcircuit (neuron cluster)  axon bundle
  • Macrocircuit (multiple neuronal clusters)  join together via projection neurons to form a macrocircuit
summary
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
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 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
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
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 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
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
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
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
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 FC31 Gal4 vector with Gateway sites
  • Inject into FC31 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
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 l att sites (L, R) for in vitro recombination
f c31 integration
FC31 Integration
  • Single host genomic site with recipient cassette
    • Avoids position effects that can affect gene regulation
  • Uses phage FC31 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
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
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
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