Genetic analysis of behavior
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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