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