Genetic Analysis of Behavior

1. Genetic Analysis of Behavior

2. 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

3. Adult Brain • Adult brain • Brain regions • Protocerebrum • Deutocerebrum • Tritocerebrum • Optic lobes

4. 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

5. Stereotypic Formation of pNBs • pNB addition is continuous; no obvious waves

6. Stereotypic Formation of pNBs • Mapping • (A) Dpn protein (blue) • (B-H) svp-lacZ (brown) and en protein (blue)

7. 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

8. 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

9. Larval Brain Organization • Neurons  cortex • Axons  neuropile • Compartments separated by glia? • Neuropile compartments  synaptic connections • NB  neuron cluster  axons with similar synaptic targets

10. Larval Brain Neuron Clusters pNB  neurons  axon bundle

11. Larval Brain Neuron Cluster • NB • GMCs • Neurons

12. Larval Brain Axon Compartments • Microcircuit (neuron cluster)  axon bundle • Macrocircuit (multiple neuronal clusters)  join together via projection neurons to form a macrocircuit

13. 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?

14. 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

15. 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

16. 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

17. 4C-Gal4 Expression • Expressed of 4C-Gal4 is in 200 neurons, possibly including Sim+ CX cells

18. 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

19. Generate Single pNB Gal4 Lines: AS-C Gene Regulation • AS-C genes • Deletion and transgenic analysis indicate NB and SOP-specific enhancers

20. 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

21. 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

22. 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

23. 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

24. 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)

25. 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

26. 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