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-shibire ts  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 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

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 att sites (L, R) for in vitro recombination

24. 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)

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