1. A Nematode Neuron Identity Crisis Bruce Wightman Muhlenberg College

2. The nervous system is comprised of many different types of neurons

3. Homeodomain transcription factors regulate neuron identity in the vertebrate spinal cord

4. The nematode C. elegans DNA Sequence known Rich genetics Simple nervous system Defined anatomy

5. The C. elegans nervous system

6. Axon anatomy can be examined by neuron-specific promoter :: GFP fusions Aequorea victoria GFP Nematode neuron-specific promoter

7. The AVK axons can be visualized with an flp-1::gfp transgene AVKL GFP flp-1 (Courtesy of C. Li)

8. The fax-1 gene is required for AVK axon pathfinding AVKL Wild Type AVKL fax-1 (gm83)

9. fax-1 is also required for normal levels of flp-1::gfp expression Wild-Typefax-1(gm83) AVKL AVKR AVKL AVKR

10. The flp-1 gene encodes a protein precursor for FMRFamide-like peptide neurotransmitter NC SADPNFLRF SQPNFLRF ASGDPNFLRF SDPNFLRF AAADPNFLRF PNFLRF AGSDPNFLRF Post-synaptic neuron Pre-synaptic neuron flp-1

11. FMRFamide is reduced in AVK neurons of fax-1 mutants anti-FMRFamide immunostaining

12. fax-1 functions in both axon pathfinding and neuro- transmitter expression FAX-1 FMRFamide axon pathfinding

13. fax-1 encodes a protein that is a member of the nuclear hormone receptor family N C DBDLBD downstream gene NHR’s regulate transcription and bind lipophilic hormones The FAX-1 DBD contains two zinc- fingers Zn 2+ C C CC C C CC (J. Much)

14. FAX-1 is a member of a subclass of NHR’s that function in nervous system development Estrogen Receptor Retinoic Acid Receptor NHR-111 TLX * NHR-67 TLL * FAX-1 * PNR* C. elegans Drosophila H. sapiens * functions in nervous system

15. Mutations in human PNR cause defects in photoreceptor identity Normal retina Enhanced S-cone syndrome retina PNR is also known as NR2E3 (J. Tanis)

16. fax-1 may regulate transcription of genes involved in speci- fication of neuron identity pathfinding gene flp-1 Other neuron- specific gene

17. Examining FAX-1 expression using antibodies and immunofluroescence staining Anti-FAX-1 antibodiesImmunofluorescence staining (N. Carmean) (B. Ebert)

18. fax-1 is expressed in 18 neurons including the AVKs mid-stage embryo adult AVKL AVKR AVK AIY RIC DVA Fax-1 Phenotypes: Unc, Che, Fab, Wandering AVB AVA AVE MI RICRAVBR AVAR AVER

19. fax-1 may regulate transcription of genes involved in speci- fication of AVK neuron identity AVK path- finding gene flp-1 Other AVK- specific gene

20. ncs-1::gfp expression in AVK requires fax-1 neuron-specific calcium sensor wild-type ncs-1::gfp fax-1 (gm83) ncs-1::gfp AVKL AVKR (Gomez, et al., 2001)

21. Summary of gene expression requirements AVK Require fax-1 flp-1 ncs-1 Cells:

22. What is the function of fax-1 in neurons other than the AVK’s? DVA AVK AIY RIC AVB AVA AVE MI

23. The RIC neurons have normal anatomy and expression of tbh-1 in fax-1 mutants tyrosine  -hydroxylase (for octopamine biosynthesis) wild-type and fax-1 (gm83) (M. Alkema and H.R. Horvitz, unpublished) RIC

24. Expression of NMDA glutamate receptor subunits in AVA and AVE requires fax-1 AVB fax-1 (gm83) nmr-1::gfp RIM AVA/E AVB RIM AVG AVB RIM AVG wild-type nmr-1::gfp AVB RIM AVA AVE Both nmr-1 and nmr-2 show the same pattern

25. Expression of non-NMDA glutamate receptor subunits does not require fax-1 AVA and AVE axons appear normal in fax-1 mutants: fax-1 not required for axon pathfinding in AVA/E wild-type glr-1::gfp fax-1 (gm83) glr-1::gfp AVA/E

26. Summary of gene expression requirements AVKAVA/E Require fax-1 flp-1 ncs-1 nmr-1 nmr-2 Cells:

27. Which genes may function upstream of fax-1?

28. The homeodomain gene unc-42 may function upstream of fax-1 fax-1 UNC-42 AVK unc-42 is expressed in the AVK, AVA, AVE and other neurons unc-42 mutants have AVK path- finding defects that are similar to fax-1 mutant defects (Wightman et al, 1997; Baran et al., 1999)

29. fax-1 is not expressed in some neurons of unc-42 mutants Wild-type unc-42 (e419) FAX-1 is absent in AVB and two anterior neurons and reduced in AVK DVA AVK AIY RIC AVB AVA AVE MI AVA AVE RIC AVK wild-type FAX-1 unc-42(e419) FAX-1 AVA/E AVB RIC AVK RIC DVA

30. unc-42 is also required for flp-1::gfp expression Percentage of AVK neurons Express flp-1::gfp fluorescence Wild-Type100 fax-1 (gm83) 65 unc-42 (e419) 0 unc-42 (e419); fax-1 (gm83) 0 (S. Mathieson)

31. Summary of gene expression requirements AVKAVA/E Require fax-1 Require unc-42 flp-1 ncs-1 fax-1 flp-1 nmr-1 nmr-2 Cells:

32. Expression of ncs-1 also depends on unc-42 wild-type ncs-1::gfp unc-42(e419) ncs-1::gfp AVKL AVKR

33. Summary of gene expression requirements AVKAVA/E Require fax-1 Require unc-42 flp-1 ncs-1 fax-1 flp-1 ncs-1 nmr-1 nmr-2 Cells:

34. What about unc-42 and fax-1 in the AVA and AVE interneurons?

35. Expression of non-NMDA glutamate receptor subunits in AVA/E requires unc-42 Expression of NMDA glutamate receptor subunits in AVA/E does NOT require unc-42 (Baran et al., 1999; Brockie et al., 2001) wild-typeunc-42(e419)fax-1(gm83) nmr-1++- nmr-2++- glr-1+-+ glr-4+-ND glr-5+-+ Axon pathfinding by AVA and/or AVE appears defective in unc-42 mutants

36. Summary of gene expression requirements AVKAVA/E Require fax-1 Require unc-42 flp-1 ncs-1 fax-1 flp-1 ncs-1 nmr-1 nmr-2 glr-1 glr-4 glr-5 Cells:

37. Models for the specification of neuron identity by unc-42 and fax-1 UNC-42 FAX-1 flp-1 Pathfinding genes ncs-1 AVK AVA/E UNC-42 FAX-1 Pathfinding genes glr-1 nmr-1 nmr-2 glr-4 glr-5

38. Approaches to identification of FAX-1 downstream targets (R. Haviland, R. Lombel, E. Murphy, K. Reinert, E. Smith, Y. Vidgop) Other neuron- specific genes

39. Is expression of FAX-1 sufficient to change gene expression? heat-shock promoter fax-1 (N. Bianco, T. Sundaresan) anti-FAX-1 hs::fax-1; fax-1(gm83)

40. Participants fax-1 Sepi Bazel Nick Bianco Nicole Carmean Sara Carr Bryan Ebert David Garbe Rebecca Haviland Chirag Kalola Rebecca Lombel Jason Much Elissa Murphy Gwen Sarver Dennis Slade Eric Smith Tilak Sundaresan Kristy Reinert Jessica Tanis Yelena Vidgop unc-20 Peter Alff Jeff Doto Brook Kohrt Son Nguyen Suchi Pandey Doug Prechtel Matt Stein Other genes David Brightbill Andrea Cerrone Kelly Klampert Sheila Mathieson Mark Alkema, Cori Bargmann, Edouard DeCastro, Gian Garriga, Oliver Hobert, Christine Li, A.V. Maricq, Ruth Yu