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A Nematode Neuron Identity Crisis

A Nematode Neuron Identity Crisis. Bruce Wightman Muhlenberg College. The nervous system is comprised of many different types of neurons. Homeodomain transcription factors regulate neuron identity in the vertebrate spinal cord. The nematode C. elegans. DNA Sequence known Rich genetics

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A Nematode Neuron Identity Crisis

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  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 Nematode neuron-specific promoter GFP Aequoreavictoria

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

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

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

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

  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 FMRFamide FAX-1 axon pathfinding

  13. fax-1 encodes a protein that is a member of the nuclear hormone receptor family DBD LBD C N NHR’s regulate transcription and bind lipophilic hormones The FAX-1 DBD contains two zinc- fingers C C C C Zn2+ Zn2+ downstream gene C C C C (J. Much)

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

  15. Mutations in human PNR cause defects in photoreceptor identity Enhanced S-cone syndrome retina Normal 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 Other neuron- specific gene flp-1

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

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

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

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

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

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

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

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

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

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

  27. Which genes may functionupstream of fax-1?

  28. The homeodomain gene unc-42 may function upstream of fax-1 AVK UNC-42 fax-1 • 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 unc-42(e419) FAX-1 wild-type FAX-1 AVB RIC AVA/E AVA/E RIC AVK AVK Wild-type AVB DVA AVA RIC AVE MI AIY AVK unc-42 (e419) DVA AVA RIC AVE AVK FAX-1 is absent in AVB and two anterior neurons and reduced in AVK

  30. unc-42 is also required for flp-1::gfp expression Percentage of AVK neurons Express flp-1::gfp fluorescence Wild-Type 100 fax-1 (gm83) 65 unc-42 (e419) 0 unc-42 (e419); fax-1 (gm83) 0 (S. Mathieson)

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

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

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

  34. What about unc-42 and fax-1in 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 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 (Baran et al., 1999; Brockie et al., 2001)

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

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

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

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

  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

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