Unexpected Diversity in the Innate Immune System of the Purple Sea Urchin

1. Unexpected Diversity in the Innate Immune System of the Purple Sea Urchin L. Courtney Smith Department of Biological Sciences George Washington University Washington DC 202-994-9211 Strongylocentrotus purpuratus

2. Deuterostomes Animal Phylogeny and the Evolution of Immunity • Chordates • Vertebrates • Mammals • Birds • Reptiles • Amphibians • Fish • Sharks & Rays • Agnathans • Lampreys • Hagfish Protostomes Arthropods Molluscs Annelids Protochordates Ascidians Echinoderms Sea Urchins

3. Sea Urchin Genome Annotation • Incomplete Complement System • Missing Classical and Terminal pathways • Duplication of Alternative pathway • Large gene families • Toll-like receptors, 222 gene models (humans have ~11) • expressed in coelomocytes (immune cells) • NOD-like receptors, 203 gene models (humans have ~20) • expressed in gut • Scavenger receptors, 218 gene models • expressed in coelomocytes • C-type lectins, 408 gene models (humans have ~100) • encoding 108 small lectins, 300 mosaic proteins (Cohen & Smith, unpublished) Highly Diverse Innate Immune System Hibino et al., Dev Biol 300:349, 2006 TSUGSC, Science 314:941,2006 Rast et al., Science 314:952, 2006

4. Gene Expression in Sea Urchin Immune Cells Responding to LPS 185/333 All others Of 1025 randomly chosen ESTs, 72% matched to two unknown sequences. EST333 (Smith et al., 1996) DD185 (Rast, Pancer & Davidson, 1999) Nair et al., Physiol Genomics 2005.

5. Alignment of 185/333 cDNAs 300 nt Gaps define blocks of sequence called elements. Alternative splicing? Terwilliger et al., Physiol Genomics 2006

6. 185/333 Gene Structure 407bp Leader Elements 1 - 25 E2 < 2 kb Quantitative PCR estimates 80 - 120 alleles per genome Terwilliger et al., Physiol Genomics 2006

7. Gene Element Patterns Exon 1 Exon 2 elements 9 12 16 21 Pattern Source Group 10 1 2 3 4 5 6 7 8 11 13 14 15 17 18 19 20 22 24 25 Int. L 23 # 1 3 B A b A 6* 1 S A b 5* B 1 S b 2 C 1 S b 3d 5 S B b B 3b 5 B B b 6* 2 S D b 7* 1 S C b C 3 2 S C b 3* 1 S C b 4* 1 S D b D C 1 28 B b 5* 3 C C c 6 1 C C a 3f 6 C E a 3c 1 C C a 2 14 B E a E 7* E 1 C b 8* 1 C E b 9* 1 C E c 1* 1 C B b F 2* 4 C B b 2* 1 C E a 1f 12 B E b 0 1c 1 S C b 1e 1 S D b 300 bp Intron elements A B C D E 2 9 1 3 4 5 8 6 7 Buckley & Smith, in prep 100 bp

8. 185/333 Sequence Diversity • Element diversity is a result of SNPs and indels • Sequences of identical genes are rarely identified within an individual sea urchin • Identical genes are never shared among sea urchins. • Some element sequences are shared among dissimilar genes

9. Intron Elements Exon 2 Elements 57 46 67 L Element Scrambling 12 3 11 11 2 2 4 4 9 11 37 3 3 7 3 8 5 2 2 2 19 1 13 1 1 1 3 20 3 61 21 1 1 1 14 2 38 unique genes from animal 10 1 1 2 2 2 2 20 9 19 2 2 2 1 1 1 19 10 1 2 1 5 11 5 17 10 10 4 16 1 1 1 1 17 16 6 11 3 17 1 1 1 1 1 1 6 19 6 3 2 6 7 2 3 2 3 12 6 3 4 3 2 34 2 3 1 2 2 4 4 6 7 6 30 4 17 3 3 6 18 13 39 8 35 1 2 1 5 3 5 3 4 5 9 13 6 9 3 13 9 5 6 7 5 9 23 1 4 6 4 2 9 2 1 14 11 17 29 10 18 10 7 25 22 8 6 4 7 3 5 13 8 4 7 8 7 41 19 18 14 21 10 1 25 1 1 5 5 12 3 1 7 1 9 9 5 6 8 5 10 2 12 14 12 15 20 28 Buckley & Smith, in prep 7

10. Conclusions • Complex immune system • Large gene families • Unknown mechanisms for diversification Do all metazoa have mechanisms for immune diversification? Future Problems • 185/333 locus structure • 185/333 protein function

11. Acknowledgments • Young-Ok Kim • Heather Del Valle • Lindsay Edwards George Washington University • David Terwilliger • Katherine Buckley • Virginia Brockton • Rebecca Easley • Macquarie University • David Raftos • Sham Nair • Nolwen Dheilly Sea urchin immunology is supported by the National Science Foundation Duke University • Tom Kepler • Supriya Munshaw Dickinson College • John Henson Medical Univ. of S. Carolina • Paul Gross