Characterization of the Zebrafish nolc1l Mutant as Potential Model for Treacher Collins Syndrome

1. Characterization of the Zebrafish nolc1l Mutant as Potential Model for Treacher Collins Syndrome Annie Lee Mentors: James Lister and Rita Shiang

2. Treacher Collins Syndrome

3. Treacher Collins/Franceschetti Syndrome 1 (TCOF1) Treacle Possible functions? • Mutation  Stabilization of p53 protein • Underexpression rate of proliferation apoptosis • Overexpression apoptosis Cell Growth Homeostasis

4. Neural crest cells

5. Zebrafish, Daniorerio • Advantages • Disadvantages Transparent embryo Fast development Large number of embryos Development outside More distantly related from human genes than mice

6. Nucleolar and coiled-body phosphoprotein 1-like (nolc1l) • Ortholog of TCOF1 • Previous research: • Knockdowns using morpholino • Limitation: not heritable

7. Retrovirus-mutated zebrafish • Retrovirus-mutated zebrafish by chance • Mutation: heritable

8. Objectives • Nolc1l mutant zebrafish = good model? • Possible rescue by mutating p53?

9. Identify mutants • Obtain genomic DNA by fin clipping • PCR for the presence of nolc1l mutation Mutant/ carrier Wildtype/ non-carrier

10. Identify mutants Mutant: 346 bp; 2,3,4,7,8,9,10,11,12,13 Wildtype: 5,6

11. Nolc1l mutant zebrafish nolc1l/+ nolc1l/+

12. in situ hybridization • Identify phenotypic differences • Probes: neural crest cells, jaw cartilage, cartilage cells

13. MO injected embryos

14. Nolc1l mutant zebrafish nolc1l/+ nolc1l/+ ¼ Homozygous wildtype strong expression ½ Heterozygotes  mild expression ¼ Homozygous mutant  little/no expression

15. in situ hybridization Crestin goosecoid Goosecoid close up sox9a Dlx2a foxi3 tbx2b foxa3 cb356 WHY?

16. Tcof1 probe Homozyous wildtype Heterozygote mutant Homozyous wildtype close up Heterozygote Close up

17. Pcna probe • Proliferating cell nuclear antigen • Proliferating cells Homozyouswildtype Heterozygote mutant Homozyouswildtype close up Heterozygote Close up

18. Possible Rescue- p53 mutation nolc1l nolc1l p53 stabilization of p53

19. 2 Heterozygous zebrafish P53/+ nolc1l/+ P53/+ nolc1l/+

20. PN Pn pN pn PN PPNN PPNn PpNN PpNn Pn PPNn PPnn PPNn Ppnn pN PpNN PPNn ppNN ppNn pn PpNn Ppnn ppNn ppnn Dihybrid cross • P: p53 WT • p: p53 Mutant • N: nolc1l WT • n: nolc1l mutant nn: nolc1l mutant pp: p53 mutant

21. PN Pn pN pn PN PPNN PPNn PpNN PpNn Pn PPNn PPnn PPNn Ppnn pN PpNN PPNn ppNN ppNn pn PpNn Ppnn ppNn ppnn Dihybrid cross • P: p53 WT • p: p53 Mutant • N: nolc1l WT • n: nolc1l mutant nn: nolc1l mutant pp: p53 mutant NO RESCUE: 4/16 mutants RESCUE: 3/16 mutants

22. Easier Way? mdm2 mdm2 mdm2 UNLESS zebrafish is p53 mutant p53 p53 p53 p53 p53 p53 p53 mdm2 Apoptosis

23. Morpholino Injection NO rescue: ¼ mutant Yes rescue: NO mutants PN Pn pN pn PN PPNN PPNn PpNN PpNn Pn PPNn PPnn PPNn Ppnn pN PpNN PPNn ppNN ppNn pn PpNn Ppnn ppNn ppnn

24. Ratios 21 embryos that lacked cell death 13 embryos with swim bladder 8 embryos without swim bladder • Swim bladder is not the most effective way to examine mutation • Could try using in situ hybridization or genotyping for the mutants

25. Conclusion • Nolc1l mutated zebrafish may be a good model • In situ hybridization: tcof1 and pcna1 • p53 rescue is still inconclusive

26. Works Cited [1]Dambly-Chaudiere, C., et al. "The Lateral Line of Zebrafish: A Model System for the Analysis of Morphogenesis and Neural Development in Vertebrates." Biology of the cell / under the auspices of the European Cell Biology Organization 95.9 (2003): 579-87. Print. [2]Dixon, J., et al. "Increased Levels of Apoptosis in the Prefusion Neural Folds Underlie the Craniofacial Disorder, Treacher Collins Syndrome." Human molecular genetics 9.10 (2000): 1473-80. Print. [3]Golling, G., et al. "Insertional Mutagenesis in Zebrafish Rapidly Identifies Genes Essential for Early Vertebrate Development." Nature genetics 31.2 (2002): 135-40. Print. [4]Gonzales, B., et al. "The Treacher Collins Syndrome (TCOF1) Gene Product is Involved in Pre-rRNA Methylation." Human molecular genetics 14.14 (2005): 2035-43. Print. [5]Hayano, T., et al. "Proteomic Analysis of Human Nop56p-Associated Pre-Ribosomal Ribonucleoprotein Complexes. Possible Link between Nop56p and the Nucleolar Protein Treacle Responsible for Treacher Collins Syndrome." The Journal of biological chemistry 278.36 (2003): 34309-19. Print. [6]Holser, T. M. "A new model for Treacher Collins Syndrome: characterizing the zebrafish tcof1 ortholog." Master’s Thesis, Virginia Commonwealth University. 2007. [7]Holser, T.M., Lee, A., Patel, A., Shiang, R., Lister, J.A. “Knockdown of the zebrafish nolc1l (nucleolar and coiled-body phosphoprotein 1-like) gene induces p53-dependent cell death and craniofacial defects.” Manuscript in preparation, 2010 . [8]Jegalian, B. G., and E. M. De Robertis. "Homeotic Transformations in the Mouse Induced by Overexpression of a Human Hox3.3 Transgene." Cell 71.6 (1992): 901-10. Print. [9]Meier, U. T., and G. Blobel. "Nopp140 Shuttles on Tracks between Nucleolus and Cytoplasm." Cell 70.1 (1992): 127-38. Print. [10]Mogass, M., et al. "Genomewide Analysis of Gene Expression Associated with Tcof1 in Mouse Neuroblastoma." Biochemical and biophysical research communications 325.1 (2004): 124-32. Print. [11]Patel, A. D. "Characterization of zebrafish models of Treacher Collins syndrome." Master’s Thesis, Virginia Commonwealth University, 2008. [12]Schulte-Merker, S., et al. "Expression of Zebrafish Goosecoid and no Tail Gene Products in Wild-Type and Mutant no Tail Embryos." Development (Cambridge, England) 120.4 (1994): 843-52. Print. [13]Splendore, A., et al. "Parental Origin of Mutations in Sporadic Cases of Treacher Collins Syndrome." European journal of human genetics : EJHG 11.9 (2003): 718-22. Print. [14] The Treacher Collins Syndrome Collaborative Group, Positional cloning of a gene involved in the pathogenesis of Treacher Collins syndrome, Nat. Genet. 12(1996) 139-136. [15]Thisse, C., and B. Thisse. "High-Resolution in Situ Hybridization to Whole-Mount Zebrafish Embryos." Nature [16] Langheinrich, U., Hennen, E., Stott, G. and Vacun, G. “Zebrafish as a model organism for the identification and characterization of drugs and genes affecting p53 signaling.”(2002) Curr. Biol., 12, 2023-2028.