1. Tiffany Hough The N-terminal region of centrosomal protein 290 (CEP290) restores vision in a zebrafish model of human blindness. Baye, L.M., Patrinostro, X., Swaminathan, S., Bck, J.S., Zhang, Y., Stone, E.M., Sheffield, V.C., & Slusarski, D.C. (2011). The N-terminal region of centrosomal protein 290 (CEP290) restores vision in a zebrafish model of human blindness. Human Molecular Genetics. 20 (8): 1467-1477.

2. Outline  Background  Objectives  Experimental Approach & Results  Conclusions  Critique and Future Research

3. Background Cilia-based Disorders *Lebers congenital amaurosis Meckel Gruber Syndrome Joubert Syndrome Senor-Loken Syndrome Bardet-Biedl Syndrome CEP290: 12q21.33 54 exons, 2479 AA, 25 predicted domains, motifs, & localization signals 30% of LCA Most common mutation results in stop codon giving alternative splicing

4. Objectives Observe developmental and functional roles of CEP290 in zebrafish Test different regions of CEP290 for localization and ability to restore knockout function Test ability to bind NPHP2 (Senor-Loken)

5. Experimental Design & Results: Outline 1. Developmental Expression 2. Gene Targeting & Gross Morphant Phenotypes 3. BBS Phenotypes 4. Characteristics of LCA 5. Human Truncated Constructs Localize Normally 6. Vision Rescue 7. Binding with NPHP2

6. Experimental Approach & Results: Developmental Expression RT-PCR demonstrates maternal inheritance and presence throughout all stages of development (A) Whole mount in situ hybridization show: Ubiquitous expression at 8- somite stage especially in KV (B,C) 5dpf: gut, notochord, brain, eye, ear (F) 5 dpf retina: ganglion cell layer, inner nuclear layer, photoreceptor cell layer (H)

7. Experimental Approach & Results: Gene Targeting & Gross Morphant Phenotypes Most common mutation in LCA Used antisense oligonucleotide (Morpholino) MO to disrupt splicing and result in intron inclusion.(A) Body curvature defects (B) in dose dependent manner RT-PCR with cDNA Shows some wild type present: hypomorphic (C) Percentages of embryos with body curvature defects based on MO dose Experimental group Phenotype (percentage) n Straight Bent Curly Wild-type 100 0 0 129 cep290 MO (5 ng) 60 6 34 205 cep290 MO (8 ng) 40 5 55 282 cep290 MO (10 ng) 13 7 80 208

8. Experimental Approach & Results: BBS Phenotypes 10 ng MO dose All BBS genes have these characteristics Same size or smaller KV than notochord (C,D) occurred in dose dependent manner (D) Treatment of dark adapted 5dpf embyos with epinephrine Delayed melanosome transport (H)

9. Experimental Approach & Results: Characteristics of LCA 8ng MO dose Histological analysis using hematoxylin & eosin staining Regardless of curly/straight phenotype at 3dpf: Fully laminated retina (C,D) Photorecerptor outer segments in central retina normal (G,H) At 5dpf outer segments disorganized (wavy, K,L) Decreased visual acuity Wt = 3.53 responses crx positive control = 2.00 responses Morphant = 2.46 responses

10. Experimental Approach & Results: Human truncated constructs localize normally N-terminal fragment (A blue, 1059AA) – spans CEP290 mutation being studied C-terminal fragment (A green, 1765-2479AA) – spans mutations in rd16 mouse and rdAC cat Injected mRNA encoding myc- tagged fusion constructs & immunohistochemistry at 50% epiboly(B,E,H) Centrin fused with gfp for centrioles (C,F,I) Paracentriolar & cytoplasmic localization (D,G)

11. Experimental Approach & Results: Vision Rescue No change in KV defects C-terminal treatment significantly reduced melanosome transport delays Experimental groupKV (%)MT (min) ± SE Vision (responses) ± SE Wild-type5.21.51 ± 0.073.53 ± 0.17 control MO (10 ng)8.31.52 ± 0.063.48 ± 0.18 cep290 MO (8 ng)18.2 ++ 2.10 ± 0.10**2.46 ± 0.12** cep290 MO (8 ng) + N terminus (1.8 ng) 22.6 ++ 2.05 ± 0.10**3.48 ± 0.13 cep290 MO (8 ng) + C terminus (0.6 ng) 15.4 ++ 1.82 ± 0.072.44 ± 0.22**

12. Experimental Approach & Results: Binding with NPHP2 NPHP5 shown to interact with N-terminal region of CEP290 Mutations in NPHP2 and NPHP5 have been implicated in vision impairment Transient transfection and co- immunoprecipitation assays followed by SDS-PAGE and Western blot N-terminal region interacts with NPHP2

13. Conclusions CEP290 is present throughout development and is expressed in multiple tissues The mutation model resulted in abnormal body curvature, decreased KV size, delayed melanosome transport, and vision impairment Both constructs localize normally The N-terminal region is sufficient to rescue vision and can bind with NPHP2, suggesting domain and tissue specificity for this region of the protein Supported hypothesis of the mutation being hypomorphic: presence of wild type in models and dose dependence of effects

14. Future Research & Critique Identify NPHP complexes and proteins with which they interact during proper vision: Molecular pathway examined in a tissue specific manner Identify other proteins that interact specifically with CEP290 to help explain rescue by N- terminus Identify the specific mechanism of N-terminal region of CEP290 Test other models, such as a mouse, with this mutation for rescue. Negates other research in other models (rd16 mouse and rdAc cat) Didn’t explain how this mutation models BBS or why this was important. What mutations result in BBS? Otherwise a very strong paper which demonstrated known phenotypes for this mutation and introduces a promising target for retroviral gene therapy.