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Zebrafish as a Model to Investigate the Disease Mechanisms of Infantile Neuronal Ceroid Lipfuscinosis http://www.devbio.uga.edu/gallery/images/embryo4LRG.jpg Nicole Brant and Dr. Wendy Boehmler, Department of Biology, York College of Pennsylvania http://www.bcm.edu/cain_foundation/noframes/html/pages/staff/n eurons%20confocal%20mu1a%20dcx.jpg Introduction •Infantile neuronal ceroid lipfuscinosis (INCL) is a devastating neurodegenerative disorder that destroys neurons in certain tissues of the central nervous system such as the thalamus, cortex, and cerebellum. •INCL is caused by a mutation in the PPT1 gene which encodes palmitoyl protein thioesterase 1. •A whole-mount in situ hybridization was performed on 24, 48, and 72 hpf embryos. Collected 24, 48, and 72 hpf embryos, stored them in MeOH, made the sense and antisense probes and reagents Permeabilization of the embryos and hybridization of the RNA probe •PPT1 is a lysosomal enzyme that plays a major role in the degradation of lipid-modified proteins. •How PPT1 deficiency leads to selective central nervous system degeneration is unclear. Washes Washes Staining and washing, stored embryos and took pictures •Neurons have an altered endoplasmic reticulum morphology. •Microarray analyses on INCL brain tissues shows an upregulation of inflammatory genes. •The post-mitotic nature of neurons may contribute to their selective degeneration. Incubation with antiDIG antibody alkaline phosphase Results •Zebrafish, a small vertebrate, are becoming the organism of choice for modeling diseases and for drug discovery. •The genome is fully sequenced. •Breeding pairs can produce hundreds of embryos. •Embryos develop ex utero, develop rapidly, and are transparent Figure 1: RT-PCR analysis of PPT1 from adult zebrafish. The PPT1 gene was amplified from adult zebrafish brain, gut, and muscle RNA. The primers used encompassed the predicted ATG start codon and stop codon to produce an amplicon of 1033 base pairs (bp). Primer sequences: forward 5’AGATTGAATAATGGCTCCACC3’ and reverse 5’TATCTGAGACGGTAGTTACGA3’. Objectives 1. Determine if there is expression of the PPT1 gene in adult zebrafish tissues. 2. Determine if there is spatial and temporal expression of the PPT1 gene during zebrafish development. Methods •Searches of expressed sequence tag databases (http://www.ncbi.nlm.nih.gov/) led to the identification of a potential zebrafish PPT1 homolog (Gene Accession number NM213339). •RT-PCR was conducted using RNA collected from adult zebrafish and embryos. Figure 2: RT-PCR analysis of PPT1 from 24, 48, and 72 hpf embryos. The PPT1 gene was amplified from 24, 48, and 72 hpf embryo RNA. Primers encompassed the entire gene to produce the predicted 1033bp amplicon. SC B A 24 hpf D B Collected tissues from the brain, eye, gut, heart, and muscle and 24, 48, and 72 hours post fertilization (hpf) embryos SC RNA isolation with phenol/chloroform extraction B 48 hpf E E B PCR Denature for 30 sec. at 94oC Annealing for 30 sec. at 48oC Elongation for 1 min. at 72oC 30 cycles Zebrafish PPT1 gene was successfully cloned into a pDrive and sequenced 24 hpf E 48 hpf SC Reverse Transcription cDNA MASPGCLWLLAVA-LLPWTCASRALQHLDPPAPLPLVIWHGMGDSCCNPLSMGAIKKMVE 59 MAPPAAFRLLSVSGCLLLLCGT----SWASNGTVPLVIWHGMGDSCCNPLSMGAIKKMVE 56 **.*..: **:*: * *.: . ..:************************** Human Zebrafish KKIPGIYVLSLEIGKTLMEDVENSFFLNVNSQVTTVCQALAKDPKLQQGYNAMGFSQGGQ 119 QEVSGIYVLSLMIGKSVFEDTENGFLMDVNKQVSFVCDQLAKDPKLKEGYNAMGFSQGAQ 116 :::.******* ***:::**.**.*:::**.**: **: *******::**********.* Human Zebrafish FLRAVAQRCPSPPMINLISVGGQHQGVFGLPRCPGESSHICDFIRKTLNAGAYSKVVQER 179 FLRAVAQRCPDPPMRNLISVGGQHQGVYGLPRCPGESSHICDWIRKQLNSGAYTDAVQKH 176 **********.*** ************:**************:*** **:***:..**:: Human Zebrafish LVQAEYWHDPIKEDVYRNHSIFLADINQERGINESYKKNLMALKKFVMVKFLNDSIVDPV 239 LVQAQYWHDPLNDDLYKKYSLFLADINQERVVNETYKKNLMSLNKFVMVKFLQDSIVDPV 236 ****:*****:::*:*:::*:********* :**:******:*:********:******* Human Zebrafish DSEWFGFYRSGQAKETIPLQETSLYTQDRLGLKEMDNAGQLVFLATEGDHLQLSEEWFYA 299 DSEWFGFYKAGQAEELETLQESPIYKEDRLGLAAMDSAGKLVFLASEGDHLQFTREWFNE 296 ********::***:* .***:.:*.:***** **.**:*****:******::.*** Human Zebrafish HIIPFLG 306 NLLSYLL 303 :::.:* Figure 4: Comparison of zebrafish and human PPT1. Human and zebrafish PPT1 amino acid sequences were aligned using CLUSTALW. Dashes in sequences allow optimal alignment for amino acid insertions/deletions. Identical amino acids are highlighted by asterisks and conserved are highlighted by dots. •While the gene mutation has been identified, there is still a need to investigate the neurobiology of the disease course throughout early development and to assess potential therapeutic treatments. •Mouse models of INCL have provided significant clues to the biological basis of the disease, however such studies can be slow, laborious, and expensive to perform. Human Zebrafish Table 1: Markers and Genes Syntenic with PPT1 Genes in Zebrafish and Humans. Zebrafish Human Gene Name Chromosome Chromosome PPT1 dnajc8 flj10504 mhcuaa trh3 tpm3b ctps hdac1 flj14490 rbb4 fuca1 bmp8 col9a2 ilf2 sfpq pum1 cap1 rpl11 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Conclusion 1.The data presented here represents the first analysis of temporal and spatial expression of the PPT1 gene in developing zebrafish embryos. 2.PPT1 is also expressed in adult zebrafish in the brain, gut, and muscle. 3.Sequence comparisons and syntenic analysis supports the identification of a novel zebrafish PPT1 homolog. Future Experiments •Perform a morpholino knockdown of PPT1 gene to assess any gross morphological abnormalities in development. C 72 hpf F E 72 hpf Figure 3: Expression of the palmitoyl protein thioesterase 1 gene. Lateral views of 24 (A, D), 48 (B, E), and 72 (C, F) hpf embryos. Note the sense probe (A, B, C) and the antisense probe (D, E, F). B-brain, E-eye, SC-spinal cord Literature Cited •Cooper, Jonathan D., Russel, Claire and Mitchison, Hannah, M. 2006. Progress towards understanding disease mechanisms in small vertebrate models of neuronal ceroid lipofuscinosis. Biochimica et Biophysica Acta 873-889. •Woods, I. G., Wilson, C., Friedlander, B., Chang, P., Reyes, D. K., Nix, R., Kelly, P. D., Chu, F., Postlethwait, J. H., and W. S. Talbot. 2005. The zebrafish gene map defines ancestral vertebrate chromosomes. Genome Research 15:13071314. Acknowledgements I would like to thank Dr. Boehmler for all of her time and guidance.