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Zebrafish as a Model to Investigate the Disease Mechanisms
of Infantile Neuronal Ceroid Lipfuscinosis
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Nicole Brant and Dr. Wendy Boehmler,
Department of Biology, York College of Pennsylvania
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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.