Zebrafish models for the functional genomics of neurogenetic disorders☆
Edor Kabashia,b, Edna Brusteinb, Nathalie Champagnea,b, Pierre Drapeaub,⁎
aCenter for Excellence in Neuromics, CHUM Research Center and the Department of Medicine, Université de Montréal, Montréal, QC, Canada
bDepartment of Pathology and Cell Biology and Groupe de recherche sur le système nerveux central, Université de Montréal, Montréal, QC, Canada
a b s t r a c ta r t i c l ei n f o
Received 4 December 2009
Accepted 22 September 2010
Available online 29 September 2010
In this review, we consider recent work using zebrafish to validate and study the functional consequences of
mutations of human genes implicated in a broad range of degenerative and developmental disorders of the
brain and spinal cord. Also we present technical considerations for those wishing to study their own genes of
interest by taking advantage of this easily manipulated and clinically relevant model organism. Zebrafish
permit mutational analyses of genetic function (gain or loss of function) and the rapid validation of human
variants as pathological mutations. In particular, neural degeneration can be characterized at genetic, cellular,
functional, and behavioral levels. Zebrafish have been used to knock down or express mutations in zebrafish
homologs of human genes and to directly express human genes bearing mutations related to
neurodegenerative disorders such as spinal muscular atrophy, ataxia, hereditary spastic paraplegia,
amyotrophic lateral sclerosis (ALS), epilepsy, Huntington's disease, Parkinson's disease, fronto-temporal
dementia, and Alzheimer's disease. More recently, we have been using zebrafish to validate mutations of
synaptic genes discovered by large-scale genomic approaches in developmental disorders such as autism,
schizophrenia, and non-syndromic mental retardation. Advances in zebrafish genetics such as multigenic
analyses and chemical genetics now offer a unique potential for disease research. Thus, zebrafish hold much
promise for advancing the functional genomics of human diseases, the understanding of the genetics and cell
biology of degenerative and developmental disorders, and the discovery of therapeutics. This article is part of
a Special Issue entitled Zebrafish Models of Neurological Diseases.
© 2010 Elsevier B.V. All rights reserved.
Zebrafish are used as a model for a wide variety of human diseases,
including cancer, cardiovascular disorders, angiogenesis, hemophilia,
osteoporosis, diseases of muscle, kidneyand liver, and, last but not the
least, disorders of the central nervous system. In recent years,
zebrafish have been used to study neurodegenerative disorders.
Herewereviewzebrafishmodelsof CNSdiseaseswithan emphasison
studies of degenerative neurological diseases. We also discuss our
recent efforts in extending these approaches to psychiatric disorders
of development. Although unlikely to yield accurate models for
complex psychiatric disorders, genetic insights from studies of
zebrafish are pertinent in helping to identify relevant molecular and
cellular mechanisms of human pathology and, at this level, promise
insight to the development of therapeutics. In particular, the in vivo
biological validation of variants identified in human genetic and
genomic studies provides an important step in defining the
pathological nature of mutations.
Apart from being a vertebrate with common organs and tissues
such as a brain and spinal cord with conserved organization, the
attractiveness of zebrafish as a model lies in its biology and genetics.
Zebrafish have large clutches of externally fertilized and transparent
eggs, which develop rapidly and in synchrony, with neurogenesis
starting around 10 h post-fertilization (hpf), synaptogenesis and the
first behaviors around 18 hpf and hatching around 52 hpf. Within
1 day of development, many pertinent features of the CNS appear and
can be studied in relativelysimple populations of identifiable neurons.
For example, the spinal cord is divided into 30 somites each
containing fewer than a dozen cell types that form relatively simple
circuits. The rapid development of the zebrafish embryo allows for the
study of embryonic-lethal mutations as larvae can survive on their
yolk for up to a week, allowing studies of gene expression and
function throughout early developmental stages.
In addition to its advantageous biology, the second major
advantage of the zebrafish as a model is the simplicity and
effectiveness of manipulating gene expression for cell biological
observations in living embryos with relevance to human pathology.
The zebrafish genome is sequenced and, although not completely
annotated, over 80% of gene structures are available and show a high
degree of synteny (nearest neighboring genes on the chromosomes)
across vertebrate species as well as 50–80% homology with most
human sequences. Homologs for most human genes can be identified
Biochimica et Biophysica Acta 1812 (2011) 335–345
☆ This article is part of a Special Issue entitled Zebrafish Models of Neurological
⁎ Corresponding author. Department of Pathology and Cell Biology, Université de
Montréal, C.P. 6128, Succ. Centre-ville, Montréal, Québec H3C 3J7, Canada. Tel.: +1 514
343 7087; fax: +1 514 343 5755.
E-mail address: firstname.lastname@example.org (P. Drapeau).
0925-4439/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
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of-function approaches. However, the identification of a human
homolog in zebrafish is complicated by the fact that a large number
of genes are duplicated. This phenomenon is explained by whole
genome duplication that occurred during evolution . The amino
acid identity compared to the human gene is always higher for one of
the two genes duplicated, indicating that the duplication appeared
before the species divergence. Therefore, when a gene is knocked
down, it is important to consider that the duplicated gene can
compensate for the loss of function of the targeted homolog. Also, the
functioncouldbe partitioned betweenthetwogenesorsimply lostfor
one of the genes. Comparative sequence analysis allows identifying
the zebrafish homolog of a human gene. Three major genome
browsers, NCBI (www.ncbi.nlm.gov), Ensembl (www.ensembl.org)
and UCSC (/genome.ucsc.edu/), can be use to search homologous gene
sequences. In many cases, the homologous gene predictions can be
found directly from either HomolGene (NCBI) or Orthologue Prediction
(Ensembl). We have foundthat about half of thehundredor so human
synaptic genes on chromosome X (described later) have a predicted
fish ortholog, compared to ~30% in Drosophila and Caenorhabditis
elegans using the same databases. If the homologous gene is not
annotated or if the transcript prediction is not complete, a BLAST
search using nucleotide or protein sequence against the whole
genome assemblies (especially scaffolds based on BAC sequencing)
can allow the identification of homologous genes. TargetP and SignalP
algorithms (www.cbs.dtu.dk/services/) can be used for signal peptide
and cleavage site prediction. An additional phylogenetic analysis and
multiple alignments of protein sequences (Clustal W) help to confirm
the orthology. The syntenic regions can also be compared using Multi
Contig View (Ensembl). For additional information, the Sanger
Institute (www.sanger.ac.uk) has excellent resources about zebrafish
sequence analysis. The sequence identity between zebrafish genes
and their human homologs is higher for ubiquitously expressed genes
than for highly specific genes such as some synaptic genes. The
divergence is mainly found in domains important for membrane
targeting as signal peptides and often in intracellular domains that
contain signaling motifs. Nonetheless, in general, the exon/intron
boundaries and regulatory sequences as well as many other types of
functional domains are evolutionarily conserved between the species.
knockdown by injection of selective anti-sense morpholino oligonu-
cleotides (AMOs) . ZFIN (zfin.org) has the principal database about
AMO gene knockdown and phenotype. The parameters to consider in
AMO design are the same as that for any oligonucleotide molecule: CG
content, stem and loop structure, and the oligo length . Gene-Tools
Inc. (see www.gene-tools.com) offers an AMO design service to the
researchers purchasing their morpholinos. We recommend always
checking if the AMO can bind to off-target sequences by doing a BLAST
against NCBI or Ensembl database. AMOs can be designed to target the
ATG start site to block translation initiation or they can be used to block
splice sites to produce truncated transcripts. The latter have the
advantage of sparing maternal transcripts and permit RT-PCR amplifi-
cation and quantification of the gene knockdown, which is particularly
useful when an antibody is unavailable. Further, the splice AMO can be
used to mimic splicing defects, nonsense or truncation mutations
related to disease. AMOs resist degradation by ribonucleases and are
stable for several days, and in our hands when adequately controlled,
they usually generate selective phenotypes (discussed below). Howev-
er, AMOs must be injected intracellularly as they are more of a
gene knockdown in zebrafish than interfering RNAs. Intracellular
injection is feasible in the zebrafish embryo during the first 1–2 hpf as
the blastomeres are open to the yolk prior to the 4th cell division. Free-
hand injection into the yolk near the blastomere border (Fig. 1) allows
for the injection of a hundred eggs or so in 1–2 h, yielding a sufficient
number of treated embryos to permit statistical analyses of phenotypes
and thus providing a genetic read-out often only 1 day later.
Replacing targeted zebrafish genes with human genes can for
many genes be done as simply as by injection of human mRNA
simultaneously with the AMO as the latter is designed to specifically
target the zebrafish and not the human sequence. We suggest using
the zebrafish homologous cDNA if your gene of interest has a highly
localized expression pattern like that of synaptic genes. Otherwise,
human, mouse, or rat cDNA can be used and easily obtained
commercially. NCBI gives the link to cDNA clones for certain genes.
The gene can be subcloned in expression vectors like pCS2  or
pcGlobin to synthesize the 5′-capped mRNA thatwill be injected in
embryos. The pcGlobin vector has the advantage to have 5′UTR and 3′
UTR from Xenopus, and we also note that pcGlobin gives a better yield
of mRNA. The cDNA can be tagged to MYC, flag, or HA epitopes to
follow the expression in fish. However, large DNA constructs such as
plasmids often yield chimeric expression patterns in limited numbers
of cells presumably as not all dividing progenitors retain the large
constructs. About half of the human genes we have tested permit
partial rescue of knockdown phenotypes, which then allows for
comparison between wild-type human mRNA and mRNA bearing
disease-related mutations. Thus, in the space of a few weeks, each
human variant can be validated in knockdown embryos once a clear
phenotype is recognized. Finally and as described below, recent
transgenic technologies permit the creation of stable, targeted (cell-
specific), and inducible lines for better spatially and temporally
controlled and more detailed analyses of transgenic function.
2. Degenerative brain disorders
Degenerative disorders of the brain, including Alzheimer's, Par-
to have been studied using zebrafish [6,7]. Amyloid-beta plaques are
the strongest biological marker of Alzheimer's disease and the
production of amyloid-beta is regulated by the presenilins 1 and 2
(PS1, PS2). Surprisingly, wt zebrafish zPS1 was found to promote
aberrant amyloid-beta42 secretion when expressed in HEK 293 cells,
mutation of one residue in zPS1 abolished this activity . Truncation
of zPS1 due to loss of exons 8 and 9 upon injection of splice acceptor
and possibly the expression of other Alzheimer's-related genes .
Zebrafish zPS2 is expressed at later stages and is regulated by zPS1
[11,12], but its role in amyloidogenesis in zebrafish is unclear.
Zebrafish possess two homologues of amyloid precursor protein
(APP) with 63–66% homology to their human counterpart , and
their double knockdown causes a convergence–extension develop-
mental defect that is rescued by human APP mRNA but not by a
Swedish mutation related to Alzheimer's disease . The zebrafish
APPs possess a functional gamma-secretase complex to produce
amyloid-beta  and inhibition of gamma-secretase  or knock-
down of Pen-2 , another member of the presinilin complex, blocks
Notch signaling to produce a severe neurogenic phenotype. Early
studies also examined the tau protein in zebrafish as tau is present in
human neurofibrillary tangles, mutations of tau are implicated in
dementia and tau may also contribute to Alzheimer's disease. The
zebrafish studies have shown that expression of human tau (driven
transiently upon injection of a construct with a neural-specific variant
of the GATA2 promoter) results by 2 days in disruption of cytoskeletal
structure, tau trafficking, and hyperphosphorylated fibrillar tau
staining . More recently, stable transgenic zebrafish expressing
mutated P301L human tau and a fluorescent reporter (driven pan-
neuronally from the HuC promoter) have permitted in vivo imaging of
of novel therapeutic molecules . These recent developments with
zebrafish tau and amyloid open the possibility of further screens for
E. Kabashi et al. / Biochimica et Biophysica Acta 1812 (2011) 335–345
in HuC transgenics  or amyloid-beta load in amyloid transgenics
Research into Parkinson's disease has also used zebrafish as a
model . Zebrafish embryos treated with MPTP, a neurodegener-
ative chemical that reproduces some of the effects of idiopathic
Parkinson's disease in mammalian models, demonstrated a loss of
dopaminergic neurons, which could be rescued using the monoamine
oxidase-B inhibitor deprenyl [23–25]. Half a dozen genes have been
identified in Parkinson's disease , and several of these have been
studied to date in zebrafish. For example, in zebrafish, the mRNA for
the ubiquitin processing gene UCH-L1 was detected by 1 day in the
ventral region of the midbrain and hindbrain and in the ventral
diencephalon where it was co-expressed with markers of dopami-
nergic neurons . The zebrafish DJ-1 protein has high (N80%)
homology with human and mouse DJ-1 (of unknown function) and is
expressed throughout the body . Knockdown of DJ-1 in the
zebrafish did not affect the number of dopaminergic neurons, but
zebrafish embryos were more susceptible to oxidative stress and had
elevated SOD1 levels, while simultaneous knockdown of DJ-1 and p53
caused dopaminergic neuronal loss. Recently zebrafish Parkin,
another ubiquitin processing gene, was identified (62% homology
with human PARKIN) and AMO abrogation of its activity leads to a
significant decrease in the number of ascending dopaminergic
neurons in the posterior tuberculum, which is homologous to the
substantia nigra in humans . Screens for compounds promoting
(like MPTP) or preventing (such as caffeine) the Parkinsonian
phenotype in zebrafish  should now be facilitated by the
availability of an enhancer trap line expressing GFP from the vesicular
monoamine transporter 2 promoter .
In zebrafish, the huntingtin (Htt) gene implicated in Huntington's
disease has a predicted 70% identity with the human protein  and
its knockdown disrupts a number of features  and causes massive
neuronal apoptosis due to reduced BDNF expression by 1 day of
development but not earlier . It was recently observed that
injecting mRNA coding for the N-terminal fragment of Htt with
different length polyQ repeats led to developmental abnormalities
and apoptosis in the embryos as early as 1 day later . Embryos
expressing a Q102 mRNA  or a Q56 plasmid  developed
inclusions in the cytoplasm. These Huntington's disease models are
being used to screen for novel therapeutics that could prevent
aggregate formation and clearance, such as anti-prion compounds
, or embryonic death, such as blockers of autophagy .
Another CNS disorder that has been studied recently using
zebrafish is epilepsy  as clonus-like seizures are induced by
convulsant agents [39–42], which has permitted forward mutagenesis
screens and the isolation of seizure-resistant mutants . Zebrafish
are now being used to screen for compounds that suppress seizures
. Furthermore, injection of mRNA with a mutation of the human
PRICKLE1 gene implicated in epilepsy disrupts normal function when
overexpressed in zebrafish , demonstrating the usefulness of
zebrafish for validating mutations of human genes causing brain
Our group has recently collaborated on the discovery of the
MEDNIK syndrome, a rare and severe autosomal recessive neurocu-
taneous disorder manifested by mental retardation, enteropathy,
deafness, neuropathy,ichthyosis, and keratodermiathat is often lethal
[46,47]. Affected individuals bear an A to G mutationin acceptor splice
site of exon 3 of the AP1S1 gene, which leads to a premature stop
codon (Fig. 2A ). The AP1S1 gene encodes the small subunit σ1A of
the first (AP-1) of four ubiquitous clathrin adaptor proteins [48–51].
Each one of the adaptor protein complexes is assembled from four
subunits, and the σ subunit, the one affected in the MEDNIK
syndrome, is part of the AP complex core and is suggested to
contribute to its stabilization. Also, together with the μ subunit, the σ
subunit is possibly involved in protein cargo selection [50,51]. To
demonstrate thatthe mutationin the humanAP1S1 geneindeed alters
the biological function of this gene and underlies the MEDNIK
syndrome, we knocked down the function of the homologous Ap1s1
gene in zebrafish. The zebrafish Ap1s1 protein shares 91% identity
with the human protein. To inhibit zebrafish Ap1s1 mRNA translation,
two types of AMOs were designed (Fig. 2A ). The first AMO
targeted the N-terminal, while the second AMO targeted the acceptor
splice site of intron 2 (Fig. 2A), imitating the mutation found in
MEDNIK patients. Both AMOs caused similar, severe morphological
and behavioral deficits. The 48 hpf KD larva was smaller and had
reduced pigmentation compared to the wild-type(WT) larva. Further,
blocking Ap1s1 translation caused disorganized skin formation in
general, particularly affecting fin morphology (Fig. 2B; for further
information on the epithelial disorders, see Ref. ). In addition to
the morphological deficits, the 48 hpf KD larva responded abnormally
Fig. 1. Rescue of knockdown phenotypes by human mRNA allows for the validation of disease-related variants.
E. Kabashi et al. / Biochimica et Biophysica Acta 1812 (2011) 335–345
to touch by coiling the tail instead of swimming away. The
compromised touch response of the KD larva could be attributed to
a massive reduction (by half) in the spinal neuron population,
specifically due to a loss in the interneuron population. The specificity
of the AMOeffect wasfurtherconfirmed both by Westernblotting and
immunolabeling analysis (Fig. 2C and D). Most importantly, we could
rescue the knockdown phenotype by co-injecting the AMO with
human AP1S1 wild-type mRNA (Fig. 2B and D), which is not targeted
by the Ap1s1 AMO. In contrast, co-injection of AMO with mutated
human AP1S1 mRNA missing exon 3 (Fig. 2A) failed to rescue the skin
and behavioral deficits, suggesting loss of function of this truncated
form of protein (Fig. 2C) and confirming the pathogenic nature of the
mutation found in MEDNIK patients. Further, co-injection of AMO and
an additional human AP1S1 mRNA isoform found in MEDNIK patients,
containing a cryptic splice acceptor site located 9 bp downstream of
the start of the 3rd exon, rescued the phenotype (Fig. 2A). The latter
result suggests that the predicted in frame protein, lacking only 3
amino acids (Fig. 2C), is functional and may explain the viability of the
MEDNIK patients. However, the low expression level of the alterna-
tively spliced RNA (~10%) is insufficient to sustain normal develop-
ment and function, further emphasizing the importance of AP1S1 in
normal development. Our in vivo zebrafish study of Ap1s1 function
revealed its importance for appropriate neurogenesis and skin
development. Accordingly, we could speculate that MEDNIK, a novel
neurocutaneous syndrome, is caused by impaired development of
various neural networks in the spinal cord and in the brain, explaining
the multifunctional human deficits such as ataxia, peripheral
neuropathy, and mental retardation, which are concomitant with a
perturbation in epithelial cell development in the skin and in the
digestive system. We speculate that these generalized effects of AP1S1
Fig. 2. Evaluating a mutation found in human AP1S1 gene (MEDNIK syndrome) using zebrafish. (A) An illustration of the human AP1S1 gene to show the location of the mutation
found inMEDNIK patients. An Ato G mutation inthe acceptor splice site of exon3 resulted inskipping of thisexon and led to a premature stop codon. The use of an alternative cryptic
splice site located 9 bp downstream of the start of the 3rd exon resulted in mRNA lacking 9 bp. The location of the two different morpholinos targeting either ATG or exon 3 acceptor
splice site of the zebrafish ap1s1 gene is indicated in green. The inset on the right depicts the clathrin adaptor protein 1 (AP-1) and its 4 subunits. It is the small subunit σ1 (red),
which is mutated in MEDNIK patients. (B) Ap1s1 knockdown (KD) in zebrafish using morpholino oligo nucleotides targeting the N-terminal or the splice site (splice site, intron 2)
resulted in a similar characteristic morphological phenotype (48 h post-fertilization), which is rescued by co-injection of the wild-type human AP1S1 (rescue+WT HmRNA) or the
mRNA lacking 9 bp (rescue+−9 bp HmRNA), but not by the mutated human mRNA missing exon 3 (rescue+−exon3HmRNA). (C) Illustration of the predicated human AP1S1
proteins: normal protein containing 55AA (WT, black); truncated protein containing 19AA (red), the result of skipping exon 3; and a protein missing 3AA, a result of the alternative
splicing (blue). Western blot analysis of Ap1s1 proteins from human (left) and zebrafish (right) to show that Ap1s1 protein is hardly expressed in MEDNIK patients and in ap1s1
knockdown (KD) zebrafish larva. To normalize the Western blot analysis, proteins extracted from WT, KD, and CTRL larvae were incubated with anti-actin. CTRL=control,
WT=wild type. (D) Localization of Ap1s1 in skin cells of zebrafish wholemounts is illustrated using anti-Ap1s1 antibody immunofluorescence. Cell membrane (polygonal) and a
well-defined perinuclear ring can be nicely observed in both normal (WT) and rescued larvae (rescue+WTHmRNA), whereas only a residual and diffuse staining could be observed
in knockdown (KD) larvae (modified from Ref. ).
E. Kabashi et al. / Biochimica et Biophysica Acta 1812 (2011) 335–345
mutation are due to a widespread deficit in vesicular transport and
3. Degenerative spinal cord disorders
Zebrafish have also been proven to be particularly effective as a
model for motor dysfunction . Our group has worked extensively
on motor neuron diseases, including the most common of these
disorders, amyotrophic lateral sclerosis (ALS). The advantage of
studying motor neuron diseases in zebrafish consists in the rapid
development of the spinal cord and allowing analysis of motor neuron
branching patterns as early as 24 hpf. In addition, responses to touch
and swimming can be monitored following hatching around 48 hpf
. SOD1 (ALS1) is known to cause ALS in 10–20% of familial ALS
cases mainly through an autosomal dominant toxic gain of function
and has 70% amino acid identity to the zebrafish homologue.
Overexpression of mutant SOD1 in zebrafish leads to short motor
axons with premature branching . Interestingly, several other
genes implicated in motoneuron degeneration show a similar
phenotype when tested in zebrafish. Alsin (ALS2) is the gene mutated
in juvenile ALS through autosomal recessive mode of action, which
leads in most cases to protein truncation. Corresponding to this, in
zebrafish embryos, knockdown of Alsin (61% homology) by AMO
causes shortening of motor axons and also loss of neurons in the
spinal cord . ELP3 was identified through association studies
performed in ALS patients and in a Drosophila screen for genes
important for neuronal survival . Knockdown of this gene (ELP3)
in zebrafish caused increased branching and shortened motor axons
. Recently, TDP-43 was found enriched in inclusion bodies from
spinal cord autopsy tissue obtained from ALS patients . Moreover,
about 30 mutations have been identified in a considerable number of
ALS patients, suggesting that this protein plays an important role in
disease pathogenesis [58,59]. The zebrafish ztardbp gene product
(TDP-43) has 73% homology to the human protein. Knockdown of
TDP-43 (tardbp) in zebrafish embryos led to motor neuron branching
and motility deficits (Fig. 3). This AMO phenotype was rescued by co-
expressing human wild-type (WT) TDP-43 (which is not targeted by
the AMO) (Fig. 3). However, three ALS-related mutations of human
TDP-43 identified in both in SALS and FALS cases (A315T, G348C,
A382T) failed to rescue these phenotypes, indicating that these
mutants are pathogenic (Fig. 3). A similar phenotype was observed
when mutant (but not wt) TDP-43 was overexpressed, with the
G348C mutation being the most penetrant (Fig. 3). These results
indicate that TDP-43 mutations cause motor defects, suggesting that
both a loss as well as a gain of function may be involved in the
molecular mechanism of pathogenesis . Finally, it is interesting
to note that a number of groups generated ALS2 knockout mice yet
failed to observe motor deficits associated with motor neuron
degeneration and concluded that ALS2 was not an important gene
for ALS [55,60]. However, the first three exons of ALS2 were left intact
in these mice. We found that the motor phenotype in zebrafish upon
ALS2 KD could be partially rescued upon overexpression of the
alternative transcript of the first three exons of ALS2, indicating that
complete disruption could in fact be pathogenic in ALS . This
nicely illustrates the usefulness of zebrafish knockdown models for
more complete disruption of gene function in some circumstances.
Zebrafish have been also widely used to study the functional role
of gene mutationsin another motorneuron disease, hereditary spastic
paraplegia (HSP). Over 30 loci are known for this disorder, and at least
20 genes have been identified to cause spastic paraplegia . Our
group identified missense variants in the KIAA0196 gene at the SPG8
locus and validated these mutations using the zebrafish model (87%
homology to the human protein) . Knockdown of the zebrafish
homolog of KIAA0196 gene caused a curly-tail phenotype coupled
with shorter motor axons. This phenotype could be partially rescued
by the human WT KIAA0196 but not the two mutants identified in
HSP patients . Recently, SLC33A1 was identified as the gene
responsible for SPG42 . Knockdown of this gene in zebrafish
embryos using AMO caused a curly-tail phenotype and defective axon
outgrowth from the spinal cord . Finally, knockdown of spastin
(SPG4) caused widespread defects in neuronal connectivity and
extensive CNS-specific apoptosis .
Spinal muscular atrophy (SMA) is an autosomally recessive motor
neuron disorder caused by mutations in the survival motor neuron
gene (SMN1). In a series of publications, Beattie's laboratory has
demonstrated that knockdown of the SMN1 in zebrafish embryos
(49% homology to human protein) causes motor axon outgrowth and
pathfinding defects in early development [52,65]. Further, in a series
of elegant rescue experiments, this group was able to show that a
conserved region in exon 7 of the SMN1 gene (QNQKE) is critical for
axonal outgrowth and that the plastin 3 (PLS3) may be important for
axonal outgrowth since overexpression of plastin 3 rescued the
phenotype caused by SMN1 knockdown [66,67]. Finally, transgenic
expression of homozygous deletion found in SMA patients that leads
to truncated SMN1 protein as well as knocking out the Smn1 gene in
zebrafish caused deficits in the neuromuscular junction formation and
death at the larval stage .
4. Developmental disorders
Although this developmental model is proving useful in the
study of late-onset degenerative diseases, zebrafish are just starting
to be used in the study of developmental brain diseases. Interesting
speculations have been made on the potential of zebrafish for
modeling developmental psychiatric disorders such as autism ,
but the lag in advancing these models is perhaps because the
clinical phenotypes are so subtle and specific to humans suffering
from disorders such as autism, schizophrenia, non-syndromic
mental retardation, and others that zebrafish may appear as a
doubtful model. So far (and very recently), the only genes linked to
schizophrenia to be studied in zebrafish using AMO methods are
DISC1 and NRG1 . The receptor tyrosine kinase Met has been
implicated in cerebellar development and autism, and knockdown
of either Met alone or both of its Hgf ligands (two genes in
zebrafish) together using ATG or splice junction AMOs results in
abnormal cerebellar and facial motoneuron development .
However, neither the human homologs of these genes nor their
disease-related mutations have been tested in zebrafish. Our new
ongoing genomics approach, based on large-scale re-sequencing of
human synaptic genes in patients suffering from some of these
disorders, is identifying mutations that can be validated, if not
exactly modeled, in zebrafish.
As a part of our “Synapse to Disease” project, we are using a
combination of AMO knockdown and gene overexpression to
functionally validate novel synaptic gene mutations identified in
large cohorts of patients with autism, schizophrenia, or non-
syndromic mental retardation. Hundreds of synaptic genes were
selected based on published studies and databases. Many genes were
chosen because they are X-linked  as there are many more
affected males than females in autism . Other genes that were
selected are those of the glutamate receptor complex because of their
association to neurodevelopmental diseases . So far, the exonic
regions of over 400 genes have been sequenced, and 15 de novo
mutations (present in the patients and not their parents) were
genetically validated (manuscript submitted). We have identified
deleterious rare de novo mutations in Shank3, IL1RAPL1, NRXN1, and
KIF17 genes, and all of these genes have orthologs in zebrafish. We
have identified a de novo mutation in the Shank3 gene in a patient
with autism  as Shank3 deletions or duplications were previously
identified in patients with autism . Shank3 encodes a scaffolding
protein found in excitatory synapses directly opposite to the pre-
synaptic active zone. The Shank proteins link ionotropic and
E. Kabashi et al. / Biochimica et Biophysica Acta 1812 (2011) 335–345
metabotropic glutamate receptor complexes together and to the
cytoskeleton to regulate the structural organization of dentritic
spines. The splice mutation identified in the Shank3 gene would
result in a truncated protein lacking the Homer, Cortactin, and SAM
domains important for spine induction and dentritic targeting .
The identity to human protein is overall 65% and 62% for shank3a and
shank3b, respectively, but when we compare every individual
functional domain to the human one, the amino acids identity reach
80% and is over 90% for the PDZ domain. The phenotype of Shank3
knockdown zebrafish was not reported in the literature yet, and we
observed major motility deficits that could be rescued by the rat
mRNA but not by some of the mutations .
We also found de novo mutations in IL1RAPL1 in autistic patients
. IL1RAPL1 and 2 are plasma membrane proteins that belong to a
class of the interleukin-1 receptor family characterized by a 150-aa C-
terminal domain thatinteracts withNeuronal Calcium Sensor-1(NCS-
1) [79,80]. A recent study from  used IL1RAPL1-deficient mouse to
show that IL1RAPL1 controls inhibitory network during cerebellar
development. In one patient, a de novo frameshift mutation in
IL1RAPL1 causes a premature stop of the translation (I367fs) resulting
in a truncated protein, lacking the intracellular TIR domain and the C-
terminal domain interacting with NCS-1 . Moreover, a large
deletion of exons 3 to 7 of IL1RAPL1 in three brothers with autism
and/or non-syndromic mental retardation was also identified in our
study. Mutations resulting in deletion of the TIR and the C-terminal
domains were previously identified in patients with non-syndromic
mental retardation [82,83], suggesting that those domains are
important to the protein function. Two genes encode IL1RAPL1 in
zebrafish (il1rapl1a and il1rapl1b) and share 69% and 75% identity,
respectively, with human protein. The divergence is mainly in the
signal peptide (il1rapl1a: 42%, b: 47%). Recently, a study using
zebrafish il1rapl1a showed that the mRNA is constantly expressed
during early embryonic development, indicating that it is maternally
provided . In this study, they also showed that knockdown of the
other gene, il1rapl1b, using an ATG AMO caused a selective loss of
synaptic endings in olfactory neurons and that il1rapl1b C terminus
and TIR domains regulate the synaptic vesicles accumulation and
morphological remodeling of axon terminals during synapse forma-
tion . These observations suggest that il1rapl1b may be essential
for synapse formation and indicate how a loss-of-function IL1RAPL1
mutant could affect brain neurodevelopment and its possible
association to autism and non-syndromic mental retardation. To
determine more specifically the function of IL1RAPL1 in the context of
schizophrenia, we knocked down the expression of zebrafish il1rapl1a
with an ATG AMO and observed a severe phenotype with incomplete
development of the embryo, which could not be rescued by human
mRNA. These preliminary (unpublished) results indicate that
IL1RAPL1 plays an important function during early embryonic
development. Further work, such as with splice junction AMOs that
Fig. 3. Motor phenotype of gain and loss of function of TDP-43 in zebrafish. (A) A schematic representation of TDP-43 protein showing the functional domains, localization of FLAG
(3 kDa) and myc tags (12 kDa), and sites where the G348C mutation found in ALS patients was introduced by site-directed mutagenesis. (B) Locomotor phenotype. Zebrafish
embryos develop a touch-evoked escape response as seen when embryos are injected with WT human TDP-43 RNA. Expression of TDP-43 G348C RNA causes a deficiency in the
touch-evoked response. A similar phenotype is observed when the zf tdp-43 expression was knocked down using a specific AMO. This phenotype was rescued with expression of WT
but not G348C RNA. (C) Motor axonal deficits. The behavioral (motor) phenotype was selectively associated with a shortening of the axon and premature branching in motor
neurons (see G348C TDP-43 and tdp-43AMO) (modified from Kabashi et al., accepted at Hum. Mol. Genet.).
E. Kabashi et al. / Biochimica et Biophysica Acta 1812 (2011) 335–345
spare maternal transcripts that are essential for early development, is
required in order to develop a pathogenic validation.
Neurexins are predominantly pre-synaptic cell-adhesion mole-
cules. They can induce pre-synaptic differentiation by interacting with
neuroligins. There are three neurexin genes (NRXN1, 2, and 3), each of
which encodes two major variants (alpha and beta). Only NRXN1 gene
was shown to be disrupted using CNV analysis in autism [85,86]. More
recently, NRXN1 gene has been involved in CNV found in SCZ patients
[87,88]. We have identified an insertion of 4 bp predicted to cause a
frameshift with a premature stop affecting the two major variants
alpha and beta of NRXN1 in SCZ patient. The protein is predicted to
miss the transmembrane and intracellular domains. Two orthologs of
NRXN1 have been identified in zebrafish (Nrxn1a and 1b) with an
identity to the human protein over 70%. The greatest variability is
found in the signal peptide (Nrxn1a, 27%; 1b 32%). An expression
analysis showed that all three Nrxn genes are expressed during
zebrafish embryonic development and some specific isoforms of
Nrxn1a expressed at different stage of the development . These
results indicate the potential for developing zebrafish models of
neurexin mutations. However, the fact that there exist thousands of
isoforms of Nrxn1 and the amino acids of the signal peptide are
divergent could make the functional validation in zebrafish more
Kinesin 17 (KIF17) is a member of the kinesin superfamily
containing a motor domain for ATP hydrolysis. KIF17 binds to the
scafolding Mint1 to transport the NMDA receptor subunit 2B (NR2B)
to the dendrites along microtubules [90,91]. In addition, the K+
channel Kv4.2, a major regulator of dendritic excitability, is also
transported to the dentrites by KIF17 . In mice, overexpression of
Kif17 enhances spatial and working memory . The expression of
KIF17 was shown to be altered in a mouse models for Down syndrome
(trisomy 21) leading to learning deficit . We have identified a
nonsense mutation resulting in a protein that lacks the tail domain in
a SCZ patient. This domain was shown to interact with Mint1
following its phosphorylation by CaMKII . One orthologous
sequence was identified in zebrafish with an identity of 61% compare
to the human amino acids. The divergence was mainly observed in the
motifs important for cargo binding. The zebrafish KIF17 is widely
expressed in the nervous system and retina . By using low doses
splicing AMO, this group demonstrates that KIF17 is essential for
vertebrate photoreceptor development and seems to have little effect
on the global zebrafish development. At higher doses of either an ATG
or a splice junction AMO that micked the effect of the de novo
nonsense truncation mutation in schizophrenia, we observed a
characteristic dose-dependent morphological trait with stunted,
curly embryos . This validates that the nonsense mutation is a
loss-of-function pathogenic effect.
Together, these results with several zebrafish developmental
genes indicate the potential usefulness of this model for validating
brain disease mutations, but clearly each gene requires its own set of
carefully designed experiments. Whereas some human genes can be
more difficult to study in zebrafish, with others, one can observe
faithful disease phenotypes that can be characterized in detail such as
for AP1S1 in MEDNIK and ALS2 in ALS and thus provide novel insights
zebrafish that are not as obvious in knockout mice, which may reflect
a lower degree of compensation in zebrafish, perhaps because their
reproductive biology is based more on the quantity of offspring rather
than necessarily on the quality of developmental compensation.
5. Future directions
Several approaches have the potential of further advancing
zebrafish models of human CNS disorders and making them uniquely
useful, in particular targeted expression, multigenic analysis and
5.1. Targeted expression
Most of the approaches taken to date to express foreign genes in
zebrafish are based on injection of mRNA for transient expression
throughout the embryo. This simple approach is valid for ubiqui-
tously and early expressed genes such as AP1S1, SOD1, TARDBP and
members of the ubiquination and beta-secretase complexes dis-
cussed above. However, mRNA injection risks expressing genes
ectopically, such as in the case of synaptic genes, and is usually only
efficient at embryonic stages, prior to mRNA degradation that occurs
within a few days. The latter thus limits the time course for
phenotypic analysis, which is particularly problematic for studies of
neurogeneration in (often) adult-onset human diseases. More
specific expression patterns can be achieved by stable transgenesis
using the efficient Tol2 transposon system , but this is best suited
for generating long-term models (rather than for preliminary or
large-scale screens) as it is a slower process that requires the raising
of founder fish and their out-crossing to verify stable and specific
transgenic lines. A number of promoters are available for neural-
specific transgenic expression. For example, the α-tubulin promoter
 was the first used to drive pan-neuronal expression and, as
described above, the HuC promoter expressed in post-mitotic
neurons  and a neural-specific variant of the GATA2 promoter
 are also effective at early stages. More selective subsets of
neurons can be targeted using more restrictive promoters such as the
dopaminergic-specific vesicular monoamine transporter 2 promoter
. In the spinal cord, which is an important region for studies of
motor dysfunction and synaptic transmission, the developmentally
conserved transcriptional code  has permitted the generation of
transgenic lines for selective types of neurons. These include
motoneurons: HB9 , Isl-1 , commissural neurons: Evx1
, mostly glycinergic neurons: Pax2.1 ; vGlyT2 , mostly
glutamatergic neurons: Alx/Vsx2 , as well as sensory neurons:
Ngn1  and oligodendrocytes: Nkx2.2a , olig2  and
However, these stable transgenic models also have their limita-
tions, and here, we consider three major ones that can be at least
partially avoided depending on necessity: ectopic expression, toxic
expression, and genetic background. Although promoter-based stable
transgenic lines confer more selective expression patterns, rarely do
they perfectly recapitulate the natural expression patterns as often
only subsets of enhancer elements are used in generating the
transgenic constructs. Indeed, this is a limitation of the lack of a
knock-in technology in zebrafish, requiring the integration of novel
constructs in a wild-type background (considered below). However,
perfectly faithful expression patterns are not always essential as
expression in incomplete or expanded subsets of neurons can be
useful to study. An approach that provides a more accurate expression
pattern is the use of bacterial artificial chromosomes (BACs), such as
with the amyloid line mentioned above . In principle (although
we are unaware of its practice with zebrafish), humanized models
could be generated based on transgenesis with human BACs, as is
commonly done with mice. However, vertebrate genes can be
hundreds of kilobases in size and are not always completely contained
within single BACs. As an alternative, pufferfish (Takifugu rubripes)
BACs can be used as the intergenic regions are much smaller, an
approach we have taken in generating an Evx1 transgenic line ,
although even in this case less than 80% of the neurons labeled by a
selective antibody to evx1 also expressed GFP, indicating that not all
evx1 cells transgenically express GFP.
A second issue in considering stable transgenic lines is toxicity, as
many of the transgenes bearing disease-related mutations can be
embryonic-lethal. A simple approach is to drive expression from a
heat shock promoter , although this may lose in generalized
spatial expression what it gains in restricted temporal activation. A
powerful alternative is the use of combinatorial expression systems,
E. Kabashi et al. / Biochimica et Biophysica Acta 1812 (2011) 335–345
such as the cre-loxP systemdeveloped in mice andthe yeast Gal4-UAS
binary system popular for invertebrate genetics, both of which have
found applications in zebrafish [111,112]. Examples are the Evx1-Gal4
 and HuC-Gal4  lines described earlier. The latter drives
transcription in both directions from the UAS in order to express two
constructs simultaneoulsy, such as a gene of interest (in this case
human P301L tau) and a reporter (dsRed) in the same cells. Another
technique for expressing more than one gene from the same construct
is to use the self-cleaving viral 2A peptide as a linker between the
genes of interest .
A final concern is the creation of transgenic lines in a genetic
background containing the endogenous gene as (in the absence of
knock-in technology) this results in the addition of another gene. One
possibility that is rarely used is to create a transgenic in a knockout
background. In principle, this can be done if mutants have been
isolated in screens for related phenotypes or by TILLING (Targeted
Induced Local Lesions in Genomes)  available through a new
consortium (see https://webapps.fhcrc.org/science/tilling/). A new
technology is the targeted lesioning of genes by zinc finger nucleases
(ZFN) , although this is a complex and expensive technology that
at the moment is beyond the reach of small laboratories. Preliminary
success in targeting genes in zebrafish is encouraging, and the
possibility to use homologous recombination, rather than non-
homologous end joining, to repair ZFN-induced double-strand breaks
may permit the development of a knock-in approach for zebrafish
. Much works remain to be done before this enticing possibility
becomes practical, but nonetheless, a number of transgenic
approaches are now available for refined studies of gene expression
and function in zebrafish, including human genes bearing disease-
5.2. Multigenic analysis
Although highly penetrant mutations of single genes are often
implicated in CNS disorders, such as the majority considered in this
review, most diseases are multigenic in nature, requiring multiple
weakly penetrating mutations in predisposing genetic backgrounds in
order to produce the disease. Existing genetic models most easily
address single gene mutations (or rather have focused on these
simple cases), but clearly, the future of these models lies in their
ability to provide insights to the nature of genetic interactions in
disease. The zebrafish may prove to be particularly amenable to
multigenic analysis by a combination of the approaches described
above. The use of two or even three AMOs is a simple solution once
each AMO is validated. For example, in the context of Parkinson's
disease, simultaneous knockdown of DJ-1 and p53 was found to cause
dopaminergic neuronal loss  and knockdown of both Hgf ligands
was necessary to induce Met-related cerebellar defects . Also,
photoactivatable caged AMOs can be used to release these at specific
time points . This approach seems promising, but unfortunately,
caged AMOs are not available commercially yet. Another major
limitation of the AMO is the necessityto inject in embryo at 1 or 2 cells
stage. An interesting alternative would be to use the vivo-AMOs that
allow making gene knockdown and splice modification in adult
animals. The vivo-AMOs are modified AMO able to enter cells by
endocytosis. They could be conjugated with a dendrimeric octagua-
nidine (Gene Tools) or a cell-penetrating peptide linked to phosphor-
odiamidate (PPMO) (AVI BioPharma Inc.). Vivo-AMOs were shown to
be effective in mice [118,119], and they are currently tested in adult
By performing AMO injections in mutants or transgenics, either
stable or inducible, it should in principle be possible to readily test
several genes simultaneously or in different combinations, bestowing
zebrafish with a polyvalence that is difficult to envisage for other
models such as mice. In the case of mutant and transgenic back-
grounds, comparative microarray screening  becomes feasible,
particularly as multiple conditions (e.g., different mutant alleles)
permit internal verification and comparison of the array results. A
pertinent example is the recent microarray analysis  of three
alleles of the neurogenic mindbomb mutant affecting Notch signaling
for which each allele affected the expression of hundreds of genes but
less than 100 were commonly affected. Thus, zebrafish have a unique
potential for rapidly analyzing multiple genes and dissecting complex
5.3. Chemical genetics
While zebrafish retain advantages for studying the genetics,
biology, and pathobiology of disease genes of interest, they also
have the major advantage of being the only vertebrate model
amenable to large chemical genetic screens . Small chemical
libraries of over 10,000 molecules, including approved drugs and
randomly synthesized organic molecules of unknown function, have
been screened in zebrafish models of cancer  and cardiovascular
disorders , and some of these are in clinical testing. With the
numerous models of brain diseases being developed in zebrafish, this
approach holds much potential for drug discovery. This is particularly
attractive in zebrafish as functional chemical genetic screens in vivo
with living embryos offer the potential of discovering therapeutics
without prior knowledge or need for rational screening designs. Thus,
zebrafish are a unique model ranging from molecular genetic
validation to drug discovery with important applications on the
horizon for brain diseases, including many complex and untreatable
 J.N. Volff, Genome evolution and biodiversity in teleost fish, Heredity 94 (2005)
 A. Nasevicius, S.C. Ekker, Effective targeted gene ‘knockdown’ in zebrafish, Nat.
Genet. 26 (2000) 216–220.
 J.D. Moulton, Y.L. Yan, Using morpholinos to control gene expression, Curr.
Protoc. Mol. Biol. (2008) Chapter 26, Unit 26 28.
 D.L. Turner, H. Weintraub, Expression of achaete-scute homolog 3 in Xenopus
embryos converts ectodermal cells to a neural fate, Genes Dev. 8 (1994)
 H. Ro, K. Soun, E.J. Kim, M. Rhee, Novel vector systems optimized for injecting in
vitro-synthesized mRNA into zebrafish embryos, Mol. Cells 17 (2004) 373–376.
 J.D. Best, W.K. Alderton, Zebrafish: an in vivo model for the study of neurological
diseases, Neuropsychiatr. Dis. Treat. 4 (2008) 567–576.
 P.W. Ingham, The power of the zebrafish for disease analysis, Hum. Mol. Genet.
18 (2009) R107–R112.
 U. Leimer, K. Lun, H. Romig, J. Walter, J. Grunberg, M. Brand, C. Haass, Zebrafish
(Danio rerio) presenilin promotes aberrant amyloid beta-peptide production
and requires a critical aspartate residue for its function in amyloidogenesis,
Biochemistry 38 (1999) 13602–13609.
 S. Nornes, M. Newman, G. Verdile, S. Wells, C.L. Stoick-Cooper, B. Tucker, I.
Frederich-Sleptsova, R. Martins, M. Lardelli, Interference with splicing of
presenilin transcripts has potent dominant negative effects on presenilin
activity, Hum. Mol. Genet. 17 (2008) 402–412.
 M. Newman, B. Tucker, S. Nornes, A. Ward, M. Lardelli, Altering presenilin gene
activity in zebrafish embryos causes changes in expression of genes with
potential involvement in Alzheimer's disease pathogenesis, J. Alzheimers Dis. 16
 C. Groth, S. Nornes, R. McCarty, R. Tamme, M. Lardelli, Identification of a second
presenilin gene in zebrafish with similarity to the human Alzheimer's disease
gene presenilin2, Dev. Genes Evol. 212 (2002) 486–490.
 S. Nornes, C. Groth, E. Camp, P. Ey, M. Lardelli, Developmental control of
presenilin1 expression, endoproteolysis, and interaction in zebrafish embryos,
Exp. Cell Res. 289 (2003) 124–132.
 A. Musa, H. Lehrach, V.A. Russo, Distinct expression patterns of two zebrafish
homologues of the human APP gene during embryonic development, Dev. Genes
Evol. 211 (2001) 563–567.
 P. Joshi, J.O. Liang, K. DiMonte, J. Sullivan, S.W. Pimplikar, Amyloid precursor
protein is required for convergent-extension movements during zebrafish
development, Dev. Biol. 335 (2009) 1–11.
 M. Newman, I.F. Musgrave, M. Lardelli, Alzheimer disease: amyloidogenesis, the
presenilins and animal models, Biochim. Biophys. Acta 1772 (2007) 285–297.
 A. Geling, H. Steiner, M. Willem, L. Bally-Cuif, C. Haass, A gamma-secretase
inhibitor blocks Notch signaling in vivo and causes a severe neurogenic
phenotype in zebrafish, EMBO Rep. 3 (2002) 688–694.
 W.A. Campbell, H. Yang, H. Zetterberg, S. Baulac, J.A. Sears, T. Liu, S.T. Wong, T.P.
Zhong, W. Xia, Zebrafish lacking Alzheimer presenilin enhancer 2 (Pen-2)
E. Kabashi et al. / Biochimica et Biophysica Acta 1812 (2011) 335–345
demonstrate excessive p53-dependent apoptosis and neuronal loss, J. Neuro-
chem. 96 (2006) 1423–1440.
 H.G. Tomasiewicz, D.B. Flaherty, J.P. Soria, J.G. Wood, Transgenic zebrafish model
of neurodegeneration, J. Neurosci. Res. 70 (2002) 734–745.
 D. Paquet, R. Bhat, A. Sydow, E.M. Mandelkow, S. Berg, S. Hellberg, J. Falting, M.
Distel, R.W. Koster, B. Schmid, C. Haass, A zebrafish model of tauopathy allows in
vivo imaging of neuronal cell death and drug evaluation, J. Clin. Invest. 119
 J.A. Lee, G.J. Cole, Generation of transgenic zebrafish expressing green
fluorescent protein under control of zebrafish amyloid precursor protein gene
regulatory elements, Zebrafish 4 (2007) 277–286.
 L.A. Shakes, T.L. Malcolm, K.L. Allen, S. De, K.R. Harewood, P.K. Chatterjee,
Context dependent function of APPb enhancer identified using enhancer trap-
containing BACs as transgenes in zebrafish, Nucleic Acids Res. 36 (2008)
 L. Flinn, S. Bretaud, C. Lo, P.W. Ingham, O. Bandmann, Zebrafish as a new animal
model for movement disorders, J. Neurochem. 106 (2008) 1991–1997.
 S. Bretaud, S. Lee, S. Guo, Sensitivity of zebrafish to environmental toxins
implicated in Parkinson's disease, Neurotoxicol. Teratol. 26 (2004) 857–864.
 C.S. Lam, V. Korzh, U. Strahle, Zebrafish embryos are susceptible to the
dopaminergic neurotoxin MPTP, Eur. J. Neurosci. 21 (2005) 1758–1762.
 E.T. McKinley, T.C. Baranowski, D.O. Blavo, C. Cato, T.N. Doan, A.L. Rubinstein,
Neuroprotection of MPTP-induced toxicity in zebrafish dopaminergic neurons,
Brain Res. Mol. Brain Res. 141 (2005) 128–137.
 A. Abeliovich, M. Flint Beal, Parkinsonism genes: culprits and clues, J.
Neurochem. 99 (2006) 1062–1072.
 O.L. Son, H.T. Kim, M.H. Ji, K.W. Yoo, M. Rhee, C.H. Kim, Cloning and expression
analysis of a Parkinson's disease gene, uch-L1, and its promoter in zebrafish,
Biochem. Biophys. Res. Commun. 312 (2003) 601–607.
 S. Bretaud, C. Allen, P.W. Ingham, O. Bandmann, p53-dependent neuronal cell
death in a DJ-1-deficient zebrafish model of Parkinson's disease, J. Neurochem.
100 (2007) 1626–1635.
 L. Flinn, H. Mortiboys, K. Volkmann, R.W. Koster, P.W. Ingham, O. Bandmann,
Complex I deficiency and dopaminergic neuronal cell loss in Parkin-deficient
zebrafish (Danio rerio), Brain 132 (2009) 1613–1623.
 W. Boehmler, J. Petko, M. Woll, C. Frey, B. Thisse, C. Thisse, V.A. Canfield, R.
Levenson, Identification of zebrafish A2 adenosine receptors and expression in
developing embryos, Gene Expr. Patterns 9 (2009) 144–151.
 L. Wen, W. Wei, W. Gu, P. Huang, X. Ren, Z. Zhang, Z. Zhu, S. Lin, B. Zhang,
Visualization of monoaminergic neurons and neurotoxicity of MPTP in live
transgenic zebrafish, Dev. Biol. 314 (2008) 84–92.
 C.A. Karlovich, R.M. John, L. Ramirez, D.Y. Stainier, R.M. Myers, Characterization
of the Huntington's disease (HD) gene homologue in the zebrafish Danio rerio,
Gene 217 (1998) 117–125.
 A.L. Lumsden, T.L. Henshall, S. Dayan, M.T. Lardelli, R.I. Richards, Huntingtin-
deficient zebrafish exhibit defects in iron utilization and development, Hum.
Mol. Genet. 16 (2007) 1905–1920.
 H. Diekmann, O. Anichtchik, A. Fleming, M. Futter, P. Goldsmith, A. Roach, D.C.
Rubinsztein, Decreased BDNF levels are a major contributor to the embryonic
phenotype of huntingtin knockdown zebrafish, J. Neurosci. 29 (2009)
 N.W. Schiffer, S.A. Broadley, T. Hirschberger, P. Tavan, H.A. Kretzschmar, A. Giese,
C. Haass, F.U. Hartl, B. Schmid, Identification of anti-prion compounds as efficient
inhibitors of polyglutamine protein aggregation in a zebrafish model, J. Biol.
Chem. 282 (2007) 9195–9203.
 V.M. Miller, R.F. Nelson, C.M. Gouvion, A. Williams, E. Rodriguez-Lebron, S.Q.
aggregation and toxicity in vitro and in vivo, J. Neurosci. 25 (2005) 9152–9161.
 A. Williams, S. Sarkar, P. Cuddon, E.K. Ttofi, S. Saiki, F.H. Siddiqi, L. Jahreiss, A.
Fleming, D. Pask, P. Goldsmith, C.J. O'Kane, R.A. Floto, D.C. Rubinsztein, Novel
targets for Huntington's disease in an mTOR-independent autophagy pathway,
Nat. Chem. Biol. 4 (2008) 295–305.
 S.C. Baraban, Emerging epilepsy models: insights from mice, flies, worms and
fish, Curr. Opin. Neurol. 20 (2007) 164–168.
 J.A. Tiedeken, J.S. Ramsdell, DDT exposure of zebrafish embryos enhances seizure
susceptibility: relationship to fetal p,p′-DDE burden and domoic acid exposure of
California sea lions, Environ. Health Perspect. 117 (2009) 68–73.
 S.C. Baraban, M.R. Taylor, P.A. Castro, H. Baier, Pentylenetetrazole induced
changesin zebrafish behavior, neural activity and c-fos expression, Neuroscience
131 (2005) 759–768.
 J.A. Tiedeken, J.S. Ramsdell, Embryonic exposure to domoic acid increases the
susceptibility of zebrafish larvae to the chemical convulsant pentylenetetrazole,
Environ. Health Perspect. 115 (2007) 1547–1552.
 J.A. Tiedeken, J.S. Ramsdell, A.F. Ramsdell, Developmental toxicity of domoic acid
in zebrafish (Danio rerio), Neurotoxicol. Teratol. 27 (2005) 711–717.
 S.C. Baraban, M.T. Dinday, P.A. Castro, S. Chege, S. Guyenet, M.R. Taylor, A large-
scale mutagenesis screen to identify seizure-resistant zebrafish, Epilepsia 48
 M.J. Winter, W.S. Redfern, A.J. Hayfield, S.F. Owen, J.P. Valentin, T.H. Hutchinson,
Validation of a larval zebrafish locomotor assay for assessing the seizure liability
of early-stage development drugs, J. Pharmacol. Toxicol. Methods 57 (2008)
 A.G. Bassuk, R.H. Wallace, A. Buhr, A.R. Buller, Z. Afawi, M. Shimojo, S. Miyata, S.
Chen, P. Gonzalez-Alegre, H.L. Griesbach, S. Wu, M. Nashelsky, E.K. Vladar, D.
Antic, P.J. Ferguson, S. Cirak, T. Voit, M.P. Scott, J.D. Axelrod, C. Gurnett, A.S.
Daoud, S. Kivity, M.Y. Neufeld, A. Mazarib, R. Straussberg, S. Walid, A.D. Korczyn,
D.C. Slusarski, S.F. Berkovic, H.I. El-Shanti, A homozygous mutation in human
PRICKLE1 causes an autosomal-recessive progressive myoclonus epilepsy–ataxia
syndrome, Am. J. Hum. Genet. 83 (2008) 572–581.
 T.G. Saba, A. Montpetit, A. Verner, P. Rioux, T.J. Hudson, R. Drouin, C.A. Drouin, An
atypical form of erythrokeratodermia variabilis maps to chromosome 7q22,
Hum. Genet. 116 (2005) 167–171.
 A. Montpetit, S. Cote, E. Brustein, C.A. Drouin, L. Lapointe, M. Boudreau, C.
Meloche, R. Drouin, T.J. Hudson, P. Drapeau, P. Cossette, Disruption of AP1S1,
causing a novel neurocutaneous syndrome, perturbs development of the skin
and spinal cord, PLoS Genet. 4 (2008) e1000296.
 J.S. Bonifacino, B.S. Glick, The mechanisms of vesicle budding and fusion, Cell 116
 M.S. Robinson, Adaptable adaptors for coated vesicles, Trends Cell Biol. 14
 M. Boehm, J.S. Bonifacino, Adaptins: the final recount, Mol. Biol. Cell 12 (2001)
 D.J. Owen, B.M. Collins, P.R. Evans, Adaptors for clathrin coats: structure and
function, Annu. Rev. Cell Dev. Biol. 20 (2004) 153–191.
 C.E. Beattie, T.L. Carrel, M.L. McWhorter, Fishing for a mechanism: using zebrafish to
understand spinal muscular atrophy, J. Child Neurol. 22 (2007) 995–1003.
 P. Drapeau, L. Saint-Amant, R.R. Buss, M. Chong, J.R. McDearmid, E. Brustein,
Development of the locomotor network in zebrafish, Prog. Neurobiol. 68 (2002)
 R. Lemmens, A. Van Hoecke, N. Hersmus, V. Geelen, I. D'Hollander, V. Thijs, L. Van
Den Bosch, P. Carmeliet, W. Robberecht, Overexpression of mutant superoxide
dismutase 1 causes a motor axonopathy in the zebrafish, Hum. Mol. Genet. 16
 F. Gros-Louis, J. Kriz, E. Kabashi, J. McDearmid, S. Millecamps, M. Urushitani, L.
Lin, P. Dion, Q. Zhu, P. Drapeau, J.P. Julien, G.A. Rouleau, Als2 mRNA splicing
variants detected in KO mice rescue severe motor dysfunction phenotype in
Als2 knock-down zebrafish, Hum. Mol. Genet. 17 (2008) 2691–2702.
 C.L. Simpson, R.Lemmens, K. Miskiewicz, W.J. Broom, V.K.Hansen, P.W.van Vught,
H.R. Horvitz, P.N. Leigh, C.E. Shaw, L.H. van den Berg, P.C. Sham, J.F. Powell, P.
Verstreken, R.H. Brown Jr., W. Robberecht, A. Al-Chalabi, Variants of the elongator
protein 3 (ELP3) gene are associated with motor neuron degeneration, Hum. Mol.
Genet. 18 (2009) 472–481.
 M. Neumann, D.M. Sampathu, L.K. Kwong, A.C. Truax, M.C. Micsenyi, T.T. Chou, J.
Bruce, T. Schuck, M. Grossman, C.M. Clark, L.F. McCluskey, B.L. Miller, E. Masliah,
I.R. Mackenzie, H. Feldman, W. Feiden, H.A. Kretzschmar, J.Q. Trojanowski, V.M.
Lee, Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyo-
trophic lateral sclerosis, Science 314 (2006) 130–133.
 E. Kabashi, P.N. Valdmanis, P. Dion, D. Spiegelman, B.J. McConkey, C. Vande
Velde, J.P. Bouchard, L. Lacomblez, K. Pochigaeva, F. Salachas, P.F. Pradat, W.
Camu, V. Meininger, N. Dupre, G.A. Rouleau, TARDBP mutations in individuals
with sporadic and familial amyotrophic lateral sclerosis, Nat. Genet. 40 (2008)
 J. Sreedharan, I.P. Blair, V.B. Tripathi, X. Hu, C. Vance, B. Rogelj, S. Ackerley, J.C.
Durnall, K.L. Williams, E. Buratti, F. Baralle, J. de Belleroche, J.D. Mitchell, P.N.
Leigh, A. Al-Chalabi, C.C. Miller, G. Nicholson, C.E. Shaw, TDP-43 mutations in
familial and sporadic amyotrophic lateral sclerosis, Science 319 (2008)
 S. Hadano, S.C. Benn, S. Kakuta, A. Otomo, K. Sudo, R. Kunita, K. Suzuki-
Utsunomiya, H. Mizumura, J.M. Shefner, G.A. Cox, Y. Iwakura, R.H. Brown Jr., J.E.
Ikeda, Mice deficient in the Rab5 guanine nucleotide exchange factor ALS2/alsin
exhibit age-dependent neurological deficits and altered endosome trafficking,
Hum. Mol. Genet. 15 (2006) 233–250.
 S. Salinas, C. Proukakis, A. Crosby, T.T. Warner, Hereditary spastic paraplegia:
clinical features and pathogenetic mechanisms, Lancet Neurol. 7 (2008)
 P.N. Valdmanis, I.A. Meijer, A. Reynolds, A. Lei, P. MacLeod, D. Schlesinger, M.
Zatz, E. Reid, P.A. Dion, P. Drapeau, G.A. Rouleau, Mutations in the KIAA0196 gene
at the SPG8 locus cause hereditary spastic paraplegia, Am. J. Hum. Genet. 80
 P. Lin, J. Li, Q. Liu, F. Mao, R. Qiu, H. Hu, Y. Song, Y. Yang, G. Gao, C. Yan, W. Yang, C.
Shao, Y. Gong, A missense mutation in SLC33A1, which encodes the acetyl-CoA
transporter, causes autosomal-dominant spastic paraplegia (SPG42), Am. J.
Hum. Genet. 83 (2008) 752–759.
 J.D. Wood, J.A. Landers, M. Bingley, C.J. McDermott, V. Thomas-McArthur, L.J.
Gleadall, P.J. Shaw, V.T. Cunliffe, The microtubule-severing protein Spastin is
essential for axon outgrowth in the zebrafish embryo, Hum. Mol. Genet. 15
 M.L. McWhorter, U.R. Monani, A.H. Burghes, C.E. Beattie, Knockdown of the
survival motor neuron (Smn) protein in zebrafish causes defects in motor axon
outgrowth and pathfinding, J. Cell Biol. 162 (2003) 919–931.
 T.L. Carrel, M.L. McWhorter, E. Workman, H. Zhang, E.C. Wolstencroft, C. Lorson,
G.J. Bassell, A.H. Burghes, C.E. Beattie, Survival motor neuron function in motor
axons is independent of functions required for small nuclear ribonucleoprotein
biogenesis, J. Neurosci. 26 (2006) 11014–11022.
 G.E. Oprea, S. Krober, M.L. McWhorter, W. Rossoll, S. Muller, M. Krawczak, G.J.
Bassell, C.E. Beattie, B. Wirth, Plastin 3 is a protective modifier of autosomal
recessive spinal muscular atrophy, Science 320 (2008) 524–527.
 K.L. Boon, S. Xiao, M.L. McWhorter, T. Donn, E. Wolf-Saxon, M.T. Bohnsack, C.B.
Moens, C.E. Beattie, Zebrafish survival motor neuron mutants exhibit presyn-
aptic neuromuscular junction defects, Hum. Mol. Genet. 18 (2009) 3615–3625.
E. Kabashi et al. / Biochimica et Biophysica Acta 1812 (2011) 335–345
 V. Tropepe, H.L. Sive, Can zebrafish be used as a model to study the
neurodevelopmental causes of autism? Genes Brain Behav. 2 (2003) 268–281.
 J.D. Wood, F. Bonath, S. Kumar, C.A. Ross, V.T. Cunliffe, Disrupted-in-
schizophrenia 1 and neuregulin 1 are required for the specification of
oligodendrocytes and neurones in the zebrafish brain, Hum. Mol. Genet. 18
 G.E. Elsen, L.Y. Choi, V.E. Prince, R.K. Ho, The autism susceptibility gene met
regulates zebrafish cerebellar development and facial motor neuron migration,
Dev. Biol. 335 (2009) 78–92.
 F. Laumonnier, P.C. Cuthbert, S.G. Grant, The role of neuronal complexes in
human X-linked brain diseases, Am. J. Hum. Genet. 80 (2007) 205–220.
 D.H. Skuse, Imprinting, the X-chromosome, and the male brain: explaining sex
differences in the liability to autism, Pediatr. Res. 47 (2000) 9–16.
 S.G. Grant, M.C. Marshall, K.L. Page, M.A. Cumiskey, J.D. Armstrong, Synapse
proteomics of multiprotein complexes: en route from genes to nervous system
diseases, Hum. Mol. Genet. 14 (Spec No. 2) (2005) R225–R234.
 J. Gauthier, D. Spiegelman, A. Piton, R.G. Lafreniere, S. Laurent, J. St-Onge, L.
Lapointe, F.F. Hamdan, P. Cossette, L. Mottron, E. Fombonne, R. Joober, C.
Marineau, P. Drapeau, G.A. Rouleau, Novel de novo SHANK3 mutation in autistic
patients, Am. J. Med. Genet. B Neuropsychiatr. Genet. 150B (2009) 421–424.
 C.M. Durand, C. Betancur, T.M. Boeckers, J. Bockmann, P. Chaste, F. Fauchereau, G.
Nygren, M. Rastam, I.C. Gillberg, H. Anckarsater, E. Sponheim, H. Goubran-Botros,
R. Delorme, N. Chabane, M.C. Mouren-Simeoni, P. de Mas, E. Bieth, B. Roge, D.
Heron, L. Burglen, C. Gillberg, M. Leboyer, T. Bourgeron, Mutations in the gene
encoding the synaptic scaffolding protein SHANK3 are associated with autism
spectrum disorders, Nat. Genet. 39 (2007) 25–27.
 G. Roussignol, F. Ango, S. Romorini, J.C. Tu, C. Sala, P.F. Worley, J. Bockaert, L.
Fagni, Shank expression is sufficient to induce functional dendritic spine
synapses in aspiny neurons, J. Neurosci. 25 (2005) 3560–3570.
 A. Piton, J.L. Michaud, H. Peng, S. Aradhya, J. Gauthier, L. Mottron, N. Champagne,
Dube, P. Haghighi, P. Drapeau, P.A. Barker, S. Carbonetto, G.A. Rouleau, Mutations
in the calcium-related gene IL1RAPL1 are associated with autism, Hum. Mol.
Genet. 17 (2008) 3965–3974.
 N. Bahi, G. Friocourt, A. Carrie, M.E. Graham, J.L. Weiss, P. Chafey, F. Fauchereau,
R.D. Burgoyne, J. Chelly, IL1 receptor accessory protein like, a protein involved in
X-linked mental retardation, interacts with Neuronal Calcium Sensor-1 and
regulates exocytosis, Hum. Mol. Genet. 12 (2003) 1415–1425.
 F. Gambino, A.Pavlowsky, A.Begle, J.L. Dupont, N. Bahi, R. Courjaret, R. Gardette, H.
Hadjkacem, H. Skala, B. Poulain, J. Chelly, N. Vitale, Y. Humeau, IL1-receptor
accessory protein-like 1 (IL1RAPL1), a protein involved in cognitive functions,
regulates N-type Ca2+-channeland neurite elongation,Proc. Natl. Acad. Sci. U.S. A.
104 (2007) 9063–9068.
 F. Gambino, M. Kneib, A. Pavlowsky, H. Skala, S. Heitz, N. Vitale, B. Poulain, M.
Khelfaoui, J.Chelly, P.Billuart,Y. Humeau, IL1RAPL1 controls inhibitory networks
during cerebellar development in mice, Eur. J. Neurosci. 30 (2009) 1476–1486.
 E. Tabolacci, M.G. Pomponi, R. Pietrobono, A. Terracciano, P. Chiurazzi, G. Neri, A
truncating mutation in the IL1RAPL1 gene is responsible for X-linked mental
retardation in the MRX21 family, Am. J. Med. Genet. 140 (2006) 482–487.
 A. Carrie, L. Jun, T. Bienvenu, M.C. Vinet, N. McDonell, P. Couvert, R. Zemni, A.
Cardona, G. Van Buggenhout, S. Frints, B. Hamel, C. Moraine, H.H. Ropers, T.
Strom, G.R. Howell, A. Whittaker, M.T. Ross, A. Kahn, J.P. Fryns, C. Beldjord, P.
Marynen, J. Chelly, A new member of the IL-1 receptor family highly expressed in
hippocampus and involved in X-linked mental retardation, Nat. Genet. 23
 T. Yoshida, M. Mishina, Zebrafish orthologue of mental retardation protein
IL1RAPL1 regulates presynaptic differentiation, Mol. Cell. Neurosci. 39 (2008)
 P. Szatmari, A.D. Paterson, L. Zwaigenbaum, W. Roberts, J. Brian, X.Q. Liu, J.B.
Vincent, J.L. Skaug, A.P. Thompson, L. Senman, L. Feuk, C. Qian, S.E. Bryson, M.B.
Segre, M.A.Pericak-Vance, M.L. Cuccaro, J.R. Gilbert, H.H. Wright, R.K. Abramson,C.
Betancur, T. Bourgeron, C. Gillberg, M. Leboyer, J.D. Buxbaum, K.L. Davis, E.
Hollander, J.M. Silverman, J. Hallmayer, L. Lotspeich, J.S. Sutcliffe, J.L. Haines, S.E.
Folstein,J.Piven,T.H.Wassink, V.Sheffield,D.H. Geschwind, M.Bucan,W.T.Brown,
C. Lese-Martin, J. Miller, S. Nelson, C.A. Samango-Sprouse, S. Spence, M. State, R.E.
Minshew, J. Munson, E. Korvatska, P.M. Rodier, G.D. Schellenberg, M. Smith, M.A.
Spence, C. Stodgell, P.G. Tepper, E.M. Wijsman, C.E. Yu, B. Roge, C. Mantoulan, K.
Wittemeyer, A. Poustka, B. Felder, S.M. Klauck, C. Schuster, F. Poustka, S. Bolte, S.
Feineis-Matthews, E. Herbrecht, G. Schmotzer, J. Tsiantis, K. Papanikolaou, E.
Maestrini, E. Bacchelli, F. Blasi, S. Carone, C. Toma, H. Van Engeland, M. de Jonge, C.
Kemner, F. Koop, M. Langemeijer, C. Hijmans, W.G. Staal, G. Baird, P.F. Bolton, M.L.
Rutter, E. Weisblatt, J. Green, C. Aldred, J.A. Wilkinson, A. Pickles, A. Le Couteur, T.
S. Wallace, A.P. Monaco, G. Barnby, K. Kobayashi, J.A. Lamb, I. Sousa, N. Sykes, E.H.
Cook, S.J. Guter, B.L. Leventhal, J. Salt, C. Lord, C. Corsello, V. Hus, D.E. Weeks, F.
Volkmar, M. Tauber, E. Fombonne, A. Shih, K.J. Meyer, Mapping autism risk loci
using genetic linkage and chromosomal rearrangements, Nat. Genet. 39 (2007)
 H.G. Kim, S. Kishikawa, A.W. Higgins, I.S. Seong, D.J. Donovan, Y. Shen, E. Lally, L.A.
Weiss, J.Najm, K. Kutsche, M.Descartes, L.Holt, S. Braddock, R. Troxell, L.Kaplan, F.
Volkmar, A. Klin, K. Tsatsanis, D.J. Harris, I. Noens, D.L. Pauls, M.J. Daly, M.E.
with autism spectrum disorder, Am. J. Hum. Genet. 82 (2008) 199–207.
 G. Kirov, D. Gumus, W. Chen, N. Norton, L. Georgieva, M. Sari, M.C. O'Donovan, F.
Erdogan, M.J. Owen, H.H. Ropers, R. Ullmann, Comparative genome hybridiza-
tion suggests a role for NRXN1 and APBA2 in schizophrenia, Hum. Mol. Genet. 17
 D. Rujescu, A. Ingason, S. Cichon, O.P. Pietilainen, M.R. Barnes, T. Toulopoulou, M.
Picchioni, E. Vassos, U. Ettinger, E. Bramon, R. Murray, M. Ruggeri, S. Tosato, C.
Bonetto, S. Steinberg, E. Sigurdsson, T. Sigmundsson, H. Petursson, A. Gylfason, P.I.
Olason, G. Hardarsson, G.A. Jonsdottir, O. Gustafsson, R. Fossdal, I. Giegling, H.J.
Suvisaari, A. Tuulio-Henriksson, S. Djurovic, I. Melle, O.A. Andreassen, T. Hansen, T.
Werge, L.A. Kiemeney, B. Franke, J. Veltman, J.E. Buizer-Voskamp, C. Sabatti, R.A.
Ophoff, M. Rietschel, M.M. Nothen, K. Stefansson, L. Peltonen, D. St Clair, H.
Stefansson, D.A. Collier, Disruption of the neurexin 1 gene is associated with
schizophrenia, Hum. Mol. Genet. 18 (2009) 988–996.
 A. Rissone, M. Monopoli, M. Beltrame, F. Bussolino, F. Cotelli, M. Arese,
Comparative genome analysis of the neurexin gene family in Danio rerio:
insights into their functions and evolution, Mol. Biol. Evol. 24 (2007) 236–252.
 M. Setou, T. Nakagawa, D.H. Seog, N. Hirokawa, Kinesin superfamily motor
protein KIF17 and mLin-10 in NMDA receptor-containing vesicle transport,
Science (New York, N.Y.) 288 (2000) 1796–1802.
 L. Guillaud, M. Setou, N. Hirokawa, KIF17 dynamics and regulation of NR2B
trafficking in hippocampal neurons, J. Neurosci. 23 (2003) 131–140.
 P.J. Chu, J.F. Rivera, D.B. Arnold, A role for Kif17 in transport of Kv4.2, J. Biol.
Chem. 281 (2006) 365–373.
 R.W. Wong, M. Setou, J. Teng, Y. Takei, N. Hirokawa, Overexpression of motor
protein KIF17 enhances spatial and working memory in transgenic mice, Proc.
Natl. Acad. Sci. U. S. A. 99 (2002) 14500–14505.
 R. Roberson, L. Toso, D. Abebe, C.Y. Spong, Altered expression of KIF17, a
kinesin motor protein associated with NR2B trafficking, may mediate learning
deficits in a Down syndrome mouse model, Am. J. Obstet. Gynecol. 198 (2008)
 L. Guillaud, R. Wong, N. Hirokawa, Disruption of KIF17-Mint1 interaction by
CaMKII-dependent phosphorylation: a molecular model of kinesin-cargo
release, Nat. Cell Biol. 10 (2008) 19–29.
 C. Insinna, N. Pathak, B. Perkins, I. Drummond, J.C. Besharse, The homodimeric
kinesin, Kif17, is essential for vertebrate photoreceptor sensory outer segment
development, Dev. Biol. 316 (2008) 160–170.
 M.L. Suster, H. Kikuta, A. Urasaki, K. Asakawa, K. Kawakami, Transgenesis in
zebrafish with the tol2 transposon system, Methods Mol. Biol. 561 (2009)
 D. Goldman, M. Hankin, Z. Li, X. Dai, J. Ding, Transgenic zebrafish for studying
nervous system development and regeneration, Transgenic Res. 10 (2001)
 T.M. Jessell, Neuronal specification in the spinal cord: inductive signals and
transcriptional codes, Nat. Rev. Genet. 1 (2000) 20–29.
 T. Nakano, M. Windrem, V. Zappavigna, S.A. Goldman, Identification of a
conserved 125 base-pair Hb9 enhancer that specifies gene expression to spinal
motor neurons, Dev. Biol. 283 (2005) 474–485.
 S. Higashijima, Y. Hotta, H. Okamoto, Visualization of cranial motor neurons in
live transgenic zebrafish expressing green fluorescent protein under the control
of the islet-1 promoter/enhancer, J. Neurosci. 20 (2000) 206–218.
 M.L. Suster, A. Kania, M. Liao, K. Asakawa, F. Charron, K. Kawakami, P. Drapeau, A
novel conserved evx1 enhancer links spinal interneuron morphology and cis-
regulation from fish to mammals, Dev. Biol. 325 (2009) 422–433.
 A. Picker, S. Scholpp, H. Bohli, H. Takeda, M. Brand, A novel positive
transcriptional feedback loop in midbrain–hindbrain boundary development is
revealed through analysis of the zebrafish pax2.1 promoter in transgenic lines,
Development 129 (2002) 3227–3239.
 D.L. McLean, J. Fan, S. Higashijima, M.E. Hale, J.R. Fetcho, A topographic map of
recruitment in spinal cord, Nature 446 (2007) 71–75.
 Y. Kimura, Y. Okamura, S. Higashijima, alx, a zebrafish homolog of Chx10, marks
ipsilateral descending excitatory interneurons that participate in the regulation
of spinal locomotor circuits, J. Neurosci. 26 (2006) 5684–5697.
 P. Blader, C. Plessy, U. Strahle, Multiple regulatory elements with spatially and
temporally distinct activities control neurogenin1 expression in primary
neurons of the zebrafish embryo, Mech. Dev. 120 (2003) 211–218.
 A.N. Ng, T.A. de Jong-Curtain, D.J. Mawdsley, S.J. White, J. Shin, B. Appel, P.D.
Dong, D.Y. Stainier, J.K. Heath, Formation of the digestive system in zebrafish: III.
Intestinal epithelium morphogenesis, Dev. Biol. 286 (2005) 114–135.
 J. Shin, H.C. Park, J.M. Topczewska, D.J. Mawdsley, B. Appel, Neural cell fate analysis
in zebrafish using olig2 BAC transgenics, Methods Cell Sci. 25 (2003) 7–14.
 B.B. Kirby, N. Takada, A.J. Latimer, J. Shin, T.J. Carney, R.N. Kelsh, B. Appel, In vivo
time-lapse imaging shows dynamic oligodendrocyte progenitor behavior during
zebrafish development, Nat. Neurosci. 9 (2006) 1506–1511.
 T.A. Bayer, J.A. Campos-Ortega, A transgene containing lacZ is expressed in
primary sensory neurons in zebrafish, Development (Cambridge, England) 115
 M.E. Halpern, J. Rhee, M.G. Goll, C.M. Akitake, M. Parsons, S.D. Leach, Gal4/
UAS transgenic tools and their application to zebrafish, Zebrafish 5 (2008)
 J. Dong, G.W. Stuart, Transgene manipulation in zebrafish by using recombi-
nases, Methods Cell Biol. 77 (2004) 363–379.
 E. Provost, J. Rhee, S.D. Leach, Viral 2A peptides allow expression of multiple proteins
from a single ORF in transgenic zebrafish embryos, Genesis 45 (2007) 625–629.
 C.B. Moens, T.M. Donn, E.R. Wolf-Saxon, T.P. Ma, Reverse genetics in zebrafish by
TILLING, Brief. Funct. Genomic Proteomic 7 (2008) 454–459.
E. Kabashi et al. / Biochimica et Biophysica Acta 1812 (2011) 335–345
 J.E. Foley, J.R. Yeh, M.L. Maeder, D. Reyon, J.D. Sander, R.T. Peterson, J.K.
Joung, Rapid mutation of endogenous zebrafish genes using zinc finger
nucleases made by Oligomerized Pool ENgineering (OPEN), PLoS One 4
 I.G. Woods, A.F. Schier, Targeted mutagenesis in zebrafish, Nat. Biotechnol. 26
 X. Ouyang, I.A. Shestopalov, S. Sinha, G. Zheng, C.L. Pitt, W.H. Li, A.J. Olson, J.K.
Chen, Versatile synthesis and rational design of caged morpholinos, J. Am. Chem.
Soc. 131 (2009) 13255–13269.
 S. Wu, J.M. Storey, K.B. Storey, Phosphoglycerate kinase 1 expression responds
to freezing, anoxia, and dehydration stresses in the freeze tolerant wood frog,
Rana sylvatica, J. Exp. Zool. A Ecol. Genet. Physiol. 311 (2009) 57–67.
 P.A. Morcos, Y. Li, S. Jiang, Vivo-morpholinos: a non-peptide transporter delivers
morpholinos into a wide array of mouse tissues, Biotechniques 45 (2008)
613–614 616, 618 passim.
 C.W. Sipe, M.S. Saha, The use of microarray technology in nonmammalian
vertebrate systems, Methods Mol. Biol. 382 (2007) 1–16.
 A. Hegde, N.C. Qiu, X. Qiu, S.H. Ho, K.Q. Tay, J. George, F.S. Ng, K.R. Govindarajan,
Z. Gong, S. Mathavan, Y.J. Jiang, Genomewide expression analysis in zebrafish
mind bomb alleles with pancreas defects of different severity identifies putative
Notch responsive genes, PLoS One 3 (2008) e1479.
 L.I. Zon, R.T. Peterson, In vivo drug discovery in the zebrafish, Nat. Rev. Drug
Discov. 4 (2005) 35–44.
 J.R. Yeh, K.M. Munson, K.E. Elagib, A.N. Goldfarb, D.A. Sweetser, R.T. Peterson,
Discovering chemical modifiers of oncogene-regulated hematopoietic differen-
tiation, Nat. Chem. Biol. 5 (2009) 236–243.
 A. Mukhopadhyay, R.T. Peterson, Deciphering arterial identity through gene
expression, genetics, and chemical biology, Curr. Opin. Hematol. 15 (2008)
 E. Kabashi, L. Lin, M.L. Tradewell, P.A. Dion, V. Bercier, P. Bourgouin, D. Rochefort,
S.B. Hadj, H.D. Durham, C. Vande Velde, G.A. Rouleau, P. Drapeau, Gain and loss of
function of ALS-related mutations of TARDBP (TDP-43) cause motor deficits in
vivo, Human Mol. Gen. 19 (2010) 671–683.
 J. Gauthier, N. Champagne, R.G. Lafrenière, L. Xiong, D. Spiegelman, E. Brustein,
M. Lapointe, H. Peng, M. Côté, A. Noreau, F.F. Hamdan, A. Piton, A.M. Addington,
J.L. Rapoport, L.E. DeLisi, M.-O. Krebs, R. Joober, F. Fathalli, F. Mouaffak, A.P.
Haghighi, C. Néri, M.-P. Dubé, M.E. Samuels, C. Marineau, E.A. Stone, P. Awadalla,
P.A. Barker, S. Carbonetto, P. Drapeau, G.A. Rouleau, and the S2D team, De novo
synaptic scaffolding protein SHANK3 mutations in patients ascertained with
schizophrenia, Proc. Natl. Acad. Sci. USA 107 (2010) 7863–7868.
 J. Tarabeux, N. Champagne, E. Brustein, F.F. Hamdan, J. Gauthier, M. Lapointe, C.
Maios, A. Piton, D. Spiegelman, É. Henrion, S2D team, B. Mille, J.L. Rapoport, L.E.
DeLisi, R. Joober, F. Fathalli, É. Fombonne, L. Mottron, N. Forget-Dubois, M. Boivin,
J.L. Michaud, R.G. Lafrenière, P. Drapeau, M.-O. Krebs, G.A. Rouleau, De Novo
Truncating Mutation in KIF17 Associated with Schizophrenia, Biol. Psy. 68
E. Kabashi et al. / Biochimica et Biophysica Acta 1812 (2011) 335–345