Báo cáo khoa học: Treatment with small interfering RNA affects the microRNA pathway and causes unspecific defects in zebrafish embryos doc

Treatment with small interfering RNA affects the
microRNA pathway and causes unspecific defects
in zebrafish embryos
Xiao-Feng Zhao, Anders Fjose, Natalia Larsen, Jon V. Helvik and Øyvind Drivenes
Department of Molecular Biology, University of Bergen, Norway
MicroRNAs (miRNAs) are small RNA molecules of
 21 nucleotides in metazoan animals and plants that
influence mRNA stability and translation [1–3]. These
mature miRNAs are generated from longer primary
transcripts (pri-miRNA) in two processing steps cata-
lyzed by two related RNase III endonucleases. In ani-
mals, a nuclear microprocessor complex, containing
the RNase III enzyme Drosha and the dsRNA-binding
protein DGCR8, cleaves the pri-miRNA and exises a
stem loop of  70 nucleotides [3–6]. This precursor
miRNA (pre-miRNA) is then exported to the
cytoplasm by a nuclear transport receptor complex,
exportin-5 ⁄ RanGTP [7,8]. In the cytoplasm, a second
RNase III, Dicer, cleaves the pre-miRNA to generate
the mature miRNA [3,9,10].
The function of miRNAs and the ability to knock
down expression of specific genes by RNA interfer-
ence (RNAi) methods depend to a large extent on the
same cellular machinery [3]. One important example is
Dicer, which is required for miRNA processing as
well as cleavage of dsRNA into small interfering
RNAs (siRNAs). Moreover, mature miRNA and
Keywords
dicer; maternal mRNA; microRNA; RNA
interference (RNAi); zebrafish development
Correspondence

A. Fjose, Department of Molecular Biology,
University of Bergen, PO Box 7803,
N-5020 Bergen, Norway
Fax: +47 555 89683
Tel: +47 555 84331
E-mail:
Ø. Drivenes, Department of Molecular
Biology, University of Bergen, PO Box 7803,
N-5020 Bergen, Norway
Fax: +47 555 89683
Tel: +47 555 84325
E-mail:
(Received 7 December 2007, revised 18
February 2008, accepted 3 March 2008)
doi:10.1111/j.1742-4658.2008.06371.x
MicroRNAs (miRNAs) are generated from primary transcripts through
sequential processing by two RNase III enzymes, Drosha and Dicer, in
association with other proteins. This maturation is essential for their func-
tion as post-transcriptional regulators. Notably, Dicer is also a component
of RNA-induced silencing complexes, which incorporate either miRNA or
small interfering RNA (siRNA) as guides to target specific mRNAs. In ze-
brafish, processed miRNAs belonging to the miR-430 family have previ-
ously been shown to promote deadenylation and degradation of maternal
mRNAs during early embryogenesis. We show that injection of one-cell-
stage zebrafish embryos with siRNA causes a significant reduction in the
endogenous levels of processed miR-430 and other miRNAs, leading to
unspecific developmental defects. Coinjection of siRNA with preprocessed
miR-430 efficiently rescued development. This indicates that the abnormali-
ties generally observed in siRNA-treated zebrafish embryos could be due to
inhibition of miR-430 processing and ⁄ or activity. Our results also suggest

that the miRNA pathway in mammals, under some experimental or thera-
peutic conditions, may be affected by siRNA.
Abbreviations
GFP, green fluorescent protein; hpf, hours postfertilization; miRNA, microRNA; MMB, mesencephalic–metencephalic boundary; MZdicer,
maternal–zygotic dicer; pre-miRNA, precursor miRNA; pri-miRNA, primary transcript of microRNA; RISC, RNA-induced silencing complex;
RNAi, RNA interference; siGFP, siRNA specific for green fluorescent protein coding sequence; siRNA, small interfering RNA; TRBP,
transactivating response RNA-binding protein.
FEBS Journal 275 (2008) 2177–2184 ª 2008 The Authors Journal compilation ª 2008 FEBS 2177
siRNA are assembled into the RNA-induced silencing
complexes (RISCs) miRISC and siRISC, respectively
[3]. Both of these effector complexes contain, in addi-
tion to similar Argonaute proteins, Dicer and other
factors such as the transactivating response RNA-
binding protein (TRBP) [11,12]. Interestingly, it has
also been demonstrated that TRBP associates with
Dicer to facilitate generation of siRNAs and mature
miRNAs [13,14].
RNAi has been employed extensively as a research
tool in several animal models, including Caenorhabd-
itis elegans, Drosophila melanogaster and mice, as well
as mammalian cells [15–18]. RNAi targeting of specific
genes has also been demonstrated in zebrafish
(Danio rerio) cell lines [19]. However, experimental
studies on zebrafish embryos have revealed substantial
unspecific defects of RNAi that have prevented further
use of this technology for gene function analyses [19–
22]. By circumventing these problems, studies on
zebrafish maternal–zygotic dicer (MZdicer) mutants,
which do not process pre-miRNA, have uncovered
extensive embryonic abnormalities reflecting the impor-

tance of miRNAs in developmental processes [23].
Remarkably, the brain morphogenesis defects in MZ
dicer mutants could be suppressed by injection of
preprocessed miR-430 family miRNAs, which are the
only abundant miRNAs during the first hours after
the transition from maternal to zygotic gene expression
[23]. Further studies of the developmental regulatory
function of miR-430 revealed that these miRNAs
accelerate deadenylation and degradation of several
hundred different types of maternal mRNAs, leading
to a sharpening of the maternal-to-zygotic transition
[24].
We have investigated the possible connection
between the miRNA pathway and the unspecific
defects caused by RNAi in zebrafish embryos. Accom-
panying the induction of unspecific defects, the treat-
ment with different siRNAs was also shown to
significantly reduce the levels of processed miR-430
and other miRNAs. Moreover, we demonstrated, by
coinjecting siRNA with preprocessed miR-430, that
most of the morphological abnormalities could be pre-
vented. Hence, the unspecific defects generally caused
by RNAi in zebrafish embryos are mainly due to inhi-
bition of miR-430, which has been shown previously
to have an essential role in the clearance of maternal
mRNAs [24]. These observations may have implica-
tions for the development of new RNAi techniques in
zebrafish. In addition, our results suggest a need for
investigating whether treatment of mammalian cells
with larger amounts of siRNA may also cause some

inhibition of the miRNA pathway.
Results
Different siRNAs cause similar unspecific defects
We have previously characterized the structure and
embryonic expression of zebrafish six3a (originally
named six3 [25]), and more recently we have identified
genomic and cDNA sequences representing eri1,a
zebrafish homolog of the enhanced RNA interference-1
(eri-1) gene in C. elegans (see Experimental proce-
dures). Using specific siRNAs (siEri1 and siSix3a) to
target the mRNAs expressed from these two genes, we
observed similar but not identical embryonic defects
(Fig. 1C–F). Following injection of sufficient amounts
of siRNA (see Experimental procedures), most
embryos at 28 h postfertilization (hpf) displayed tail
truncations and loss of distinct morphological features
at the mesencephalic–metencephalic boundary (MMB;
Table 1). In addition, enlarged heart cavities occurred
at lower frequencies in these embryos (not shown).
Notably, we also observed tail truncations and MMB
defects in embryos treated with siRNA specific for
green fluorescent protein (GFP) coding sequences (siG-
FP; Fig. 1G,H; Table 1), suggesting that these malfor-
mations are general consequences of siRNA treatment.
siGFP
siSix3a
siEri1
WT
AB
CD

EF
GH
*
*
*
Fig. 1. Injection of different siRNAs causes brain and tail defects.
Light micrographs are shown for wild-type (WT) and siRNA-injected
zebrafish embryos. At the 28 hpf stage, the zebrafish embryos
injected with siRNAs targeting eri1 (siEri1), six3a (siSix3a) and GFP
(siGFP) show tail and brain defects. The MMB is easily visible in
wild-type embryos (arrowhead), whereas this morphological con-
striction is missing in embryos injected with the different siRNAs
[stars in (D), (F) and (H)].
siRNA and unspecific defects X F. Zhao et al.
2178 FEBS Journal 275 (2008) 2177–2184 ª 2008 The Authors Journal compilation ª 2008 FEBS
Consistent with this assumption, equal numbers of
molecules of the same GFP sequence caused very few
abnormalities when injected as ssRNA (sense and anti-
sense in separate experiments) or dsRNA without the
2bp3¢-overhangs (Table 1).
siRNAs affect the endogenous levels of miRNAs
The defects caused by siRNA injections showed a
resemblance to the abnormalities reported previously
for MZdicer mutants, which are due to failure in the
processing of miR-430 [23]. Interestingly, the MZdicer
phenotype also included tail and MMB defects [23].
Therefore, it seemed plausible that miRNA processing
could be affected by the injection of siRNAs. To inves-
tigate this possibility, we analyzed by northern blotting
the levels of processed miR-430b and three additional

miRNAs expressed in early embryos [26,27]. This anal-
ysis showed that all three siRNAs caused reductions in
the amounts of mature miRNAs at 12 hpf (Fig. 2A).
Although the level of processed miR-430b seemed to
be most strongly affected, we also detected significant
reductions for the other three miRNAs tested. Further-
more, we generally observed stronger reductions for
the largest dosage of siRNA (500 pg). These results
were reproducible, and for miR-430b the level was
reduced by as much as 70% (supplementary Fig. S4).
The effects of siRNAs on miRNA levels were less
prominent at later embryonic stages, as revealed by
analyses at 24 hpf, where a significant reduction in the
amount of the mature form was detected only for
miR-430b (supplementary Fig. S1).
To examine whether the reduced amounts of pro-
cessed miRNAs could be due to a unique feature of
siRNAs, we analyzed the possible effects following
microinjection of other types of RNA molecules con-
taining the same GFP sequence. As expected from the
minimal effects on embryonic development caused by
injection of equal numbers of these GFP-specific RNA
molecules (Table 1), we did not detect any reduction in
the level of processed miR-430b at 12 hpf (Fig. 2B).
Consistent with the common MMB defects of the three
siRNAs, expression of the pax2a gene, which is an
Table 1. Analysis of the efficiencies of different siRNAs and
related RNA molecules in inducing MMB defects. asGFP, antisense
strand of GFP; dsGFP, double-stranded GFP without 3¢-overhangs;
sGFP, sense strand of GFP.

Embryos analyzed MMB defects (%)
250 pg of siGFP 134 129 (96.3)
250 pg of siEri1 87 76 (87.4)
400 pg of siSix3a 122 100 (82.0)
250 pg of dsGFP 114 11 (9.6)
125 pg of sGFP 109 2 (1.8)
125 pg of asGFP 126 15 (11.9)
A
B
C
Fig. 2. Injection of different siRNAs affects the levels of miRNAs.
(A) The endogenous levels of the processed forms of four different
miRNAs were analyzed at the 12 hpf stage by northern blotting.
Treatment with each of the three different siRNAs caused reduc-
tion of the levels of all four miRNAs as compared to the wild-type
(WT). (B) Northern blot analysis of the endogenous level of mature
miR-430b at 12 hpf following injection of different types of RNA
molecules corresponding to the same GFP sequence. Single-
stranded and double-stranded molecules without the characteristic
features of siRNA did not affect the level of miR-430b, as com-
pared to non-injected (WT) and buffer-injected embryos. (C) Endog-
enous levels of mature miR-430b were reduced at 12 hpf following
injection of miR-206. The different amounts (pg) injected are indi-
cated for each type of RNA. asGFP, antisense strand of GFP;
dsGFP, double-stranded GFP without 3¢-overhangs; sGFP, sense
strand of GFP.
X F. Zhao et al. siRNA and unspecific defects
FEBS Journal 275 (2008) 2177–2184 ª 2008 The Authors Journal compilation ª 2008 FEBS 2179
important midbrain marker [28,29], was also clearly
reduced in the affected region of the brain (supplemen-

tary Fig. S2).
Coinjection with preprocessed miR-430b can
prevent unspecific defects
Microinjection of preprocessed miR-430 has previously
been shown to rescue tail and brain defects of MZdicer
mutants [23]. Similarly, we investigated whether coin-
jection of miR-430b could rescue the effects of siRNA
treatment. In these experiments, we also performed
control injections with miR-206, and a mutated version
of miR-430b (miR-430b-mis) that lacks the power to
rescue MZdicer embryos [23]. Consistent with the
results of Giraldez et al. [23], the dsRNA molecules of
preprocessed miR-430b did not induce any embryonic
defects (Fig. 3A,B; Table 2). However, preprocessed
miR-206 duplexes clearly affected the morphologies
of both the tail and MMB of injected embryos
(Fig. 3C,D; Table 2), indicating that treatment of
zebrafish embryos with miRNA duplexes may
generally induce the same kind of unspecific defects as
siRNAs. In support of this assumption, we also
observed reduced levels of mature miR-430b in
miR-206-injected embryos (Fig. 2C).
Coinjection of preprocessed miR-430b efficiently res-
cued the siEri1-induced defects (Fig. 3E,F; Table 2),
and a similar result was observed for coinjections of
miR-430b with siSix3a (supplementary Fig. S3;
Table 2). Preprocessed miR-430b also rescued embryos
from defects caused by siGFP, and the efficiency was
clearly improved with a higher dosage (Fig. 3K,L;
Table 2). By contrast, coinjection of miR-430b-mis,

which has two point substitutions in the 5¢-seed region
[23], did not rescue the MMB or tail defects
caused by any of the three gene-specific siRNAs
(Fig. 3G,H,M,N; supplementary Fig. S3; Table 2).
Similarly, siEri1-induced defects were not rescued by
coinjection with miR-206 duplexes (Fig. 3I,J; Table 2).
These results show that unspecific defects induced by
siRNA, which correlate with a significant reduction of
the endogenous level of mature miR-430b, can be pre-
vented by coinjection of this particular miRNA. If
inhibition of miR-430 activity by siRNAs occurs also
at the level of miRISC assembly and ⁄ or function,
miR-430b coinjection would be expected to reduce this
effect as well (see Discussion).
Discussion
The rapidly growing knowledge on RNAi and miRNA
has revealed many common factors and interconnec-
A
B
C
D
E
F
G
H
I
J
K
L
M

N
Fig. 3. Rescue of siRNA-induced abnormalities by coinjection
of miR-430b. The effects of injecting preprocessed duplexes of
miRNAs alone and in combination with siRNAs were analyzed.
Whereas embryos were not significantly affected by injection of
miR-430b (A, B), injection of miR-206 caused similar tail and MMB
defects as siRNA injections (C, D) (see Fig. 1).The tail ⁄ MMB
defects caused by injections of the two different siRNAs, siEri1
and siGFP (see Fig. 1), were rescued by coinjection of miR-430b
alone (E, F, K, L) but not by the mutated variant miR-430b-mis (G,
H, M, N). Coinjection of miR-206 did not rescue the tail ⁄ MMB
defects caused by siEri1 (I, J). Arrowheads and stars indicate the
presence and absence of an MMB, respectively.
siRNA and unspecific defects X F. Zhao et al.
2180 FEBS Journal 275 (2008) 2177–2184 ª 2008 The Authors Journal compilation ª 2008 FEBS
tions between these two pathways. Recently, it has also
been shown that modulation of the processing of miR-
NAs is an important feature of their regulatory func-
tion and may be directly connected to cell signaling
[30–32]. However, in relation to the extensive use of
RNAi as a tool to knock down the expression of spe-
cific genes, the possible influence on miRNA process-
ing, which may cause various side effects, has been
analyzed only recently [33]. In this study, we have
investigated these aspects in zebrafish, where RNAi
experiments have previously been shown to result in
high frequencies of unspecific defects [19–22]. Because
of these problems, RNAi has not become a useful
technique for studying gene function in zebrafish. As
an alternative, morpholino antisense oligonucleotides

have been extensively used for transient knockdown of
gene expression in zebrafish embryos and larvae
[34,35]. However, unspecific effects can be a problem
with this method as well, and it cannot be further
developed as a transgenic technique with the possibili-
ties of achieving tissue-specific and ⁄ or long-term
knockdown of the targeted genes.
In Drosophila and mouse, transgenic RNAi tech-
niques have been developed to facilitate tissue-specific
or inducible knockdown [16,17]. In principle, this strat-
egy can also be used in zebrafish, but it may not be
feasible, due to the unspecific effects associated with
RNAi. Although the reason why treatment with siR-
NA causes a high frequency of general abnormalities
in zebrafish has remained unclear, some clues regard-
ing this issue have been obtained from studies of the
MZdicer mutation [23]. The MZdicer mutant embryos,
which display several defects similar to those caused
by siRNAs, were rescued by injection of preprocessed
miR-430 miRNAs [23]. Remarkably, further investiga-
tions on the mRNA targets of miR-430, which are the
only abundant miRNAs before gastrulation, demon-
strated that miR-430 is essential for efficient removal
of maternal mRNAs during the maternal-to-zygotic
transition [24]. Hence, considering the common factors
in the RNAi and miRNA pathways, and the impor-
tance of miR-430 at early stages of zebrafish develop-
ment, we assumed a possible involvement of miR-430
in the unspecific defects caused by siRNA treatment.
Using siRNAs corresponding to sequences in two

endogenous genes (eri1 and six3a) and the exogenous
reporter gene GFP, we investigated the possibility that
miRNAs may in some way be influenced by the siR-
NAs introduced into zebrafish embryos. By northern
analysis of miR-430b and three additional miRNAs,
we found a general reduction in the levels of processed
miRNAs in embryos treated with siRNAs. Injection of
other types of RNA molecules, such as ssRNAs and
dsRNAs without the 3¢-overhangs, which contained
the same sequence, did not cause any general abnor-
malities, and the levels of mature miRNAs were not
affected. These results suggest that the characteristic
features of siRNAs are critical for reducing the levels
of processed miRNAs, particularly miR-430, and this
may lead to the unspecific defects observed in zebrafish
embryos.
If this interpretation is correct, it will be natural to
ask how injection of siRNAs can possibly interfere
with the endogenous levels of mature miRNAs.
Although correctly sized siRNAs ( 21 bp) are not cut
by Dicer, which is the enzyme responsible for the last
processing step of miRNAs, siRNAs are known to be
assembled into effector complexes (siRISCs) containing
Argonaute proteins as well as Dicer and other factors
[3]. One of the additional factors is TRBP, which
together with Dicer facilitates generation of siRNAs
and mature miRNAs from dsRNAs and pre-miRNAs,
respectively [13,14]. Accordingly, the injection of large
amounts of siRNAs would affect the availability of
these factors for processing of pre-miRNAs. Thus, the

most plausible explanation is that the observed reduc-
tion of mature miRNAs is due to inhibition of pre-
miRNA processing by siRNAs competing for binding
to Dicer, TRBP, and ⁄ or other limiting factors. How-
ever, since our northern blot analysis did not reveal a
concomitant increase of pre-miRNAs, we cannot
exclude other possibilities, such as enhanced degrada-
tion of mature miRNAs.
When discussing the relevance of miR-430 to the
unspecific defects caused by siRNAs, it should be
noted that these miRNAs are most abundant during
early stages of zebrafish development [26,36,37]. Follow-
Table 2. Analysis of the influence of siRNAs and miRNAs on MMB
morphology. Coinjection of miR-430b efficiently rescued MMB
defects caused by siRNA treatment. The MMB defects were not
significantly rescued by miR-206 and the mutated variant miR-430b-
mis.
Embryos
analyzed
MMB
defects
(%)
250 pg of miR-430b 98 0 (0)
250 pg of miR-206 91 91 (100)
250 pg of siEri1 + 250 pg of miR-430b 111 9 (8.1)
250 pg of siEri1 + 250 of pg miR-430b-mis 115 97 (84.3)
250 pg of siEri1 + 250 pg of miR-206 110 110 (100)
250 pg of siGFP + 250 pg of miR-430b 88 55 (62.5)
250 pg of siGFP + 450 pg of miR-430b 46 21 (45.7)
250 pg of siGFP + 250 pg of miR-430b-mis 109 107 (98.2)

400 pg of siSix3a + 250 pg of miR-430b 91 7 (7.7)
400 pg of siSix3a + 250 pg of miR-430b-mis 112 101 (90.2)
X F. Zhao et al. siRNA and unspecific defects
FEBS Journal 275 (2008) 2177–2184 ª 2008 The Authors Journal compilation ª 2008 FEBS 2181
ing siRNA injection, we detected a > 50% reduction
of processed miR-430b, and this would be at least par-
tially equivalent to the conditions in MZdicer embryos,
in which processing of pre-miRNAs does not occur
[23]. Similar to the rescue of MZdicer mutants by
miR-430b [23], we observed efficient rescue when
siRNAs were coinjected with the preprocessed duplex
form of this particular miRNA. In contrast, coinjec-
tion of another early embryonic miRNA (miR-206) or
a mutated version of miR-430b (miR-430b-mis) did
not give any rescue. These results are entirely consis-
tent with the documented role of mature miR-430 in
promoting deadenylation and degradation of maternal
mRNAs, which is required for a normal maternal-to-
zygotic transition [24]. Because of this crucial function
of miR-430, development of zebrafish embryos is likely
to be affected by treatment with siRNAs. Our results
from experiments with three different siRNAs suggest
that this effect is a general phenomenon in zebrafish.
However, some variations with respect to the abilities
of different siRNAs to cause unspecific defects and
reduced levels of mature miRNAs suggest a certain
degree of sequence dependence. This may simply reflect
differences in the binding affinities of various siRNAs
to one or more factors that are shared between the
RNAi and miRNA pathways.

Relevant to this issue, we also noted that single
injections of miR-206 duplexes, in contrast to miR-
430b, caused a high frequency of brain and tail abnor-
malities, as well as a reduction of the endogenous level
of processed miR-430b. These observations indicated
that the level of the mature form of miR-430 was
affected by miRNA injection but was compensated by
the introduction of preprocessed miR-430b. Therefore,
we conclude that injected miRNA duplexes (with inter-
nal mismatches) can probably affect the endogenous
concentrations of mature miRNAs in the same way as
siRNA duplexes.
The importance of miR-430 was confirmed by coin-
jection of preprocessed miR-430b duplexes, which
apparently rescued most of the unspecific defects
caused by siRNAs. However, at lower doses of
siRNAs, when the endogenous level of mature
miR-430 was less affected, we also observed relatively
high frequencies of unspecific defects. This could reflect
a particularly high sensitivity to changes in the concen-
tration of this important miRNA, but it seems more
likely that siRNAs may cause an additional block of
miRNA function at the level of the effector complex
miRISC. Hence, excess amounts of siRNAs may effi-
ciently compete with miR-430 (and other miRNAs)
for binding to Dicer, Argonaute proteins and ⁄ or other
factors of this complex, and prevent interaction with
the mRNA targets. For the same reason, coinjection
of preprocessed miR-430b duplexes would be expected
to reduce this inhibition.

The results reported here suggest that siRNAs
injected into zebrafish embryos compete for limiting
factors that are required in the miRNA pathway. By
contrast, a recent study of systemic administration of
synthetic siRNA in mouse and hamster did not reveal
any effect on miRNA levels or activity in the liver [33].
However, a more complete investigation is required to
analyze whether or not treatment of mammals with
higher doses of siRNA can inhibit the endogenous
miRNA pathway in particular tissues and ⁄ or during
embryogenesis. Another issue, which is also relevant to
therapeutic use of siRNA in humans, concerns the
possible sensitivity to changes in miRNA levels or
activity. Negative side effects reflecting such sensitivity
have already been reported from experiments where
short hairpin RNAs were expressed at high levels in
the liver of mice [34]. This treatment was shown to sat-
urate the nuclear exportin-5 transporter, leading to
reduction of the levels of processed miRNAs and
lethality.
Experimental procedures
Isolation and analysis of genomic DNA and cDNA
Two zebrafish eri1 expressed sequence tags (BQ285328 and
BI888174), reported previously [38], were subjected to
blast analysis against the zebrafish genomic database at
ENSEMBL, and a genomic region containing the eri1 locus
was identified (GenBank accession number BX511222).
Using genscan [39], webgene [40] and eri1 expressed
sequence tag alignments, we identified a putative eri1 cod-
ing region composed of seven exons spanning a genomic

region of 7542 bp. Using primers located in the putative
5¢-region and 3¢-region, ERI1F1 (5¢-AAA CCA GAT GTG
AGT GTT TCT GA-3¢) and ERI1R1 (5¢-CAC AAC ATG
GCA GGT TTT CA-3¢), we isolated the complete eri1
coding sequence by PCR using adult zebrafish cDNA as
template.
Injections of siRNA and miRNA duplexes
Adult fish were kept at 28.5 °C on a natural 14 h light ⁄ 10 h
dark cycle, and all embryos were obtained from natural
mating. The siRNAs targeting eri1, six3a and GFP (see
below) were designed using the Dharmacon siRNA design
center ( and pur-
chased from MWG Biotech (Ebersberg, Germany).
Embryos were injected in the yolk at the one-cell stage,
with an average injection volume of 2 nL, which contained
siRNA and unspecific defects X F. Zhao et al.
2182 FEBS Journal 275 (2008) 2177–2184 ª 2008 The Authors Journal compilation ª 2008 FEBS
250 pg of siRNA and⁄ or miRNA. In the case of six3a,a
larger amount of siRNA (400 pg) was required to achieve a
high frequency of defects (Table 1). Following injection,
embryos were incubated at 28.5 °C in E3 medium.
Oligonucleotide sequences
The sequences are given in the 5¢-to3¢-direction: siEri1,
UCAGUGAUCCGGUGUAUAA(TT); siSix3a, CUAUCA
GGAGGCCGAGAAA(TT); siGFP, AAGCUGACCCU
GAAGUUCA(TT); dsGFP, AAGCUGACCCUGAAGU
UCA; sGFP, AAGCUGACCCUGAAGUUCA(TT);
asGFP, UGAACUUCAGGGUCAGCUU(TT).
Northern blot probes: miR-430b, BIO-GUACCC
CAACUUGAUAGCACUUU; miR-206, BIO-CCACATG

CTTCCTTATATTCCATA; miR-17a-1, BIO-ACTACCTG
CACTGTAAGCACTTTG; miR-19b, BIO-TCAGTTTT
GCATGGATTTGCACA. miR-206 duplex: AAUGUAA
GGAAGUGUGUGGGU; CCACACACUUCCUUACAA
UUU.
miR-430b duplex: AAAGUGCUAUCAAGUUGGG
GU; CCCAACUUGAUAGCACUAUUU. miR-430b-mis
duplex, as described in [23]: AAAGACCUAUCAAG
UUGGGGT; CCCAACUUGAUAGGUCUAUTT.
Northern blot analysis
Total RNA was isolated from wild-type and injected
embryos at 12 hpf and 24 hpf using Trizol (Invitrogen,
Carlsbad, CA, USA). Five micrograms of total RNA was
separated on a 15% denaturing polyacrylamide gel contain-
ing 8 m urea, and was blotted according to standard proce-
dures. Biotin-labeled probes were purchased from MWG
Biotech. Prehybridization and hybridization were carried out
in 0.25 m sodium phosphate (pH 7.2), 7% SDS, and 0.5%
sodium pyrophosphate. After hybridization, the membrane
was washed in 2 · SSC and 1% SDS at 37 °C. The biotin
signal was detected using the Chemiluminescent Nucleic
Acid Detection Module kit (Pierce, Rockford, IL, USA).
Acknowledgements
We thank Dr Hee-Chan Seo for technical advice and
the Faculty of Mathematics and Natural Sciences at
the University of Bergen for special support.
References
1 Nilsen TW (2007) Mechanisms of microRNA-mediated
gene regulation in animal cells. Trends Genet 23, 243–
249.

2 Pillai RS, Bhattacharyya SN & Filipowicz W (2007)
Repression of protein synthesis by miRNAs: how many
mechanisms? Trends Cell Biol 17, 118–126.
3 Rana TM (2007) Illuminating the silence: understanding
the structure and function of small RNAs. Nat Rev Mol
Cell Biol 8, 23–36.
4 Denli AM, Tops BBJ, Plasterk RHA, Ketting RF &
Hannon GJ (2004) Processing of primary microRNAs
by the Microprocessor complex. Nature 432, 231–235.
5 Gregory RI, Yan KP, Amuthan G, Chendrimada T,
Doratotaj B, Cooch N & Shiekhattar R (2004) The
microprocessor complex mediates the genesis of
microRNAs. Nature 432, 235–240.
6 Han JJ, Lee Y, Yeom KH, Kim YK, Jin H & Kim VN
(2004) The Drosha–DGCR8 complex in primary micr-
oRNA processing. Genes Dev 18, 3016–3027.
7 Yi R, Qin Y, Macara IG & Cullen BR (2003) Exportin-
5 mediates the nuclear export of pre-microRNAs and
short hairpin RNAs. Genes Dev 17, 3011–3016.
8 Lund E, Guttinger S, Calado A, Dahlberg JE & Kutay
U (2004) Nuclear export of microRNA precursors.
Science 303, 95–98.
9 Grishok A, Pasquinelli AE, Conte D, Li N, Parrish S,
Ha I, Baillie DL, Fire A, Ruvkun G & Mello CC
(2001) Genes and mechanisms related to RNA interfer-
ence regulate expression of the small temporal RNAs
that control C-elegans developmental timing. Cell 106,
23–34.
10 Hutvagner G & Zamore PD (2002) A microRNA in a
multiple-turnover RNAi enzyme complex. Science 297,

2056–2060.
11 Chendrimada TP, Gregory RI, Kumaraswamy E, Nor-
man J, Cooch N, Nishikura K & Shiekhattar R (2005)
TRBP recruits the Dicer complex to Ago2 for micro-
RNA processing and gene silencing. Nature 436, 740–
744.
12 Haase AD, Jaskiewicz L, Zhang HD, Laine S, Sack R,
Gatignol A & Filipowicz W (2005) TRBP, a regulator
of cellular PKR and HIV-1 virus expression, interacts
with Dicer and functions in RNA silencing. EMBO Rep
6, 961–967.
13 Saito K, Ishizuka A, Siomi H & Siomi MC (2005) Pro-
cessing of pre-microRNAs by the Dicer-1–Loquacious
complex in Drosophila cells. PLoS Biol 3, 1202–1212.
14 Kok KH, Ng MHJ, Ching YP & Jin DY (2007) Human
TRBP and PACT directly interact with each other and
associate with dicer to facilitate the production of small
interfering RNA. J Biol Chem 282, 17649–17657.
15 Grishok A (2005) RNAi mechanisms in Caenorhabditis
elegans. FEBS Lett 579, 5932–5939.
16 Dietzl G, Chen D, Schnorrer F, Su KC, Barinova Y,
Fellner M, Gasser B, Kinsey K, Oppel S, Scheiblauer S
et al. (2007) A genome-wide transgenic RNAi library
for conditional gene inactivation in Drosophila. Nature
448, 151–156.
17 Gao X & Zhang PM (2007) Transgenic RNA interfer-
ence in mice. Physiology 22, 161–166.
X F. Zhao et al. siRNA and unspecific defects
FEBS Journal 275 (2008) 2177–2184 ª 2008 The Authors Journal compilation ª 2008 FEBS 2183
18 Martin SE & Caplen NJ (2007) Applications of RNA

interference in mammalian systems. Annu Rev Genom 8,
81–108.
19 Gruber J, Lampe T, Osborn M & Weber K (2005)
RNAi of FACE1 protease results in growth inhibition
of human cells expressing lamin A: implications for
Hutchinson–Gilford progeria syndrome. J Cell Sci 118,
689–696.
20 Wargelius A, Ellingsen S & Fjose A (1999) Double-
stranded RNA induces specific developmental defects in
zebrafish embryos. Biochem Biophys Res 263, 156–161.
21 Oates AC, Bruce AEE & Ho RK (2000) Too much
interference: injection of double-stranded RNA has
nonspecific effects in the zebrafish embryo. Dev Biol
224, 20–28.
22 Zhao ZX, Cao Y, Li M & Meng AM (2001) Double-
stranded RNA injection produces nonspecific defects in
zebrafish. Dev Biol 229, 215–223.
23 Giraldez AJ, Cinalli RM, Glasner ME, Enright AJ,
Thomson JM, Baskerville S, Hammond SM, Bartel DP
& Schier AF (2005) MicroRNAs regulate brain mor-
phogenesis in zebrafish. Science 308, 833–838.
24 Giraldez AJ, Mishima Y, Rihel J, Grocock RJ, Van
Dongen S, Inoue K, Enright AJ & Schier AF
(2006) Zebrafish MiR-430 promotes deadenylation
and clearance of maternal mRNAs. Science 312, 75–
79.
25 Seo HC, Drivenes O, Ellingsen S & Fjose A (1998)
Expression of two zebrafish homologues of the murine
Six3 gene demarcates the initial eye primordia. Mech
Dev 73, 45–57.

26 Chen PY, Manninga H, Slanchev K, Chien MC, Russo
JJ, Ju JY, Sheridan R, John B, Marks DS, Gaidatzis D
et al. (2005) The developmental miRNA profiles of
zebrafish as determined by small RNA cloning. Genes
Dev 19, 1288–1293.
27 Kloosterman WP, Steiner FA, Berezikov E, de Bruijn
E, van de Belt J, Verheul M, Cuppen E & Plasterk
RHA (2006) Cloning and expression of new micro-
RNAs from zebrafish. Nucleic Acids Res 34, 2558–2569.
28 Krauss S, Johansen T, Korzh V & Fjose A (1991)
Expression of the zebrafish paired box gene Pax[Zf-B]
during early neurogenesis. Development 113, 1193–2206.
29 Lun K & Brand M (1998) A series of no isthmus (noi)
alleles of the zebrafish pax2.1 gene reveals multiple sig-
naling events in development of the midbrain–hindbrain
boundary. Development 125, 3049–3062.
30 Obernosterer G, Leuschner PJF, Alenius M & Martinez
J (2006) Post-transcriptional regulation of microRNA
expression. RNA 12, 1161–1167.
31 Thomson JM, Newman M, Parker JS, Morin-Kensicki
EM, Wright T & Hammond SM (2006) Extensive post-
transcriptional regulation of microRNAs and its impli-
cations for cancer. Genes Dev 20, 2202–2207.
32 Martello G, Zacchigna L, Inui M, Montagner M,
Adorno M, Mamidi A, Morsut L, Soligo S, Tran U,
Dupont S et al. (2007) MicroRNA control of Nodal
signalling. Nature 449, 183–188.
33 John M, Constien R, Akinc A, Goldberg M, Moon
YA, Spranger M, Hadwiger P, Soutschek J, Vornlocher
HP, Manoharan M et al. (2007) Effective RNAi-medi-

ated gene silencing without interruption of the endoge-
nous microRNA pathway. Nature 449, 745–748.
34 Grimm D, Streetz KL, Jopling CL, Storm TA, Pandey
K, Davis CR, Marion P, Salazar F & Kay MA (2006)
Fatality in mice due to oversaturation of cellular
microRNA ⁄ short hairpin RNA pathways. Nature 441,
537–541.
35 Ekker SC & Larson JD (2001) Morphant technology in
model developmental systems. Genesis 30, 89–93.
36 Heasman J (2002) Morpholino oligos: making sense of
antisense? Dev Biol 243, 209–214.
37 Kloosterman WP & Plasterk RHA (2006) The diverse
functions of MicroRNAs in animal development and
disease. Devl Cell 11, 441–450.
38 Kennedy S, Wang D & Ruvkun G (2004) A conserved
siRNA-degrading RNase negatively regulates RNA
interference in C. elegans. Nature 427, 645–649.
39 Burge C & Karlin S (1997) Prediction of complete gene
structures in human genomic DNA. J Mol Biol 268,
78–94.
40 Milanesi L, D’Angelo D & Rogozin IB (1999) Gene-
Builder: interactive in silico prediction of gene structure.
Bioinformatics 15, 612–621.
Supplementary material
The following supplementary material is available
online:
Fig. S1. Northern blot analysis of the effects of siRNA
injections on endogenous levels of different miRNAs.
Fig. S2. Expression of the pax2a gene at the MMB is
affected by siRNA injection.

Fig. S3. Rescue of siSix3a-induced MMB and tail
defects by coinjection of miR-430b.
Fig. S4. Changes in the endogenous level of miR-430b
following injection of different amounts of siGFP.
Each column represents the average level of the mature
form of miR-430 (relative to the wild-type), obtained
from three different experiments.
This material is available as part of the online article
from
Please note: Blackwell Publishing are not responsible
for the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corre-
sponding author for the article.
siRNA and unspecific defects X F. Zhao et al.
2184 FEBS Journal 275 (2008) 2177–2184 ª 2008 The Authors Journal compilation ª 2008 FEBS