Targeted Inhibition of miRNA Maturation
with Morpholinos Reveals a Role for miR-375
in Pancreatic Islet Development
Wigard P. Kloosterman1, Anne K. Lagendijk1, Rene ´ F. Ketting1*, Jon D. Moulton2, Ronald H. A. Plasterk1
1 Hubrecht Laboratory-KNAW, Utrecht, The Netherlands, 2 Gene Tools, Philomath, Oregon, United States of America
Several vertebrate microRNAs (miRNAs) have been implicated in cellular processes such as muscle differentiation,
synapse function, and insulin secretion. In addition, analysis of Dicer null mutants has shown that miRNAs play a role in
tissue morphogenesis. Nonetheless, only a few loss-of-function phenotypes for individual miRNAs have been described
to date. Here, we introduce a quick and versatile method to interfere with miRNA function during zebrafish embryonic
development. Morpholino oligonucleotides targeting the mature miRNA or the miRNA precursor specifically and
temporally knock down miRNAs. Morpholinos can block processing of the primary miRNA (pri-miRNA) or the pre-
miRNA, and they can inhibit the activity of the mature miRNA. We used this strategy to knock down 13 miRNAs
conserved between zebrafish and mammals. For most miRNAs, this does not result in visible defects, but knockdown of
miR-375 causes defects in the morphology of the pancreatic islet. Although the islet is still intact at 24 hours
postfertilization, in later stages the islet cells become scattered. This phenotype can be recapitulated by independent
control morpholinos targeting other sequences in the miR-375 precursor, excluding off-target effects as cause of the
phenotype. The aberrant formation of the endocrine pancreas, caused by miR-375 knockdown, is one of the first loss-
of-function phenotypes for an individual miRNA in vertebrate development. The miRNA knockdown strategy
presented here will be widely used to unravel miRNA function in zebrafish.
Citation: Kloosterman WP, Lagendijk AK, Ketting RF, Moulton JD, Plasterk RHA (2007) Targeted inhibition of miRNA maturation with morpholinos reveals a role for miR-375 in
pancreatic islet development. PLoS Biol 5(8): e203. doi:10.1371/journal.pbio.0050203
MicroRNAs (miRNAs) have a profound impact on the
development of multicellular organisms. Animals lacking the
Dicer enzyme, which is responsible for the processing of the
precursor miRNA into the mature form, cannot live [1–3].
MiRNA mutants have been described only for Caenorhabditis
elegans and Drosophila, reviewed in . From these studies, it is
clear that invertebrate miRNAs are involved in a variety of
cellular processes, such as developmental timing [5,6],
apoptosis [7,8], and muscle growth . Analysis of conditional
Dicer null alleles in mouse has indicated a general role for
miRNAs in morphogenesis of the limb, skin, lung epithelium,
and hair follicles [10–13]. Overexpression studies in mouse
have implicated specific vertebrate miRNAs in cardiogenesis
and limb development [14,15]. In zebrafish, embryos lacking
both maternal and zygotic contribution of Dicer have severe
brain defects . Strikingly, the brain phenotype of maternal-
zygotic Dicer zebrafish can be restored by injection of miR-
430, the most abundant miRNA in early zebrafish develop-
ment. Despite all these studies describing functions for
miRNAs in development, no vertebrate miRNA mutant has
been described to date. Genetically, it is challenging to obtain
mutant miRNA alleles in zebrafish, because their small size
makes them less prone to mutations by mutagens, and for
many miRNAs, there are multiple alleles in the genome or
they reside in families of related sequence.
Temporal inhibition of miRNAs by antisense molecules
provides another strategy to study miRNA function. 29-O-
methyl oligonucleotides have been successfully used in vitro
and in vivo to knock down miRNAs [16–18]. Morpholinos are
widely applied to knock down genes in zebrafish development
 and have recently been used to target mature miR-214 in
zebrafish . However, off-target phenotypes are often
associated with the use of antisense inhibitors.
Here, we show that morpholinos targeting the miRNA
precursor can knock down miRNAs in the zebrafish embryo.
Several independent morpholinos can knock down the same
miRNA, and these serve as positive controls to filter out off-
target effects. Morpholinos can block miRNA maturation at
the step of Drosha or Dicer cleavage, and they can inhibit the
activity of the mature miRNA. We show that inhibition of
miR-375, which is expressed in the pancreatic islet and
pituitary gland of the embryo , results in dispersed islet
cells in later stages of embryonic development, whereas no
effects were observed in the pituitary gland. The morpholino-
mediated miRNA knockdown strategy presented here, is an
extremely fast and well-controlled method to study miRNA
function in development.
Academic Editor: James C. Carrington, Oregon State University, United States of
Received October 13, 2006; Accepted May 22, 2007; Published July 24, 2007
Copyright: ? 2007 Kloosterman et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Abbreviations: dpf, days postfertilization; GFP, green fluorescent protein; hpf,
hours postfertilization; LNA, locked nucleic acid; miRNA, microRNA; MO,
morpholino oligonucleotide; RT-PCR, reverse transcriptase PCR
* To whom correspondence should be addressed. E-mail: firstname.lastname@example.org
PLoS Biology | www.plosbiology.orgAugust 2007 | Volume 5 | Issue 8 | e203 1738
P PL Lo oS S BIOLOGY
Morpholinos Targeting the Mature miRNA Deplete the
Embryo of Specific miRNAs
Since it is difficult to obtain a genetic mutant for a miRNA
in zebrafish, we looked for alternative strategies to deplete
the embryo of specific miRNAs. Antisense molecules such as
29-O-methyl and locked nucleic acid (LNA) oligonucleotides
have been used to inhibit miRNAs in cell lines [16,18,22],
Drosophila embryos , and adult mice . We tried to use
these molecules to inhibit the function of endogenous
miRNAs in the zebrafish embryo. Although they can be used
to suppress the effects of miRNA overexpression ,
injection of higher concentrations required to obtain good
knockdown of endogenous miRNAs resulted in toxic effects,
when injecting 1 nl solution at a concentration of approx-
imately 10 lM and 50 lM for LNA and 29-O-methyl
oligonucleotides, respectively (unpublished data). Therefore,
we switched to morpholinos because these are widely used to
inhibit mRNA translation and splicing in zebrafish embryos
, and have also been shown to target miRNAs in the
embryo [2,20,24]. We injected 1 nl of 600 lM morpholino
solution with a morpholino complementary to the mature
miR-206 in one- or two-cell–stage embryos. Subsequently,
embryos were harvested at 24, 48, 72, and 96 hours
postfertilization (hpf), and subjected to in situ hybridization
and Northern blotting (Figure 1A and 1B). This analysis
showed that the mature miRNA signal is suppressed up to 4 d
after injection of the morpholino. The knockdown effect was
specific for this miRNA; parallel in situ analysis of the same
embryos with a probe for miR-124 did not show any effects
on expression of this miRNA (Figure 1B). Thus, miRNA
detection can be specifically and efficiently suppressed
during embryonic and early larval stages of zebrafish
development using morpholinos antisense to the mature
The zebrafish embryo can be used to monitor the effect of
miRNAs on green fluorescent protein (GFP) reporters fused
to miRNA target sites . To determine the effect of a
morpholino in this assay system, we constructed a GFP
reporter for miR-30c and tested it in the presence and
absence of a mature miR-30c duplex. Injected miR-30c
silences this GFP reporter, which is in line with previous
reports using similar strategies in the embryo (Figure 1C)
[2,20,24]. Co-injection of the miR-30c duplex and a morpho-
lino targeting mature miR-30c rescues the reporter signal,
whereas injection of a control morpholino did not reverse the
silencing by miR-30c. These data indicate that a morpholino
can block the activity of a mature miRNA duplex in a
functional assay .
There are three possible explanations for the observed
reduction in the detection signal for a miRNA that is targeted
by a morpholino. First, the hybridization of a morpholino
could disturb isolation of the miRNA. Second, the morpho-
lino could destabilize the miRNA. Third, the morpholino
could inhibit the maturation of the miRNA.
To examine the effect of a morpholino on the isolation of a
mature miRNA, we incubated a mature miR-206 duplex and a
control duplex (miR-205) with a morpholino against miR-206
in vitro. After isolation, samples were analyzed by Northern
blotting for the presence of miR-206 and miR-205. We could
still detect miR-206, indicating that there is no effect of the
morpholino on the RNA isolation procedure (Figure 1D).
However, when morpholino and miRNA duplex were
incubated together in vitro and loaded on a denaturing gel
without isolation, we observed a decrease in the signal for
miR-206, indicating that the morpholino can bind to the
miRNA in vitro and still does so in the denaturing gel.
Next, we wanted to know whether a morpholino could
affect the stability of a mature miRNA in vivo. Therefore, we
injected a mature miR-206 and a control duplex (miR-205)
together with a morpholino against miR-206 in the embryo.
After incubation for 8 h, RNA was isolated and subjected to
Northern blot analysis to probe for injected miR-206 and
injected miR-205. In contrast to the data obtained for
endogenous miR-206, there was no decrease observed in the
amount of injected miR-206 in the morpholino-injected
embryos (Figure 1D) (endogenous miR-206 is not yet ex-
pressed at this stage).
Since these data show that there is no effect of a
morpholino on miRNA isolation or stability, we conclude
that morpholinos deplete the embryo of miRNAs by inhibit-
ing miRNA maturation. If this is the case, then we expect
morpholinos targeting other regions of the miRNA precursor
to act as well as the morpholinos designed against the mature
miRNA, and this is indeed what we find (see next section).
Morpholinos Targeting the miRNA Precursor Interfere
with Primary miRNA Processing
Injection of antisense oligos in embryos might result in off-
target effects. Thus, phenotypic data retrieved from antisense
knockdown experiments should be treated with caution. In
Drosophila, 29-O-methyl oligo–mediated knockdown of embry-
onically expressed miRNAs caused defects that clearly
differed from the phenotype of the corresponding knockout
fly [9,23]. In sea urchin experiments, off-target effects of
morpholino knockdowns are well documented, though low
incubation temperatures favor off-target interactions .
To filter out off-target effects, we sought a control strategy
that would allow us to compare effects of morpholinos with
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Morpholino-Mediated miRNA Knockdown
The striking tissue-specific expression patterns of microRNAs
(miRNAs) suggest that they play a role in tissue development.
These small RNA molecules (;22 bases in length) are processed
from long primary transcripts (pri-miRNA) and regulate gene
expression at the posttranscriptional level. There are hundreds of
different miRNAs, many of which are strongly conserved. Vertebrate
embryonic development is most easily studied in zebrafish, but
genetically disrupting miRNA genes to see which miRNA does what
is technically challenging. In this study, we interfere with miRNA
function during the first few days of zebrafish embryonic develop-
ment by introducing specific antisense morpholino oligonucleotides
(morpholinos have been used previously to interfere with the
synthesis of the much larger mRNAs). We show that morpholinos
targeting the miRNA precursor can block processing of the pri-
miRNA or directly inhibit the activity of the mature miRNA. We also
used morpholinos to study the developmental effects of miRNA
knockdown. Although we did not observe gross phenotypic defects
for many miRNAs, we found that zebrafish miR-375 is essential for
formation of the insulin-secreting pancreatic islet. Loss of miR-375
results in dispersed islet cells by 36 hours postfertilization,
representing one of the first vertebrate miRNA loss-of-function
independent sequences targeted to the same miRNA. Because
our data on morpholinos targeting the mature miRNA
suggested that miRNA biogenesis might be affected, we
designed morpholinos targeting the Drosha and Dicer
cleavage sites of the precursor miRNA (Figure 2A). We
decided to test this strategy on miR-205, since it is expressed
relatively early, and there are only two, but identical, copies
in the fish genome. Four different morpholinos were
designed to inhibit miR-205 biogenesis: two targeting the
Drosha cleavage site complementary to either the 59 or 39 arm
of the stem, and two morpholinos similarly targeting the
Dicer cleavage site (Figure S1). These morpholinos were
injected under similar conditions as described for miR-206
and compared to the morpholino targeting mature miR-205.
Interestingly, all five morpholinos induced complete or near-
complete loss of miR-205 (Figure 2B).
Many miRNAs are highly expressed during later stages of
embryonic development . Therefore, we tested how long
the effect of the morpholinos would last. Although for this
series of morpholinos the knockdown is best at 24 hpf, the
effect is still significant up to 72 hpf (Figure 2C).
Next, we tested a similar series of morpholinos against the
miR-30c precursor and analyzed miR-30c expression by
Northern blotting (Figure S2). However, we only observed
knockdown for the morpholino targeting mature miR-30c,
but not for the other four morpholinos targeting the miR-30c
precursor. This could be because miR-30c resides in a family
of closely related species, with more sequence variability in
the regions outside of the mature miRNA. The precursors of
the family members might not all be targeted by these
morpholinos (Figure S2). Thus, not all miRNAs are equally
prone to knockdown by morpholinos that target the miRNA
To investigate the effect of morpholinos on exogenously
introduced pri-miR-205, we injected mRNA derived from a
GFP construct with pri-miR-205 in the 39 UTR. Again, we
could not detect mature miR-205 derived from this construct
after targeting by morpholinos (Figure 2D). Interestingly, the
miR-205 precursor also could not be detected in the embryos
co-injected with morpholinos, whereas pre-miR-205 could be
detected in the absence of morpholinos (Figure 2D). Because
pri-miR-205 was cloned in the 39 UTR of GFP, we monitored
GFP fluorescence after injection of this construct. In the
presence of a morpholino, GFP fluorescence increased
(Figure 2E), suggesting accumulation of the primary miRNA.
Therefore, we performed reverse transcriptase PCR (RT-
PCR) on 8-h-old embryos injected with GFP-pri-miR-205 and
a control mRNA (luciferase) (Figure 2F). In the presence of a
morpholino, the GFP-pri-miR-205 mRNA level is higher
compared to control embryos that were not injected with
morpholinos. This experiment confirms the GFP data and
shows that morpholinos targeting the miRNA precursor
inhibit Drosha cleavage.
Next, we tested whether processing of the pre-miRNA
might also be inhibited by morpholinos. Therefore, we
injected a miR-205 precursor in the one-cell–stage embryo.
Northern analysis showed that the precursor was processed
into mature miRNA in the embryo (Figure 2G). However, co-
Figure 1. Morpholinos Targeting the Mature miRNA Deplete the Zebrafish Embryo of Specific miRNAs
(A) Northern blot for miR-206 in wild-type and MO miR-206–injected embryos at 24, 48, and 72 hpf. 5S RNA serves as a loading control.
(B) In situ analysis of miR-206 and miR-124 expression in different stage embryos after injection of MO miR-206.
(C) Effect of a morpholino targeting miR-30c on a silencing assay with miR-30c and a responsive GFP sensor construct.
(D) In vivo and in vitro effects of a morpholino on the stability and RNA extraction of a synthetic miR-206 duplex. miR-205 serves as a loading control.
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Morpholino-Mediated miRNA Knockdown
Figure 2. Morpholinos Targeting the Precursor miRNA Interfere with miRNA Maturation
(A) Design of morpholinos targeting the precursor miRNA.
(B) Northern blot analysis of miR-205 in 30-h-old embryos injected with different morpholinos against pri-miR-205. 5S RNA serves as a loading control.
(C) Time series of miR-205 expression after injection of mature, no lap loop, and drosha star morpholinos against pri-miR-205.
(D) Northern blot analysis of miR-205 derived from embryos injected with a GFP-pri-miR-205 transcript and four different morpholinos targeting pri-
miR-205. Co-injected miR-206 serves as an injection and loading control. Embryos were collected 8 h after injection.
(E) GFP expression in 24-h embryos injected with morpholinos and a GFP-pri-miR-205 construct as used in (C). Pri-miR-205 is positioned just upstream of
the polyA signal in the 39 UTR of the GFP mRNA. Red fluorescent protein (RFP) serves as an injection control.
(F) RT-PCR analysis of injected GFP-pri-miR-205 mRNA with (þ) and without (?) co-injected morpholinos. Luciferase serves a an injection control.
Embryos were collected 8 h after injection.
(G) Northern analysis of the effect of morpholinos on an injected miR-205 precursor. Embryos were collected 8 h after injection.
WT, wild type.
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Morpholino-Mediated miRNA Knockdown
injection of the overlap loop and non-overlapping loop
morpholinos blocked processing completely. There was only
a little effect of morpholinos targeting the Drosha cleavage
site, probably because they only partially overlap the
A similar analysis was performed for miR-375, which is
expressed in the pancreatic islet and pituitary gland , and
has two copies in the zebrafish genome, which differ in the
regions outside the mature miRNA.
Overlap loop and loop morpholinos were designed for
both miR-375–1 and miR-375–2, and a morpholino against
the miRNA star sequence could be used to target both copies
of miR-375 simultaneously (Figure 3A). The efficacy of all
morpholinos was assessed by determining their effect on
injected pri-miR-375–1 or pri-miR-375–2 transcripts (Figure
3B). As expected, each morpholino targeted the transcript to
which it was directed. However, the star miR-375 morpholino
did not knock down miR-375 completely. In addition,
morpholino oligonucleotide (MO) miR-375 did not interfere
with processing of miR-375 from pri-miR-375–1, possibly
because this primary transcript forms a more stable hairpin.
In all cases, the lack of a signal for mature miR-375 coincided
with the absence of pre-miR-375, which could be detected in
the absence of a complementary morpholino.
Next, all morpholinos were injected separately and in
combination, and embryos were subjected to Northern
blotting to determine endogenous miR-375 expression at 24
and 48 hpf (Figure 3C). In contrast to the results obtained by
in situ hybridization (see last section), the morpholino to
mature miR-375 only slightly decreased the expression of
miR-375. However, MO miR-375 could inhibit the activity of a
mature miR-375 duplex in a GFP-miR-375-target reporter
assay (Figure 3E). The morpholinos targeting only one copy of
miR-375 reduced miR-375 expression, with the strongest
effect for the morpholinos targeting pri-miR-375–1. How-
ever, simultaneous injection of morpholinos targeting pri-
miR-375–1 and pri-miR-375–2 completely knocked down
mature miR-375, indicating that both transcripts are ex-
To further determine the contribution of each transcript
to mature miR-375 accumulation, we performed in situ
hybridization for pri-miR-375–1 and pri-miR-375–2 (Figure
3D). Both transcripts could not be detected in wild-type
embryos. However, pri-miR-375–1 was detected in the
pancreatic islet and the pituitary gland in embryos injected
with the miR-375–1 loop morpholino and the morpholino to
miR-375 star. Similarly, pri-miR-375–2 was only detected in
embryos injected with the miR-375–2 loop morpholino, the
morpholino to miR-375 star and mature miR-375. Thus, both
transcripts are expressed in the pituitary gland and the
pancreatic islet, similar to miR-1 in the developing mouse
heart . Together, this indicates that these morpholinos
inhibit primary miRNA processing and result in primary
miRNA accumulation, as we described for miR-205.
In conclusion, our data demonstrate that morpholinos
targeting the miRNA precursor can interfere with primary
miRNA processing at either the Drosha or Dicer cleavage step
and that morpholinos targeting the mature miRNA can
inhibit their activity in a functional assay. Taken together,
our data show that different morpholinos targeting the same
miRNA may serve as positive controls for miRNA knockdown
phenotypes in the embryo.
Knockdown of Many miRNAs Does Not Result in Any
Observed Developmental Defects
To identify functions for individual miRNAs in zebrafish
embryonic development, we knocked down a series of 11
conserved vertebrate miRNAs (Table S1) and analyzed their
expression after morpholino knockdown. Injected embryos
were monitored phenotypically by microscopic observation
until four days postfertilization (dpf). Knockdown of most
miRNAs resulted in loss of in situ staining for the respective
miRNA. However, we could not observe gross morphological
malformations after knockdown of these miRNAs (Figure 4A).
Therefore, we analyzed embryos injected with morpholinos
against miR-182, miR-183, or miR-140 in more detail, because
we could easily stain the tissues that express these miRNAs
(Figure 4B). Embryos injected with morpholinos against miR-
182 or miR-183, which are expressed in the lateral line
neuromasts and hair cells of the inner ear, were treated with
DASPEI, which stains hair cells. Embryos injected with a
morpholino against miR-140, which is expressed in cartilage,
were subjected to Alcian Blue staining, a cartilage marker.
However, staining of these specific cell types that express the
miRNA did not uncover any defects upon knockdown (Figure
In conclusion, knockdown of many miRNAs does not
appear to significantly affect zebrafish embryonic develop-
ment, at least not to the extent that can be visualized by the
methods used in these examples.
Knockdown of miR-375 Affects Pancreatic Islet
MiR-375 is known to be expressed in the pancreatic islet
and the pituitary gland, and was first isolated from pancreatic
beta cells [21,26]. This miRNA is conserved in vertebrates and
may regulate insulin secretion by inhibiting myotrophin .
We injected a morpholino against mature miR-375 into the
one-cell–stage embryo. This morpholino effectively knocked
down miR-375 in the first 4 d of development (Figure 5A), and
it could also block the activity of an injected miR-375 duplex,
as monitored by its effect on a GFP reporter silenced by miR-
375 (Figure 3E).
During the first 5 dpf, there was no clear developmental
defect except for a general delay in development. At around 7
dpf, approximately 80% of the injected embryos died. Next,
we analyzed the development of both the pituitary gland and
the pancreatic islet, by in situ hybridization with pit1 and
insulin markers. This analysis revealed no change in the
formation of the pituitary gland (Figure 5B). However,
analysis of insulin expression showed a striking malformation
of the islet cells in 3-d-old morphant embryos (Figure 5B).
Wild-type embryos have a single islet at the right side of the
midline, whereas the miR-375 knockdown embryos have
dispersed insulin-positive cells. The effect is sequence
specific, because a morpholino complementary to the mature
miR-375 morpholino inhibited the pancreatic islet pheno-
type (Figure 5E).
The pancreatic islet consists of four cell types, a, b, d, and
PP, expressing glucagon, insulin, somatostatin, and pancre-
atic polypeptide, respectively. Insulin is the first hormone
expressed, and somatostatin co-localizes partially with in-
sulin, whereas glucagon-expressing cells are distinct . A
more detailed analysis using somatostatin and glucagon as
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Morpholino-Mediated miRNA Knockdown
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Morpholino-Mediated miRNA Knockdown
marker genes revealed a similar pattern of scattered islet cells
in the miR-375 morphant (Figure 5C).
In zebrafish, insulin is first expressed at the 12-somite stage
in a few scattered cells located at the midline, dorsal to the
yolk . Insulin-positive cells migrate posteriorly and
converge medially to form an islet by 24 hpf. To look at the
development of the pancreatic islet in time, we collected MO
miR-375 and noninjected control embryos at different stages,
Figure 3. Specific Morpholinos Deplete the Embryo of miR-375
(A) Sequence alignment of the two miR-375 genes from zebrafish and design of morpholinos targeting the dre-miR-375–1 and dre-miR-375–2
(B) Northern blot analysis of the effect of morpholinos on the expression of miR-375 derived from injected pri-miRNA mRNAs for miR-375–1 and miR-
375–2. MO-375–1 overlap loop and loop morpholinos target exclusively the pri-miR-375–1 construct, and MO-375–2 overlap loop and loop
morpholinos target exclusively the pri-miR-375–2 construct. Co-injected miR-206 serves as a loading and injection control. Embryos were collected 8 h
(C) Northern blot analysis of the effect of morpholinos on endogenous miR-375 expression at 24 hpf and 48 hpf. MiR-206 serves as loading control.
(D) In situ hybridization for pri-miR-375–1 and pri-miR-375–2 on wild-type (WT) and morpholino-injected embryos. Arrowheads indicate the pituitary
gland and the pancreatic islet.
(E) Analysis of GFP expression in 24-h embryos injected with a miR-375 GFP sensor construct, a synthetic miR-375 duplex and MO miR-375. Red
fluorescent protein (RFP) serves as an injection control.
NIC, noninjected control.
Figure 4. Knockdown of Many miRNAs Does Not Affect Zebrafish Embryonic Development
(A) Phenotypes and in situ analysis of 3- and 4-d-old embryos after injection of morpholinos against 11 different mature miRNAs.
(B) Daspei staining of 72-h-old embryos injected with MO miR-182 and MO miR-183, and wild-type control (upper panel). Alcian Blue staining of 72-h-
old embryos injected with MO miR-140 and noninjected control (lower panel).
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Morpholino-Mediated miRNA Knockdown
and investigated the expression of insulin (Figure 5D). At the
16-somite stage, insulin-positive cells are scattered at the
midline in both noninjected and MO miR-375–injected
embryos, and a presumptive islet is formed by 24 hpf.
Subsequently, when the insulin-positive islet is moving to the
right side of the embryo in later stages, the islet breaks apart
and insulin-positive cells become scattered in morphant
embryos (Figure 5D). Also, in later stages, the phenotype
persists, although miR-375 is re-expressed at approximately 5
dpf in morpholino-injected embryos (Figure 5A).
Next, we analyzed the effect of all miR-375 control
morpholinos described in the previous section, by staining
for insulin (Figure 6A). Both the dispersion phenotype and
the knockdown were striking for embryos injected with MO
Figure 5. Knockdown of miR-375 Results in Aberrant Migration of Pancreatic Islet Cells
(A) In situ analysis of miR-375 knockdown in MO miR-375–injected embryos and noninjected controls at 24, 48, 72, and 120 hpf. Arrowheads indicate
the pituitary gland and the pancreatic islet.
(B) In situ analysis of the pancreatic islet (insulin staining) and the pituitary gland (pit1 staining) in miR-375 morphants and noninjected controls.
Arrowheads indicate the pituitary gland and the pancreatic islet.
(C) In situ analysis of pancreatic islet development in wild-type and morphant embryos using insulin, somatostatin, and glucagon as markers.
(D) Time series of insulin expression in wild-type and morphant embryos injected with MO miR-375.
(E) Insulin expression in 72-hpf embryos injected with MO miR-375 and a complementary morpholino.
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Morpholino-Mediated miRNA Knockdown
Figure 6. Specific Effects of miR-375 Knockdown on the Development of the Endocrine Pancreas
(A) In situ analysis of miR-375 and insulin expression in 72-hpf embryos injected with morpholinos against the miR-375 precursor and negative control
morpholinos for let-7 and miR-124.
(B) Expression of islet1, foxa2, and ptf1a in wild-type and miR-375 knockdown embryos. Arrows indicate the pancreatic islet.
WT, wild type
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Morpholino-Mediated miRNA Knockdown
miR-375. Injection of the overlap loop and loop morpholinos
targeting pri-miR-375–1 also resulted in scattered insulin-
positive cells at 72 hpf, although the effect was weaker
compared to MO miR-375. The miR-375–2 loop and overlap
loop morpholinos hardly induced any scattering of insulin-
positive cells, whereas the effect was very strong in embryos
injected with morpholinos to pri-miR-375–1 and ?2 simulta-
neously. The effect of the miR-375 star morpholino on
insulin-positive cells was moderate compared to MO miR-
To further prove the specificity of the pancreatic islet
phenotype, we injected two control morpholinos against let-7
and miR-124 and analyzed these for miR-375 and insulin
expression. None of these control morpholinos showed loss
of miR-375 expression or abnormal development of the islet
cells (Figure 6A).
Next, we analyzed miR-375 knockdown embryos with
markers staining the endocrine or exocrine pancreas (Figure
6B). Similar to insulin staining, islet1 expression showed
dispersed islet cells in embryos of 48 hpf and 72 hpf, but not
24 hpf. Embryos injected with MO miR-375 exhibited delayed
development of the exocrine pancreas, liver, and gut as
shown by ptf1a and foxa2 staining. At 72 hpf, these markers
showed a similar pattern in MO miR-375–injected embryos as
in noninjected embryos at 48 hpf. However, co-injection of
miR-375–1/2 loop morpholinos did not delay development of
the exocrine significantly, but these embryos still displayed
the scattered insulin-positive cells (Figure 6A). This shows
that loss of miR-375 mainly results in malformation of the
endocrine pancreas, whereas surrounding tissues that do not
express miR-375 are not affected.
Functional data on miRNAs in vertebrate development
have been obtained mainly from overexpression studies and
analysis of conditional Dicer knockouts. For example, the role
of miR-430 in zebrafish brain morphogenesis has become
clear from experiments that rescued Dicer null mutants by
injection of an miRNA duplex that mimicked a miR-430
family member .
MiRNA expression can be conveniently studied in zebrafish
embryos. However, dissecting miRNA function by disrupting
miRNA genes is difficult in zebrafish, because the miRNA is
too small to efficiently search for mutations by a target-
selected mutagenesis approach . In addition, it is unclear
what such point mutations would do to processing or
function of the miRNA.
It has been shown previously that morpholinos can target
miRNAs in the zebrafish embryo [20,24]. In a recent study,
mature miR-214 was targeted by a morpholino in zebrafish,
and this resulted in a change in somite shape, reminiscent of
attenuated hedgehog signaling . Although the phenotype
could be rescued by simultaneous inhibition of a negative
regulator of hedgehog signaling, no positive control mor-
pholinos were reported that could mimic the phenotype. In
addition, data were lacking that showed an effect of the
morpholino on endogenous miR-214 levels.
The results in this paper show that morpholinos targeting
the miRNA precursor form a reliable and efficient tool to
deplete the embryo of miRNAs during the first 4 d of
development, when most organ systems are formed and
miRNAs are expressed. We have shown that miRNA expres-
sion can be inhibited by targeting the mature miRNA, the
precursor miRNA or the primary miRNA. Our data show that
such morpholinos can inhibit miRNA processing at the
Drosha cleavage step or the Dicer cleavage step, probably by
steric blocking, although the exact mechanism is unclear. In
addition, morpholinos targeting the mature miRNA can
inhibit their activity, probably by preventing binding to a
We used morpholinos targeting the mature miRNA for a
set of 13 conserved vertebrate miRNAs to identify their
developmental functions. By microscopic analysis, we could
not observe clear defects associated with loss of 11 of these
miRNAs during the first 4 d of embryonic development,
although in situ hybridization revealed specific loss of most
knocked-down miRNAs. Because all the targeted miRNAs are
expressed in very specific tissues and we did not investigate
most morphants in much detail by marker analysis, we may
have missed subtle defects. In addition, many miRNAs reside
in families of related sequence (e.g., let-7 and miR-182), and
these should possibly be targeted simultaneously by different
morpholinos to obtain a biological effect. Furthermore, in
those instances in which miRNAs of unrelated sequence
target a similar set of mRNAs when expressed in the same
tissue , removing only one miRNA might not have a
profound impact on transcript levels or expression. Finally,
microarray analysis and computational predictions have
shown that a single miRNA may regulate hundreds of mRNAs
[30,31], but that some miRNAs act as a backup for mRNAs
that are already repressed transcriptionally . Thus,
knockdown of such miRNAs might not dramatically affect
gene expression, but ensure robustness of protein interaction
networks as for example miR-7 in Drosophila .
In zebrafish, there are two copies of miR-375, and in human
and mouse only one copy has been identified . To verify
the miR-375 knockdown phenotype, we designed control
morpholinos targeting both precursors simultaneously (MO
miR-375 star) and separately. Complete knockdown was only
observed in those instances in which both miR-375 copies
were targeted simultaneously. This also led to scattered islet
cells, proving the specificity of the phenotype. However,
knockdown with miR-375–1/2 loop morpholinos did not delay
development as seen in the knockdown with the mature miR-
375 morpholino. This shows the strength of using control
morpholinos and excludes the delayed development as a
relevant miR-375 loss-of-function phenotype. A moderate
version of the phenotype was also observed in embryos
injected with a morpholino specifically targeting miR-375–1.
Thus, a reduction in the level of miR-375 already disturbs islet
integrity. Similar to mouse miR-1 , miR-375 copies
survived evolution and are expressed similarly in time and
space, probably to ensure the high intracellular concentra-
tion of miR-375 necessary to repress many weakly binding
In a forward genetic screen, several mutants were identified
with improper development of the endocrine pancreas .
These mutants fall into three classes: (1) mutants with severely
reduced insulin expression; (2) mutants with reduced insulin
expression and abnormal islet morphology; and (3) mutants
with normal levels of insulin expression and abnormal islet
morphology. However, in all of these mutants, islet cells do
not merge into an islet from their first appearance at
PLoS Biology | www.plosbiology.orgAugust 2007 | Volume 5 | Issue 8 | e203 1747
Morpholino-Mediated miRNA Knockdown
approximately the 14-somite stage. Our miR-375 knockdown
phenotype differs from this, because in the first instance, an
islet is formed at approximately 24 hpf, but in later stages, the
islet falls apart into small groups of cells. This rules out a
general role for miR-375 in early endocrine formation as is
seen for Wnt5 , but rather indicates a role in maintenance
of tissue identity, which is assumed to be a general function of
miRNAs in development . It is as yet unclear which miR-
375 targets are involved in the phenotype. Work in cell lines
has implicated miR-375 in insulin secretion by targeting
myotrophin . The zebrafish homolog of myotrophin also
contains a seven-nucleotide seed match to miR-375 (unpub-
lished data), but future studies should reveal whether this
target or many other predicted targets are relevant to the
phenotype. The specific expression of miR-375 in the
pancreatic islet and its implication in insulin secretion make
it a candidate drug target in diabetes, e.g., to influence insulin
levels in the blood. However, our data show that if miR-375 is
used as a drug target, developmental side effects need to be
taken into account.
Materials and Methods
Morpholino and miRNA injections. Morpholinos were obtained
from Gene Tools LLC (http://www.gene-tools.com) and dissolved to a
concentration of 5 mM in water. Morpholinos were injected into one-
or two-cell–stage embryos at concentrations between 200 lM and
1,000 lM, and per embryo, one nl of morpholino solution was
RNA oligos (Table S2) were obtained from Sigma (http://www.
sigmaaldrich.com) and dissolved to a concentration of 100 lM in
distilled water. Oligos were annealed using a 5x buffer containing 30
mM HEPES-KOH (pH 7.4), 100 mM KCl, 2 mM MgCl2, and 50 mM
NH4Ac. Typically, 1 nl of a 10 lM miRNA duplex solution was
All morpholino sequences used in this study are listed in Table S1.
Construction of miR-30c and miR-375 GFP reporters and pri-
miRNA constructs. The miR-30c and miR-375 reporter constructs
were made by cloning two annealed oligos containing two perfectly
complementary miRNA target sites into pCS2 (Clontech, http://www.
clontech.com) containing a gfp gene between BamHI and ClaI
restriction sites. A construct containing pri-miR-205 was made by
amplifying a genomic region (801 base pairs) containing the miR-205
precursor (miR-205-hairpinF ggcattgaattcataaCCTCTTACCTGCAT-
GACCTG; miR-205-hairpinR ggcatttctagaGTGTGTGCGTGTATT-
CAACC). The resulting PCR fragment was cloned between XbaI
and EcoRI restriction sites of PCS2GFP. Pri-miR-375–1 and pri-miR-
375–2 constructs were made by amplifying genomic regions contain-
ing miR-375–1 and miR-375–2 precursors (WKmiR-375–1F-pCS2
attacgaattcTCAAACTCTCCACTGACTGC; and WKmiR-375–2F-
pCS2 gcccgggatccGCCCTCCCATTTGACTC; WKmiR-375–2R-pCS2
attacgaattcAATGAGTGCACAAAATGTCC), and cloning of the re-
sulting PCR fragments into the BamHI and EcoRI sites of pCS2.
mRNA was synthesized using SP6 RNA polymerase. Luciferase mRNA
was derived from pCS2 containing luciferase between BamHI and
In situ hybridization, Northern blotting, and RT-PCR. In situ
hybridization was performed as described previously . LNA
probes for miRNA detection were obtained from Exiqon (http://
www.exiqon.com) and labeled using terminal transferase and DIG-11-
ddUTP. cDNA clones for pri-miR-375–1, pri-miR-375–2, pit1, insulin,
somatostatin, and glucagon were used for antisense DIG-labeled
probe synthesis by T7 or Sp6 RNA polymerase.
For Northern blotting, total RNA was isolated from ten embryos
per sample using Trizol reagent (Invitrogen, http://www.invitrogen.
com). RNA was separated on a 15% denaturing polyacrylamide gel.
Radiolabeled DNA probes complementary to miRNAs or 5S RNA
(atcggacgagatcgggcgta) were used for hybridization at 37 8C.
Stringency washes were done twice for 15 min at 37 8C using 2 3
SSC 0.2% SDS. Alternatively, DIG-labeled LNA probes were used for
hybridization at 60 8C and stringency washes were performed at 50 8C
with 2x SSC 0.1% SDS for 30 min and 0.5x SSC 0.1% SDS for 30 min.
For RT-PCR, RNA was isolated with Trizol, treated with DNAse
(Promega, http://www.promega.com) and subsequently purified again
using Trizol. cDNA was made with a poly dT primer. Primers used for
amplification were miR-205-hairpinF and miR-205-hairpinR, and
lucF (ATGGAAGACGCCAAAAACATAAAG) and lucR (ATTACATC-
Alcian Blue and Daspei staining. For Alcian Blue staining, embryos
were fixed for 1 h at room temperature in 4% PFA in PBS, rinsed for
5 min in 50% MeOH, and stored overnight in 70% MeOH at 4 8C.
Next, embryos were incubated for 5 min in 50% MeOH and for 5 min
in 100% EtOH. Embryos were stained at room temperature with
Alcian Blue (Sigma) for 90 min with continuous shaking. Subse-
quently, embryos were rinsed in 80%, 50%, and 25% EtOH for 2 min
each and two times in water containing 0.2% Triton and neutralized
in 100% Borax solution. Finally, embryos were incubated for 60 min
in digest solution (60% Borax solution, 1 mg/ml colleganase-free and
elastase-free trypsin, 0.2% trypsin) and stored in 70% glycerol.
Staining of the hair cells was done by incubating live embryos for 5
min in a 200 lM solution of Daspei (Sigma) inþchorion. After rinsing
twice in þ chorion, embryos were anesthetized using MS222 and
mounted in methylcellulose.
Figure S1. Design of Morpholinos Targeting the miR-205 Precursor
Found at doi:10.1371/journal.pbio.0050203.sg001 (224 KB TIF).
Figure S2. Morpholino-Mediated Knockdown of miR-30c
(A) Design of morpholinos targeting the miR-30c precursor.
(B) Northern analysis of miR-30c expression in 24-h-old embryos
injected with different morpholinos targeting the miR-30c precursor.
(C) Alignment of the precursor of miR-30 family miRNAs.
Found at doi:10.1371/journal.pbio.0050203.sg002 (784 KB TIF).
Table S1. Morpholinos Used in This Study
Found at doi:10.1371/journal.pbio.0050203.st001 (21 KB XLS).
Table S2. miRNA Sequences
Found at doi:10.1371/journal.pbio.0050203.st002 (13 KB XLS).
We thank B. Ason for reading the manuscript critically and F.
Argenton and M. Hammerschmidt for providing cDNA clones for
endocrine pancreas and pituitary markers.
Author contributions. WPK, AKL, RFK, JDM, and RHAP conceived
and designed the experiments. WPK and AKL performed the
experiments and analyzed data. JDM contributed reagents. WPK,
RFK, JDM, and RHAP and wrote the paper.
Funding. This work was supported by the Council for Earth and
Life Sciences of the Netherlands Organization for Scientific
Competing interests. The authors have declared that no competing
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