Targeted inhibition of miRNA maturation with morpholinos reveals a role for miR-375 in pancreatic islet development.

Abstract

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.

Full text

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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
Introduction
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 [4]. 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 [9]. 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 [2]. 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
[19] and have recently been used to target mature miR-214 in
zebrafish [20]. 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 [21], 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
America
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: rene@niob.knaw.nl
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Results
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 [23], and adult mice [17]. 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 [24],
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
[19], 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
miRNA.
The zebrafish embryo can be used to monitor the effect of
miRNAs on green fluorescent protein (GFP) reporters fused
to miRNA target sites [24]. 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 [25].
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
Author Summary
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
phenotypes.

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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 [21]. 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
precursor.
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.
doi:10.1371/journal.pbio.0050203.g001
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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.
doi:10.1371/journal.pbio.0050203.g002
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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
precursor.
A similar analysis was performed for miR-375, which is
expressed in the pancreatic islet and pituitary gland [21], 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-
pressed.
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 [15]. 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
4B).
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
Morphology
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 [26].
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 [27]. A
more detailed analysis using somatostatin and glucagon as
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