Deficiency of ribosomal protein S19 during early
embryogenesis leads to reduction of erythrocytes
in a zebrafish model of Diamond-Blackfan anemia
Tamayo Uechi, Yukari Nakajima, Anirban Chakraborty, Hidetsugu Torihara, Sayomi Higa
and Naoya Kenmochi?
Frontier Science Research Center, University of Miyazaki, Miyazaki 889-1692, Japan
Received May 2, 2008; Revised and Accepted July 18, 2008
Ribosomes are responsible for protein synthesis in all cells. Ribosomal protein S19 (RPS19) is one of the 79
ribosomal proteins (RPs) in vertebrates. Heterozygous mutations in RPS19 have been identified in 25% of
patients with Diamond-Blackfan anemia (DBA), but the relationship between RPS19 mutations and the
pure red-cell aplasia of DBA is unclear. In this study, we developed an RPS19-deficient zebrafish by knocking
down rps19 using a Morpholino antisense oligo. The RPS19-deficient animals showed a dramatic decrease in
blood cells as well as deformities in the head and tail regions at early developmental stages. These pheno-
types were rescued by injection of zebrafish rps19 mRNA, but not by injection of rps19 mRNAs with
mutations that have been identified in DBA patients. Our results indicate that rps19 is essential for hemato-
poietic differentiation during early embryogenesis. The effects were specific to rps19, but knocking down the
genes for three other RPs, rpl35, rpl35a and rplp2, produced similar phenotypes, suggesting that these genes
might have a common function in zebrafish erythropoiesis. The RPS19-deficient zebrafish will provide a valu-
able tool for investigating the molecular mechanisms of DBA development in humans.
Diamond-Blackfan anemia (DBA) is characterized by dimin-
ished numbers of erythroid progenitors in bone marrow (BM)
in early infancy. In some cases, patients also show diverse
physical abnormalities, such as upper limb malformations,
short stature and kidney dysfunction (1,2). DNA analysis of a
DBA patient with a translocation at 19q13 identified ribosomal
protein (RP) S19 as a candidate disease gene for DBA (3). Sub-
sequently, heterozygous mutations in RPS19 were detected in
25% of 172 patients (4). RPS19 is one of 79 RPs, and is
expressed in every cell where protein synthesis occurs. It is
stillunclear howthe mutations insuchaubiquitously expressed
gene specifically affect erythropoiesis.
Several attempts have been made to address this question,
mainly using cell lines. For instance, cells with siRNA-
mediated knockdown of the RPS19 gene proliferate less (5)
and have defects in erythroid differentiation similar to those
seen in DBA patients (6). Apoptotic changes in progenitor
cells have been suggested to be a major factor for anemia
development, as evidenced by increased apoptosis in CD34þ
cells from DBA patients as well as in siRNA-treated
RPS19-deficient cells (7). Others have suggested that cell
cycle arrest at G0/G1, not apoptosis, is more critical for the
development of anemia (8). Regardless of the mechanism
involved, the question remains how a lack of RPS19 protein
disturbs the proliferation of erythroid precursors.
Recently, defects in ribosome biogenesis have been pro-
posed to be important in DBA pathogenesis. RPS19 mutations
result in the accumulation of premature ribosomal RNAs
(rRNAs) in patient-derived cells and in RPS19-deficient cell
lines (9,10). When transfected into human cell lines, RPS19
proteins with the mutations seen in DBA patients fail to
associate with ribosomes (11). Moreover, mutations in two
other RP genes, RPS24 and RPS17, have also been identified
in a small but significant number of DBA patients (12,13),
suggesting that changes in ribosomal function could be
responsible for the defective erythropoiesis in DBA.
In vertebrates, ribosomes are composed of four rRNA
species and 79 different proteins (14–16). Although RPs are
?To whom correspondence should be addressed at: Frontier Science Research Center, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki
889-1692, Japan. Tel: þ81 985859084; Fax: þ81 985859084; Email: firstname.lastname@example.org or email@example.com
# The Author 2008. Published by Oxford University Press. All rights reserved.
For Permissions, please email: firstname.lastname@example.org
Human Molecular Genetics, 2008, Vol. 17, No. 20
Advance Access published on July 24, 2008
by guest on June 3, 2013
essential for the assembly of ribosomes, not much is known
about their role, if any, during translation. Therefore, elucidat-
ing the currently unknown functions of RPs should provide
significant insight into the pathogenesis of DBA and other
ribosome-associated diseases. To date, only a single animal
model, Rps19-knockout mice, has been developed to explore
the role of RPS19 in DBA (17). However, Rps19-heterozygous
mice in this model system display no abnormalities in any
organ, including the hematopoietic system, whereas the null
mice die embryonically. Here, we report the development of
rps19-knockdown zebrafish that show marked reduction in
erythrocyte numbers during embryogenesis.
Phenotypes of rps19-knockdown embryos
Mutationsinuniversallyexpressed genes,suchasRPgenes, are
generally assumed to result in systemic abnormalities.
However, in our previous study, embryos injected with
Morpholino antisense oligos (MOs) against 20 different RP
genes showed specific phenotypes depending on the RP gene
unknown, function during early development in zebrafish. To
investigate thespecificrole oftheRPS19geneinerythropoiesis,
weknocked down thezebrafishortholog (rps19)using MOsand
analyzed the effect on the synthesis of blood cells.
The coding region of rps19 shares 78% nucleotide and 88%
amino acid identity with its human ortholog. Although gene
duplication is common in zebrafish, available information from
public databases suggests that rps19 exists as a single copy in
the genome. We targeted this gene using an MO aimed at its
translation initiation site. Following injection of this MO at the
one-cell stage, we compared the morphological features of the
morphants with wild-type siblings at about 24 h postfertilization
(hpf). At this stage, pigmentation in the retina begins to appear
and somitogenesis is completed. The rps19 morphants showed
incomplete brain subdivisions and a ventrally bent tail (Fig. 1).
We also observed other phenotypes commonly associated with
RP morphants (18), such as an incomplete yolk sac extension
and a rough surface appearance. The embryos injected with a
control MO did not display any morphological changes. All
the rps19 morphants died by 10 days postfertilization.
Reduced red blood cell synthesis in rps19-knockdown
Blood circulation in wild-type zebrafish is easily visible in the
posterior cardinal vein and the common cardial vein by 26 hpf.
However, the process was delayed by 2–3 h in the rps19 mor-
phants. Therefore, we compared the circulation pattern of the
morphants at 29 hpf with that of wild-type embryos at 26 hpf.
blood cells compared with the control embryos (Supplementary
Material, Fig. S1–S4), and this reduction continued even at
later stages of development.
When observed at 48 hpf, the heart in normal embryos
appeared red-colored due to a high density of blood cells;
however, it was almost transparent in rps19 morphants, indicat-
ing a decreased number of blood cells (Fig. 2B, arrow).
To further confirm this reduction, we performed hemoglobin
staining on the embryos. As expected, the hemoglobin staining
observed inthe cardial vein(Fig.2C,light graydots) was mark-
edly decreased in the morphants. To investigate whether this
reduction in blood cells was common in RP deficient
embryos, we carried out hemoglobin staining of 19 additional
RP morphants. All the 19 RP morphants showed varying
degrees of morphological abnormalities; however, there was
Figure 1. Morphological changes in rps19-knockdown embryos. Lateral views
of the head and trunk region in wild-types (A and B) and morphants (C–F) at
25 hpf. The brain of the rps19 morphant has improper subdivisions (dotted
curve) and a smaller otic capsule (arrow) (C) than the wild-type embryo
(A). The body trunks of the morphants show a downward bend in the tail
and a thin yolk sac extension (solid line; D). These deformities are not seen
in embryos injected with the control MO (E and F). tel, telencephalon; ot,
optic tectum; mhb, midbrain–hindbrain boundary; oc, otic capsule. Scale
bar, 200 mm.
Figure 2. Drastic reduction of blood cells in the rps19 morphants. Lateral
views of wild-type (A) and MO-injected embryos (B) at 48 hpf. The heart
of the rps19 morphant (arrow in B) is almost transparent because it has
fewer erythrocytes than the wild-type. The cardial vein region in the yolk
sac is shown in wild-type zebrafish (C) and rps19 morphants (D). Wild-type
embryos contain a high density of blood cells (light gray dots in C),
whereas the number of red blood cells is drastically reduced in red blood
cells in the rps19 morphants, as indicated by the absence of hemoglobin-
Human Molecular Genetics, 2008, Vol. 17, No. 203205
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no correlation between the staining intensity and severity of the
associated morphological defects (described later). This
suggests that defective blood cell production is a consequence
of a specific RP deficiency.
Primitive erythropoiesis defects in rps19 morphants
To determine the onset of defective erythropoiesis, we analyzed
the expression pattern of gata1, an early erythroid gene, by
whole mount in situ hybridization. Because the initiation of cir-
culation was slow in the morphants due to the developmental
delay,weexaminedgata1 expression at differentdevelopmental
stages (23–27 hpf) in wild-type and MO-injected embryos. The
gata1 expression in the intermediate cell mass (ICM) peaked
around 24 hpf in the wild-type embryos, whereas it peaked
around 26 hpf in the morphants (Fig. 3A,b and e). However,
when compared to gata1 mRNA levels at appropriate develop-
mental stages, the expression level of gata1 mRNA was not sig-
nificantly different between the wild-type embryos and the
morphants until the onset of circulation (Fig. 3A, a and d, b
and e). In contrast, when erythroblasts maturedintoerythrocytes
during circulation (19), gata1 mRNA was detected in the
yolk region of wild-type embryos, but not in the morphants,
although their gata1 expression patterns at ICM were similar
(Fig. 3A, c and f).
To further confirm the specificity of the erythroid defects
caused by rps19 deficiency, we examined the expression of
vascular and myeloid lineage genes. The expression of tie-1
(Fig. 3B) and fli-1a (data not shown) during vasculogenesis
was similar in the wild-type and MO-injected embryos. Simi-
larly, semi-quantitative RT–PCR of other myeloid genes,
including pu.1, l-plastin and mpo, did not show any significant
difference in the transcript levels at 24 hpf (Fig. 3C). To
confirm the localization of these genes, we carried out
in situ hybridization and found no obvious differences in the
expression patterns between the wild-type embryos and the
morphants (Supplementary Material, Fig. S5). Even at
48 hpf, a stage when significant reduction of erythrocytes
occurred in the morphants as judged by hemoglobin staining
(Fig. 2D), the expression level of l-plastin and mpo was
similar in both types of embryos (data not shown). These
data indicate that maturation and proliferation of the erythroid
lineage is the main defect of the morphants.
Phenotypic rescue in the morphants by rps19 mRNA
synthesized in vitro
Since the reduced blood cell phenotype was specific to
RPS19 deficiency, we examined whether a synthesized rps19
mRNA could rescue this phenotype in embryos. For this,
Figure 3. Specific reduction in the levels of erythropoietic marker genes. (A) Whole mount in situ hybridization assay for gata1 at different developmental
stages. The expression pattern of gata1 in ICM displays no significant difference until circulation starts (a, b, d and e). The gata1-expressing circulating
cells are detected in the yolk region of wild-type embryos (arrows), but not in that of the morphants (c and f). (B) Whole mount in situ hybridization assay
for tie-1. Although the morphants display a smaller body, the expression pattern of tie-1 is same as in the wild-type. (C) Semi-quantitative RT–PCR for
pu.1, l-plastin, and mpo genes. RNAs were prepared from the embryos at 24 hpf. Beta-actin was used as an internal control. WT, wild-type; MO, rps19 morphant.
Scale bars, 200 mm.
3206Human Molecular Genetics, 2008, Vol. 17, No. 20
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we incorporated a five-base change into the MO-target site of
the rps19 mRNA to prevent in vivo binding of the MO to the
synthesized mRNA (Fig. 4A). Co-injection of this mRNA
(1.0 mM) with the rps19 MO (60 mM) resulted in almost com-
plete recovery of blood cells, as observed by hemoglobin
staining and observation of gross morphology (Fig. 4B).
These results confirm that the decreased blood cell synthesis
is directly related to the RPS19 deficiency in zebrafish.
Several types of mutations, including allelic loss, point
mutations, insertions and deletions, have been identified in
the RPS19 gene of DBA patients (4,20). Among these, a mis-
sense mutation at the 62nd amino acid and a nonsense
mutation at the 94th amino acid, both in exon 4 of the gene,
occur at high frequency. We assessed whether mRNAs con-
taining these patient-type mutations could rescue the rps19
phenotype (Fig. 4A). However, when we injected these two
mutated mRNAs at a 1.0 mM concentration, which is sufficient
for rescue by non-mutated mRNA, the embryos displayed
more severe developmental defects than the rps19 morphants
(data not shown). Even in wild-type embryos, injection of
these mRNAs at the same concentration resulted in morpho-
logical abnormalities (data not shown). Injection of these
mRNAs at a lower concentration (0.5 mM) did not induce
any additional deformities, but also did not rescue the blood
cell production and other associated phenotypes of the knock-
down (Fig. 4C). These results indicate that the rps19 mutations
seen in patients remove the normal function of the RPS19
protein and therefore render them unable to rescue red
blood cell synthesis in the zebrafish displaying defective
Reduced red blood cell synthesis in other RP morphants
Recently, it was proposed that the pathogenesis of DBA might
be linked to functional changes in the translational machinery
due to defective RPs (21,22). We, therefore, investigated the
possibility that RP genes other than RPS19 could contribute
to the onset of DBA.
We examined the circulation pattern and the red blood
cell density in 20 different RP morphants at various stages of
development (26–48 hpf) by using live video imaging and
hemoglobin staining. Most of the RP morphants displayed an
initial delay in circulation compared to the wild-type embryos,
which may be due to a general effect of RP deficiency. There-
fore, we focused on whether these initial circulatory defects
recovered during later stages of development. We categorized
Figure 4. Rescue of morphological and anemia-like phenotypes by rps19 mRNA. (A) Schematic representation of the injected rps19 mRNAs. In ‘WT rps19’,
four codons (start, stop and two commonly mutated in the patients) are shown with their corresponding amino acid numbers. The ‘modified rps19’ includes silent
mutations (vertical white lines) around the start codon that prevent the binding of the Morpholino (MO; gray line on the top) to the mRNAs. ‘Missense rps19’ and
‘nonsense rps19’ mRNAs also include, respectively, an amino acid change of arginine to tryptophan at the 63rd amino acid and a premature stop codon instead of
valine at the 95th amino acid. (B) Morphological observations and hemoglobin staining of embryos coinjected with MO-resistant rps19 mRNA and rps19 MO.
The MO-injected embryos show bent tails, reduced yolk sac extensions (arrows in a) and fewer blood cells (orange dots in d). These phenotypes were rescued by
injection of 1.0 mM modified mRNA (b and e), and attained almost the same phenotype as the wild-type (c and f). (C) Morphological observations and hemo-
globin staining of embryos coinjected with MO-resistant, mutated rps19 mRNA and rps19 MO. Less of the modified mRNA (0.5 mM) still rescues the rps19
phenotypes (a and d), whereas the embryos injected with the mutated mRNAs at the same concentration (b, c, e and f) display abnormal phenotypes similar to
those in rps19 morphants.
Human Molecular Genetics, 2008, Vol. 17, No. 20 3207
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the defects into three different groups: severe, moderate and
almost normal. Our observations revealed that the circulatory
defects and blood cell reduction were consistent in three RP
morphants in addition to rps19: rpl35, rpl35a and rplp2
(Table 1). Although the morphological phenotypes differed
among them, the blood cell defects were similar to those of
the rps19 morphants, as indicated by a dramatic decrease in
hemoglobin staining (Fig. 5). Thus, these four RP genes
might have a common function in zebrafish erythropoiesis.
RPS19-deficient zebrafish as a DBA model
Since the first report of DBA in 1938 (23), not much has been
learned about its pathogenic mechanisms. Although the RPS19
gene was identified as a candidate disease gene in 1999 (3), we
still have not been able to understand this disease completely.
Recent studies of DBA and other inherited BM-related dis-
eases indicate a possible connection between the translational
machinery and BM defects (24,25). For instance, the SBDS
gene, predicted to have a role in ribosome biogenesis, is
mutated in Shwachman-Diamond Syndrome, a disease charac-
terized by exocrine pancreatic insufficiency, BM failure and
other somatic abnormalities (26,27). Similarly, in dyskeratosis
congenita and cartilage-hair hypoplasia, mutations have been
identified in the genes that encode proteins involved in ribo-
some biogenesis (28,29). However, like DBA, there is cur-
rently no direct evidence to prove the relationship between
the mutations in these genes and the resultant clinical features
of the diseases. Although the molecular mechanisms of patho-
genesis are not understood in these BM disorders, the ribo-
some seems to be a common link between them. Therefore,
elucidating the underlying mechanism in DBA may bring
new insights into other ribosome-associated diseases as well.
Studies using cell lines and cells derived from DBA patients
demonstrated that changes in basic cellular functions, such as
apoptosis and cell cycle arrest, contribute to DBA develop-
ment (7,8). However, these observations, although important,
focused on the erythroid lineages, and changes in other
tissues may have been overlooked. To investigate the mechan-
isms underlying the erythroid specificity of RPS19 depletion,
studies in animal models are necessary. A previous attempt
to use Rps19-knockout mice was unsuccessful because the
complete loss of the gene resulted in embryonic lethality,
and loss of a single allele did not produce any unusual pheno-
type (17,30). Although it is not clear why the Rps19 hetero-
zygous mice developed normally, given that DBA patients
have heterozygous mutations, it seems that Rps19 knockout
mice are not a suitable animal model for DBA. As an alterna-
tive approach, we knocked down rps19 in zebrafish. In this
study, we successfully demonstrated that the loss of rps19
function in zebrafish recapitulates the DBA phenotype of a
severe decrease in the production of erythroid cells.
However, early erythropoiesis was not disrupted, as suggested
by the normal expression of gata1 (Fig. 3). In contrast, the
level of mature erythrocytes was significantly lower in the
morphants. This may indicate the existence of an unknown
mechanism causing pure red-cell anemia. There are several
advantages to using zebrafish that make this model appropriate
for studying the molecular mechanism of DBA. First, the mor-
phology and the maturation process of erythroid cells closely
resemble those in mammals. Second, the blood cells in the
early stages of development consist mainly of erythroid pro-
genitors, which are the primary targets in DBA, making the
analysis of the hematopoietic system easier. Third, the
embryos can survive for several days even in the absence of
blood cells, and hence are suitable for anemia research (31).
Effects of mutated S19 proteins on embryogenesis
In this zebrafish system, the blood cell reduction was easily
rescued by rps19 mRNA, but not by mRNAs with DBA
patient-type mutations. Interestingly, these mutated mRNAs,
when overexpressed, led to severe phenotypes different from
those seen in rps19 morphants. A previous in vitro study
Table 1. The extent of reduction in red blood cells by RP gene knockdown
PhenotypesBlood cell circulation
Posterior caudal vein
Blood cell density
Hb stainingCardial vein
Figure 5. Reduction of blood cells in rpl35, rpl35a and rplp2 morphants.
Hemoglobin staining of cardial veins at 48 hpf. Compared to the rpl36a mor-
phants (A), a morphant that display a moderate level of blood cell recovery
(Table 1), the rpl35, rpl35a and rplp2 morphants (B–D) show a drastic
reduction in number of hemoglobin stained blood cells (orange dots).
3208Human Molecular Genetics, 2008, Vol. 17, No. 20
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demonstrated that mutated S19 proteins failed to assemble
into the ribosomes (11). It is conceivable that the defective
ribosome biogenesis may have deleterious effects on embryo-
genesis. Alternatively, mutated S19 could alter the transla-
tional efficiency in a mature ribosome, leading to abnormal
phenotypes. Although the exact mechanism is still unclear, it
is likely that expression of a defective RPS19 protein affects
morphogenesis in zebrafish in a way that differs from the
absence of RPS19 due to MO knockdown.
Other RP genes potentially involved in hematopoiesis
Many RPs are believed to have additional functions besides
their role in ribosomes (32,33). Initially, the loss of such an
extraribosomal function of RPS19 was assumed to be the
cause of the pure red-cell aplasia in DBA (3). Subsequent
studies, however, suggested that the ribosome itself has a
role in this disease. If so, involvement of other RP genes in
DBA seems plausible. Interestingly, mutations in RPS24 and
RPS17 have been identified in some DBA patients who do
not have any mutation in RPS19 (12,13). Therefore, some
RP genes may have shared functions that are important for
hematopoiesis. In this study, the morphants for rpl35, rpl35a
and rplp2 genes also displayed a severe reduction in blood
cell production. We assume that, like rps19 morphants, the
erythropoietic system is impaired in these morphants,
suggesting that these RPs have a common role in hemato-
poiesis. Recently, mutations in RPL35A were reported in
DBA patients (34), indicating the usefulness of our data for
screening other DBA candidate genes.
Ribosome defects and human diseases
In Drosophila, haploinsufficiency of any RP gene leads to the
Minute mutant, which displays thin bristles, poor fertility and
an overall developmental delay (35,36). Such phenotypes are
believed to be a consequence of decreased translational effi-
ciency due to a reduction of fully functional ribosomes.
However, Ts and Bst mutants in mice (37,38) and DBA in
humans, which are strongly linked to mutations in RP genes,
display tissue-specific phenotypes. It is conceivable that the
loss of function of a specific RP may not be critical for all
cell types. Accordingly, a lower RPS19 gene dosage may
not have the same effect on other cells. In other words,
mutations in RPS19 may lead to serious defects in the
erythroid lineage but not in other cell lineages. Recently, it
was reported that the yeast ribosomes include a subset of para-
logous RPs, and specific combinations of RPs are required for
translating specific mRNAs (39). Thus, in higher organisms,
such functionally specific RPs may impart organ-specific
translational preferences to ribosomes, and depletion of an
RP may lead to selective effects in a particular organ. Based
on this assumption, we hypothesize that, in DBA, the
mutations in the RPS19 gene affect the translational efficiency
of mRNAs essential for erythroblast differentiation. The
RPS19-deficient zebrafish model developed in this study
could be a valuable tool to explore this possibility and shed
light on the relationship between RPS19 mutations and
erythroid cell susceptibility in DBA.
MATERIALS AND METHODS
MOs were obtained from Gene Tools, LLC (Philomath, OR,
USA). The MOs were injected into one-cell-stage embryos
at a concentration of 0.5 mg/ml. The sequences of the MOs
were rps19 MO, 50-CACTGTTACACCACCTGGCATCTTG,
and control MO, 50-CACTcTTAgACgCACCTGcCATgTTG
(bases complementary to the start codon are underlined and
mispaired bases are shown in lower case). The sequences of
the 19 other RP MOs can be found in our database (http://
zebrafish.med.miyazaki-u.ac.jp). These MOs were injected
intoembryos atthe optimal
Whole mount in situ hybridizations
Digoxigenin-labeled antisense riboprobes were transcribed
from a linearized plasmid containing gata1 and a PCR-based
template containing tie-1 using DIG RNA labeling Mix and
T7 RNA polymerase (Roche). The template for the tie-1
probe was generated by PCR using the forward primer 50-C
TGGCCCTCTTTTACATTCG and reverse primer 50-TA
Total RNA was isolated from wild-type and MO-injected
embryos using TRIzol reagent (Invitrogen). Semi-quantitative
RT–PCR was performed with 0.5 mg total RNA using an
OneStep RT–PCR kit (Qiagen). The primer pairs for each
gene were as follows: pu.1, 50-CAGAGCTACAAAGCGTG-
CAG, and 50-GCAGAAGGTCAAGCAGGAAC; l-plastin,
50-GGCATACGGGAGAAAGATGA and 50-ATGTTGCTG
CCCAGTTTAGG; mpo, 50-AGGGCGTGACCATGCTATAC
and 50-CGGTGTTGTCGCAGATTATG; beta-actin, 50-GCC
CATCTATGAGGGTTACG and 50-GCAAGATTCCATACC-
mRNA synthesis for rescue experiments
Full-length rps19 (GenBank accession no. NM_200750) was
amplified by PCR using the forward primer 50-GCAAGATG
CCAGGTGGTGT and the reverse primer 50-TTATTTTA-
CACTTTCTTGCTTGCAG and cloned into a TA vector
(Promega, Madison, WI, USA). Using this TA vector as a tem-
plate, we generated the cDNA for ‘modified rps19’, which
included a silent five-base change around the MO binding
site. We used this as a template to synthesize ‘missense
rps19’ and ‘nonsense rps19’ mRNAs, corresponding to two
mutations found in DBA patients. These cDNAs were then
digested by EcoRI and cloned into a pCS2þvector (provided
by Dr Kunio Inoue, Kobe university, Japan) for in vitro tran-
scription. Capped mRNAs were synthesized from 1 mg of the
linearized template by SP6 RNA polymerase using a mMes-
sage mMachine kit (Ambion, Austin, TX, USA).
Human Molecular Genetics, 2008, Vol. 17, No. 203209
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Observation of blood cell circulation and
The embryos were grown at 28.58C and the circulating blood
cells in the posterior cardinal vein and the common cardial
vein were recorded at 26 and 29 hpf by live video imaging
using CCD camera (DP70, Olympus) attached to a stereo-
scopic microscope (SZX12, Olympus). To examine the density
of red blood cells around the cardial vein at 32–48 hpf, we
carried out hemoglobin staining using o-dianisidine as pre-
viously described (40). We divided the RP morphants into
three groups depending on the extent of recovery of the
blood cell density and circulation pattern at later stages of
This work was supported by Grants-in-Aid (20790734,
20659044, 1806457) from the Ministry of Education,
Culture, Sports, Science and Technology (MEXT) and Japan
Society for the Promotion of Science (JSPS), and funds from
The Life Science Foundation of Japan and The Naito Foun-
dation. A.C. is a research fellow of JSPS (P06457).
Supplementary Material is available at HMG Online.
We thank Dr Kunio Inoue for kindly providing the pCS2þ
vector; Dr Noriyoshi Sakai and Dr Minori Shinya for tech-
nical advice; Dr Kinta Hatakeyama, Dr Yujiro Asada and
Ms Ritsuko Sotomura for useful suggestions; and Dr Maki
Yoshihama for useful discussions.
Conflict of Interest statement. None declared.
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