Slc39a7/zip7 Plays a Critical Role in Development and Zinc Homeostasis in Zebrafish
Citation: Yan G, Zhang Y, Yu J, Yu Y, Zhang F, et al. (
Slc39a7/zip7 Plays a Critical Role in Development and Zinc Homeostasis in Zebrafish
Guang Yan 0
Yuchao Zhang 0
Junlei Yu 0
Yu Yu 0
Fan Zhang 0
Zhuzhen Zhang 0
Aimin Wu 0
Xianghua Yan 0
Yi Zhou 0
Fudi Wang 0
Yann Gibert, Deakin School of Medicine, Australia
0 1 Group of Bio-Metal Metabolism, Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences , Shanghai , People's Republic of China, 2 College of Animal Sciences and Technology, Key Laboratory of Swine Genetics and Breeding, Ministry of Agriculture, Huazhong Agricultural University , Wuhan , China , 3 Schools of Life and Food Engineering, Nanchang University , Nanchang , China , 4 Stem Cell Program and Division of Hematology/Oncology Children's Hospital Boston and Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Harvard Medical School , Boston, Massachusetts , United States of America
Background: Slc39a7/Zip7, also known as Ke4, is a member of solute carrier family 39 (Slc39a) and plays a critical role in regulating cell growth and death. Because the function of Zip7 in vivo was unclear, the present study investigated the function of zip7 in vertebrate development and zinc metabolism using zebrafish as a model organism. Principal Finding: Using real-time PCR to determine the gene expression pattern of zip7 during zebrafish development, we found that zip7 mRNA is expressed throughout embryonic development and into maturity. Interestingly, whole mount in situ hybridization revealed that while zip7 mRNA is ubiquitously expressed until 12 hours post-fertilization (hpf); at 24 hpf and beyond, zip7 mRNA was specifically detected only in eyes. Morpholino-antisense (MO) gene knockdown assay revealed that downregulation of zip7 expression resulted in several morphological defects in zebrafish including decreased head size, smaller eyes, shorter palates, and shorter and curved spinal cords. Analysis by synchrotron radiation X-ray fluorescence (SRXRF) showed reduced concentrations of zinc in brain, eyes, and gills of zip7-MO-injected embryos. Furthermore, incubation of the zip7 knockdown embryos in a zinc-supplemented solution was able to rescue the MO-induced morphological defects. Significance: Our data suggest that zip7 is required for eye, brain, and skeleton formation during early embryonic development in zebrafish. Moreover, zinc supplementation can partially rescue defects resulting from zip7 gene knockdown. Taken together, our data provide critical insight into a novel function of zip7 in development and zinc homeostasis in vivo in zebrafish.
Funding: This work was supported by research grants from the Ministry of Science and Technology of China (973 Program) (grant numbers 2009CB941400,
2011CB966200 to FW); the National Natural Science Foundation of China (grant numbers 10979071, 30970665, 31030039 to FW, 30901193 to YY); Science &
Technology Commission of Shanghai Municipality grant (grant number 10JC1416800 to FW) and Chinese Academy of Sciences Hundred Talents Program (grant
number KSCX2-YW-R-141 to FW). FW is a scholar of the Hundred Talents Program of the Chinese Academy of Sciences. The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
Zinc is an essential trace element required for DNA synthesis,
cell division, regulation of transcription, and protein synthesis.
Approximately 2000 enzymes use zinc as a catalytic cofactor ,
and zinc binding motifs are found in up to 10% of the proteins
encoded by the human genome  including
zinc-fingercontaining proteins, the most abundant protein superfamily in
the mammalian genome. In this regard, zinc is an essential
cofactor required for the activity of numerous proteins involved in
cellular signaling pathways and biological processes including
growth factors, cytokines, receptors, enzymes, and transcription
factors [3,4,5,6]. In addition, zinc has been found to play a role in
cell-mediated immunity and signal transduction, and as an
antioxidant and an anti-inflammatory agent [7,8]. It is broadly
acknowledged that numerous disorders are the result of zinc
deficiency such as poor appetite, growth retardation, skin lesions,
mental lethargy, delayed wound healing, neurosensory disorders,
and cell-mediated immune disorders [9,10,11].
Zinc homeostasis in single cells and in whole organism is
regulated by two families of zinc transporters: zinc exporters
(Slc30a/ZnT or CDF) and importers (Slc39a/
Zip)[10,11,12,13,14,15,16,17]. In addition, the tissue-specific
expression of each zinc transporter gene, the metals, hormones
and cytokines that influence their expression, and the diseases that
have been linked to their aberrant expression have been elucidated
. There are 10 ZnT family members in mammals. Deficiency
in ZnT2 or ZnT4 causes reduced zinc concentrations in milk in
mammals [18,19], while ZnT5-KO mice suffer from growth
retardation and osteogenic problems . ZnT5 and ZnT6 are
both localized to the Golgi apparatus  and uniquely form a
heterodimer . ZnT8 is specifically expressed in pancreatic
bcells and has been identified as a novel target autoantigen in
patients with type 1 diabetes . Furthermore, mutation of ZnT8
is associated with glucose intolerance and type 2 diabetes .
The Zip family can be divided into four subfamilies, named
subfamilies I and II, gufA and LIV-1, with most mammalian Zip
family members being classified into the LIV-1 subfamily. The
LIV-1 subfamily contains nine members (Zip4, Zip5, Zip6, Zip7,
Zip8, Zip10, Zip12, Zip13, and Zip14) . With the exception of
Zip7 and Zip13, a feature of the LIV-1 subfamily are a potential
metalloprotease motif (HEXPHEXGD) in the fifth
transmembrane domain (TMD V) [17,25]. Mutations in the human ZIP4
gene cause the inherited disorder acrodermatitis enteropathica
, and our previous studies identified Zip4 as a critical regulator
of zinc homeostasis via a process distinct from zinc-stimulated
endocytosis [27,28]. While Zip5 expression is restricted to many
tissues important for zinc homeostasis, including the intestine,
pancreas, liver and kidneys, abundance of Zip5 mRNA is not
altered in response to changes in zinc concentration . Rather,
Zip4 and Zip5 are both dynamically regulated by several
posttranscriptional, translational, and post-translational mechanisms
. Interestingly, zebrafish zip6/liv1 controls the
epithelialmesenchymal transition (EMT) via activation of signal transducer
and activator of transcription 3 (STAT3), suggesting that zip6/liv1
may have an important role in cell migration [31,32]. The Zip13
molecule is involved in the bone morphogenetic protein (BMP)/
transforming growth factor beta (TGF-b) signaling pathway by
controlling the nuclear localization of Smad proteins .
Understanding of the diverse functions of the Liv1 family
continues to expand as more studies are conducted in model
Zip7 (Slc39a7, Ke4) also belongs to the LIV-1 subfamily of zinc
transporters , and has been shown to play a critical role in
maintaining the intracellular balance of zinc by affecting the
redistribution of zinc from intracellular stores to the cytosol .
While ectopic expression of Zip7 in cells results in an increase in
intracellular zinc concentration , Zip7 is localized to the
membranes of endoplasmic reticulum (ER) and Golgi apparatus,
but not to the plasma membrane; suggesting that Zip7 functions to
transport zinc from the ER and Golgi to the cytosol of mammalian
cells  . In addition, recent data suggests that Zip7 acts at a
critical link in zinc-mediated tyrosine kinase signaling, and may be
involved in breast cancer progression . A recent study
identified protein kinase casein kinase II (CK2) as the kinase
responsible for Zip7 activation. CK2 could trigger cytosolic zinc
signaling pathways through phosphorylation of Zip7 and in turn,
affect proliferative responses and cell migration.
Although Zip7 has attracted much interest in numerous fields of
research and many studies have been performed in primary cells
and in cell lines, the in vivo functions of Zip7 have not been
determined due to a lack of a Zip7 gene knockout animal model.
Zip7 orthologs include CATSUP in Drosophila  and IAR1 in
Arabidopsis . Although knockout of CATSUP in Drosophila is
lethal, testing partial loss of function mutants revealed CATSUP
could downregulate tyrosine hydroxylase, a rate-limiting enzyme
for production of dopamine in the brain . Furthermore, IAR1
has been suggested to transport zinc or other metals out of the ER
and into the cytosol . In this study, we have elucidated the in
vivo role of zip7 in the zebrafish vertebrate model. Previous studies
revealed that zebrafish eyes highly express zinc transporters znt4
and zip1, and have constitutively high levels of zip7 . We
extended these findings and found that zebrafish zip7 has essential
roles in eye development. Morpholino-induced loss of zip7
(zip7MO) resulted in decreased eye and head size as well as
campylorrhachia. Together, our results suggest that zip7 may
play an important role in eye, brain, and skeletal development by
regulating zinc transport during zebrafish development.
The expression pattern of zip7 mRNA during zebrafish
We used whole-mount in situ hybridization to determine the
spatial distribution of zip7 gene expression during zebrafish
embryogenesis and found that at early stages of somitogenesis
(approximately 12hpf), zip7 mRNA was ubiquitously expressed
(Figure 1A). At 24 hpf, zip7 mRNA transcripts were still detected
in the forebrain-proximal part of retina (Figure 1B), and at later
stages of zebrafish development, embryos continued to express
zip7 mRNA around the retina (Figure 1D, 1E, and 1F). Temporal
changes in zip7 mRNA expression levels during development were
quantified by real-time PCR between 0.2 hpf and 120 hpf, and in
adult zebrafish (4 months). Analysis of the mRNA expression
pattern of zip7 (normalized to b-actin mRNA) showed that zip7
mRNA is expressed during embryogenesis and continues to be
expressed in adult zebrafish (Figure 1G). In addition, zip7 mRNA
is highly expressed in brain and eye of adult zebrafish (Figure 1H).
Silencing of zip7 causes developmental defects in
We took a gene knockdown approach to study zip7 function in
vertebrate development. zip7-MO were designed against the
zebrafish zip7 mRNA initiating ATG to block zip7 protein
translation. A zip7-GFP reporter assay confirmed that
administration of zip7-MO effectively blocked zip7 protein translation
(Figure 2A). We tested a series of doses of zip7-MO and control
MOs (4 ng, 6 ng, 8 ng, 10 ng, and 12 ng per embryo). While we
did not detect abnormal development in embryos injected with
control MO at any stage (Figure 2B), we did observe obvious
developmental defects at multiple stages (24 hpf, 48 hpf, and 72
hpf) in embryos injected with zip7-MO. Among embryos injected
with 10 ng or 12 ng zip7-MO, severe morphological abnormalities
were observed at 72 hpf. Specifically, we observed death in a
fraction of the zip7-MO-injected embryos, as well as many
embryos with delayed growth that could not hatch and displayed a
strikingly curved notochord and decreased eye size compared to
controls (Figure 2C).
We then cloned the full-length zebrafish zip7 gene and observed
that injection of 225 pg zip7 mRNA alone did not cause
phenotypic changes in embryos (data not shown). To confirm
the specificity of the zip7-MO, we co-injected 225 pg zip7 mRNA
with 10 ng zip7-MO and found that the developmental defects
described above were partially rescued (Figure 2D). Statistical
analysis (Figure 2E) showed that 72 hours after microinjection,
approximately 94% of the fish displayed smaller eyes, spine
bending, and head dysplasia compared to controls. Strikingly,
72 hours after zip7 mRNA and zip7-MO were co-injected into
fertilized eggs, the zip7 knockdown phenotype was rescued
suggesting that loss of zip7 was specifically responsible for the
developmental defects observed following zip7-MO injection.
Zinc supplementation can partially rescue development
defects resulting from zip7 silencing
We next tested whether supplementation with various
concentrations of zinc (Zn2+ 25 mM, 50 mM, 75 mM, or 100 mM) could
restore normal development in hatched embryos injected with
Figure 1. Zip7 expression during zebrafish early embryonic development in wild type embryos and in tissues of adult zebrafish. (A
F) Lateral views (anterior to the left) of WISH of zip7 expression at 12 hpf, 24 hpf, 36 hpf, 48 hpf, 60 hpf, 72 hpf, embryos. Arrows indicate the eye. (G)
The zip7 mRNA expression pattern assayed by qRT-PCR normalized to b-actin transcripts in zebrafish embryos across development and at the adult
stage. (H) Normalized zip7 mRNA expression levels in tissues of zebrafish adult. * P,0.05 versus muscle (1-way ANOVA, Dunnetts multiple
10 ng zip7-MO. Strikingly, we found that, in fact, the defects we
observed in zebrafish morphology during early embryonic
development upon zip7 knockdown could be rescued with zinc
supplementation. In this regard, while the phenotypes of embryos
at 72 hpf injected with control MO (Figure 3A) or zip7-MO
(Figure 3B) were consistent with our previous results (Figure 2), we
observed that the morphological defects in zip7-MO-injected
embryos hatching in the presence of 50 mM zinc at 72 hpf were
partially rescued (Figure 3C). Moreover, when embryos were
microinjected with zip7-MO and then cultured in different
concentrations of zinc solution, we found that in the presence of
50 mM or 75 mM zinc, approximately 80% of the zip7-knockdown
embryos showed rescue (Figure 3D). However, the phenotype of
zip7-knockdown embryos could not be restored in the presence of
25 mM zinc ion due to a failure to hatch, and concentrations
higher than 100 mM would be toxic (data not shown). Thus,
hatching in the presence of 50 to 75 mM zinc could supply an
Silencing of zip7 can result in abnormal zinc distribution
Zinc intensity maps generated with SR-XRF showed zinc
distribution and concentrations in zebrafish embryos at 72 hpf.
Data were imaged at 30 mm X-Y resolution in two- and
threedimensions using Igor Pro Folder software (Figure 4). Zinc mainly
distributes in eyes, gills, brain and yolk of wild type embryos. In
this regard, in contrast to the images of wild type (Figure 4A and
4E) or control MO-injected embryos (Figure 4B and 4F), zebrafish
with zip7-MO injection showed a loss of zinc in eyes (Figure 4C
and 4G). However, zebrafish injected with zip7-MO hatching in
the presence of zinc (50 mM) showed the presence of zinc in eyes
(Figure 4D and 4H). Quantitation and statistical analysis showed
no evident difference in the whole embryo zinc content (Figure 4I).
However, it was noteworthy that zinc content in eye of zip7-MO
Figure 3. Morphologic phenotypes of zip7-deficient embryos and Zn2+ (50 mM) rescue at 72 hpf. Lateral views (anterior to the left) of
embryos at 72 hpf. (A)Wild type with control-MO microinjection (2 nL, 10 ng). (B) Wild type with zip7-MO microinjection (2 nL, 10 ng). (C) Wild type
with zip7-MO microinjection (2 nL, 10 ng) hatching in the presence of Zn2+ (50 mM). (D) Statistical analysis of microinjection zip7-MO and Zn2+
was remarkably lower than wild type and control MO, while
addition of exogenous zinc restored it to a normal level (Figure 4J).
Together, these data indicate that zip7 could play a critical role for
zinc transportation in the eyes.
Silencing of zip7 affects the expression of other zinc
We analyzed the expression levels of two zinc transporter family
members, the zip and znt families, in zip7-MO and control MO
embryos at 3 dpf (Figure 5A and B). Compared with the control
MO embryos, the zip7 transcript level in zip7-MO embryos
decreased markedly. However, the expression of zip3, zip6, znt2,
znt5 and znt6 increased significantly. The transcript levels of other
zip and znt family members were not different between zip7-MO
and control MO embryos.
In the present study, we investigated the function of Zip7 in
development using zebrafish as a vertebrate model organism. In
the zebrafish model system, several experimental approaches can
be utilized, including RNA in situ hybridization, morpholino
injections, and analysis of mutant and transgenic fish lines.
Incorporation of these diverse approaches can lead to improved
understanding of the in vivo regulation of key molecular pathways
with conserved roles in vertebrate zinc homeostasis. For example,
results from a study of zip6/liv1 using the zebrafish model suggest
that zip6/liv1, as a downstream target of STAT3, is required for
nuclear translocation of the Zn-finger transcription factor Snail,
which regulates the epithelial-mesenchymal transition (EMT)
during early zebrafish development .
The mouse Zip7 gene was discovered while characterizing
genes in the major histocompatibility complex on chromosome 17
. Human ZIP7 was mapped to the HLA class II region on
chromosome 6 . However, the precise function of Zip7 in
whole body zinc homeostasis is not clear. Our results using
RTPCR revealed that in zebrafish, zip7 began to be expressed early in
embryonic development, and its expression continued throughout
embryonic development and into maturity. These results were
further confirmed by a whole-mount in situ hybridization assay
that showed zip7 expression gradually decreased and became
restricted to developing embryos, and in adult zebrafish was found
highly zip7 expression in the brain and eye. Previous study also
found zip7 was mainly expressed in eye, brain, ovary, liver, gill and
intestine in zebrafish. The expression pattern of zip7 may
suggest that zip7 performs a specific function in these organs. In
addition, the mouse and human ZIP7 mRNAs were also detected
in many cDNA libraries including embryo, mammary gland,
ovary, uterus, cervix, testis, prostate, tongue, larynx, stomach,
pancreas, bladder, eye, pituitary, bone, bone marrow, skin, and
peripheral nervous system . According to the human ZIP7
gene expression atlas available in BioGPS (http://www.biogps.
org/#goto = genereport&id = 7922), ZIP7 is highly expressed in
prostate, pituitary gland, retina, smooth muscle, lung and colon,
which is similar to zebrafish zip7 expression.
Furthermore, we analyzed embryos lacking zip7 using antisense
morpholino (zip7-MO) oligonucleotides (MOs), which are the most
widely used anti-sense knockdown tools in the zebrafish (Danio rerio)
community. MOs are typically employed as oligomers of 25
Figure 5. The expression of zip and znt family members in zip7-MO embryos. (A) Relative mRNA levels of zip family members in wild type
embryos and zip7-MO embryos at 3 dpf. (B) Relative mRNA levels of znt family members in wild type embryos and zip7-MO embryos at 3 dpf. For
qRTPCR analysis, results were normalized to the internal control, b-actin, and presented as relative expression level calculated by the 2DDCt method.
Results are presented as mean 6 SEM. * P,0.05, ** P,0.005.
morpholine bases that are targeted via complementary base
pairing to the RNA of interest. A neutrally-charged
phosphorodiamidate backbone results in molecules with high binding
affinity for RNA, thereby facilitating steric hindrance of proper
transcript processing or translation . In our studies, embryos
receiving injections of zip7-MO displayed retarded embryonic
development accompanied by smaller heads, smaller eyes, shorter
palates and spinal lordosis, whereas the injection of control-MO
did not result in any obvious phenotype. We also carried out
rescue experiments in which we co-injected zip7-mRNA with
zip7MO, and found that the zip7 mRNA expression was comparable
to that found in wild type embryos. These results suggest that zip7
plays a critical role during zebrafish embryonic development.
Furthermore, we explored how Zip7 affected the developmental
process in zebrafish through zinc rescue experiments. Addition of
50 mM zinc to hatching zip7-MO-injected embryos resulted in
normal development, which indicated that zip7-mediates
development through zinc+ instead of directly influencing other signal
pathways. To further verify our results, we measured the
distribution of zinc in whole embryos by Synchrotron radiation
microbeam X-ray fluorescence (SR-XRF). SR-XRF is highly
specific and sensitive for identification, characterization, and
distribution analysis of metals and nonmetals in a given cell type or
tissue . SR-XRF is one of the few techniques capable of
providing spatially resolved in vivo metal abundance data on a
submicrometer scale, without the need for chemical fixation, coating,
drying or even sectioning of samples . The SR-XRF has been
used to detect the relative contents and distributions of many trace
metals in biological samples . We found that the distribution of
zinc between zip7-MO fish and wild type fish was highly different.
Compared with the wild type fish, the distribution of zinc in
zip7MO-treated embryos was disordered and severely reduced in eyes.
Quantitation and statistical analysis showed no significant
difference in the whole embryo zinc content, while zinc content
was severely reduced in the zip7-MO eye. These data indicate that
zip7 is a critical zinc transporter in zebrafish eye and is essential for
zinc homeostasis during eye development. In contrast, the
distribution of zinc in zip7-MO embryos hatching in the presence
of 50 to 75 mM zinc resembled that observed in wild type embryos.
This result suggested zip7-MO zinc content could be restored to
normal levels by exogenous zinc supplementation, which was
critical for zebrafish embryonic development.
Taken together, our results show that Zip7 has a very vital effect
on embryonic development by regulating the absorption and
distribution of zinc, however the specific details of this process
remain unclear. We continued to detect the expression of other
zinc transporters when zip7 was silenced. qRT-PCR analysis
revealed the expression of zip3, zip6, znt2, znt5 and znt6 were
significantly increased in the absence of zip7. Zip3 localizes to cell
bodies of the retina that also express PKC (protein kinase C) .
Zip6 localizes at the plasma membrane of rat neurons, suggesting
a role for Zip6 in neuronal zinc uptake , and Zip6 has been
shown to control the epithelial-mesenchymal transition in the
zebrafish gastrula organizer . ZnT2 expression was restricted
to tissues with unique zinc requirements, such as mammary and
prostate glands, where it mainly localized to the zymogen granules.
In addition, ZnT2 expression level could be affected by dietary
zinc content [49,50]. ZnT5 and ZnT6 have both been shown to
localize to the Golgi apparatus, and often form hetero-oligomers
that function to activate alkaline phosphatases in the early
secretory pathway [21,51]. Together, these studies show that the
same tissues or organelles expressing zip7 co-express zip3, zip6,
znt2, znt5 or znt6. Silencing of zip7 could change zinc levels and
affect the expression of other zinc transporters, ultimately leading
to a more serious zinc imbalance. Exogenous zinc
supplementation could rectify this vicious circle, and other zinc transporters
could execute their normal roles. Thus, the defect caused by zip7
silencing can be compensated for. Previous studies revealed that
zinc is essential for normal cell growth and development and is
involved in protein, nucleic acid, carbohydrate and lipid
metabolism, as well as in the control of gene transcription, growth, and
differentiation. Intracellular zinc signals are classified into
transcription-independent early zinc signaling (EZS) and
transcriptiondependent late zinc signaling (LZS) . Many cytosolic proteins
may have zinc-binding potential are expected to be closely
involved in a wide range of physiological responses including
development, immune function, cancer progression, and hard and
connective tissue disorders . Zinc itself affects a variety of
signaling molecules including PKC, Ca2+/calmodulin-dependent
protein kinase II, Erk1/2, cAMP-dependent protein kinase,
protein tyrosine phosphatase, and caspase-3. In addition, zinc
also acts as an intracellular second messenger [3,5]. It has been
suggested that ZIP7 protein is localized to the Golgi apparatus and
the endoplasmic reticulum, which are critical organelles in the
redistribution of zinc from intracellular stores to the cytosol
[36,53]. As such, zinc release has downstream effects on cell
signaling and hence, zinc is indeed a second messenger. An
important component of zinc action in cells is the ability to inhibit
protein tyrosine phosphatase activity, resulting in activation of
mitogen-activated protein kinases, such as ERK1/2, c-Jun
Nterminal kinase, and p38, as well as the tyrosine kinases Src and
epidermal growth factor receptor. Thus, Zip7 is a central hub in
cell signaling, regulating cell growth and differentiation as well as
embryonic development [5,25].
In summary, our results reveal that zip7 plays indispensable
roles in maintaining zinc homeostasis and organism development
especially in eyes, brain, and gills. These findings will be helpful for
the understanding of mechanisms of zinc homeostasis and diseases
resulting from defects in proper zinc homeostasis.
Materials and Methods
Fish husbandry and embryo preparation
Adult male and female zebrafish (Danio rerio) were maintained
under a 14 hour light/10 hour dark cycle at 28.5uC with
recirculating deionized water. Embryos were collected from
natural matings and staged matings according to Kimmel .
All zebrafish experimental protocols were approved by the
Institutional Animal Care and Use Committee of the Institute
for Nutritional Sciences, Shanghai Institutes for Biological
Sciences, and Chinese Academy of Sciences.
Pooled embryos or adult tissues were homogenized in TRIzol
Reagent (Invitrogen) to extract total RNA according to the
manufacturers instructions and treated with DnaseI (Promega).
RNA concentration and purity were assessed by
spectrophotometry. 2.0 mg of RNA was reverse-transcribed with M-MLV reverse
transcriptase (Promega) and oligo (dT) 18 primers (Takara) as
recommended. PCR was performed by using CFX96TM
RealTime System (Bio-Rad) and iQTM SYBR Green Supermix
(BioRad) as described in the manufacturers manual. The reaction
proceeded as follows: 95uC for 5 min, 40 cycles of 94uC for 30 s,
60uC for 30 s and 72uC for 30s. b-actin was used as an internal
reference to normalize the PCR reaction. Primer sequences are
listed in Table 1. The primers for genes (zip1, zip3, zip4, zip6, zip9,
zip11, zip13, znt1, znt2, znt4, znt5, znt6, znt7, znt8 and znt9) have
been reported previously .
Table 1. Primers for qRT-PCR analysis.
Forward Primer (5939)
Reverse Primer (5939)
The zip7 coding region was amplified from whole-genome
cDNA and cloned into the pCS2+ vector to generate pCS2+-zip7.
Injection of zip7 mRNA (in vitro transcribed by mMESSAGE
mMACHINEH High Yield Capped RNA Transcription Kit) was
performed to rescue zip7 knockdown. For whole-mount in situ
hybridization studies, a PCR-amplified region containing 431 bp
of the last exon and the 39UTR of zip7 was inserted into the
pCS2+ vector to generate the pCS2+-zip7-probe.
Microinjection of morpholino-oligonucleotides (MOs)
Antisense morpholino oligonucletides against zip7 mRNA were
designed and synthesized by Gene Tools, LLC. The sequences of
the translational blocking zip7-MO were
59-GCGATTTGCTAAAGACCCTCATTGT-39 (23 to 222, using the nucleotide
of the start codon as the reference). The sequence of the
mismatched control MO for zip7 was
59-CCTCTTACCTCAGTTACAATTTATA-39. The dosage for morpholino injection was
10 ng per embryo.
Capped sense RNA was synthesized using the mMESSAGE
mMACHINE kit (Ambion) from pCS2+2zip7. Microinjections
were carried out using the Harvard Apparatus PLI-90
microinjector. For double zip7-MO injections and pCS2+2zip7 mRNA
and zip7-MO injections, embryos were injected separately with
1 nL of each at appropriate concentration.
GFP reporter assay
The zip7-MO designed against zip7 targeted the zip7 ATG start
site to block its translation. To assay the effectiveness of zip7-MO,
a 195-bp zip7 cDNA fragment (27 bp upstream and 165 bp
downstream of ATG start site) was fused with GFP cDNA and
cloned into PCS2+ vector. The zip7-GFP reporter construct,
which contains the zip7-MO target site, was injected to one-cell
stage, wild type embryos together with or without zip7-MO. GFP
was detected by fluorescent microscope at 24 hours after injection.
Whole mount in situ hybridization
Antisense RNA probes were synthesized with DIG RNA
Labeling Kit (AP6/T7) (Roche) from the cDNA in the pCS2+
vector. Embryos were selected at 12 hpf, 24 hpf, 36 hpf, 48 hpf, 60
hpf, or 72 hpf. Embryos beyond 24 hpf were treated with 0.003%
phenylthiourea to prevent melanization, and all the embryos were
removed from chorions. The steps of whole mount in situ
hybridization referred to Sun Y . Finally, the embryos were
photographed using the Nikon SMZ1500 Zoom
Full scale scanning of zebrafish by SR-XPF
We detected the distribution of zinc in whole embryos injected
with zip7-MO or not, and cultured with zinc or not, by SR-XRF.
The absolute contents and distribution of zinc in zebrafish
embryos 72 hpf were analyzed with SR-mXPF at the beamilin
BL15U at Shanghai Synchrotron Radiation Facility (Shanghai,
China). The continuous synchrotron X-rays were
monochromatized by a Si double crystal . A monochromatic X-ray beam
with photon energy of 12 keV was used to excite the zebrafish.
The zinc distribution in the zebrafish was continuously scanned at
a step of 30 mm for both x and y directions. Each spot was
irradiated for 1.5 s. The results were analyzed using the Igor Pro
Folder program and Originlab OriginPro 8.5 software.
We are grateful to Dr. Tingxi Lius lab at the Institute of Health Science,
Chinese Academy of Sciences, who provided reagents and scientific
discussion. We thank Ms. Ke Yang at the beamline BL15U at the
Shanghai Synchrotron Radiation Facility (SSRF) for on-site assistance. We
appreciate the encouragement and helpful comments from members of the
Conceived and designed the experiments: FW. Performed the experiments:
GY Y. Zhang JY. Analyzed the data: YY FZ ZZ AW XY Y. Zhou. Wrote
the paper: GY Y. Zhang JY FW.
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