Neurulation and neurite extension require the zinc
transporter ZIP12 (slc39a12)
Winyoo Chowanadisaia,b,1, David M. Grahamc, Carl L. Keena, Robert B. Ruckera, and Mark A. Messerlib,c
aDepartment of Nutrition, University of California, Davis, CA 95616;bCellular Dynamics Program, Marine Biological Laboratory, Woods Hole, MA 02543; and
cEugene Bell Center for Regenerative Biology and Tissue Engineering, Marine Biological Laboratory, Woods Hole, MA 02543
Edited by Yuh Nung Jan, Howard Hughes Medical Institute, University of California San Francisco, San Francisco, CA, and approved May 1, 2013 (received for
review December 19, 2012)
Zn2+is required for many aspects of neuronal structure and func-
tion.However,the regulationofZn2+in thenervoussystemremains
poorly understood. Systematic analysis of tissue-profiling microar-
ray data showed that the zinc transporter ZIP12 (slc39a12) is highly
expressed in the human brain. In the work reported here, we con-
firmed that ZIP12 is a Zn2+uptake transporter with a conserved
pattern of high expression in the mouse and Xenopus nervous sys-
tem. Mouse neurons and Neuro-2a cells produce fewer and shorter
neurites after ZIP12 knockdown without affecting cell viability. Zn2+
chelation or loading in cells to alter Zn2+availability respectively
mimicked or reduced the effects of ZIP12 knockdown on neurite
ing protein activation and phosphorylation at serine 133, which is
a critical pathway for neuronal differentiation. Constitutive cAMP
response element-binding protein activation restores impairments
in neurite outgrowth caused by Zn2+chelation or ZIP12 knockdown.
ZIP12 knockdown also reduces tubulin polymerization and increases
sensitivity to nocodazole following neurite outgrowth. We find that
ZIP12 is expressed during neurulation and early nervous system de-
velopment in Xenopus tropicalis, where ZIP12 antisense morpholino
knockdown impairs neural tube closure and arrests development
during neurulation with concomitant reduction in tubulin polymer-
ization in the neural plate. This study identifies a Zn2+transporter
that is specifically required for nervous system development and
provides tangible links between Zn2+, neurulation, and neuronal
brain development|CREB|neural tube defect|zinc deficiency|
for zinc deficiency (1). Zn2+is a nutrient that plays critical roles in
more than 1,000 proteins, including enzyme catalysis, cell signaling,
and DNA repair (2), and as a result is essential for neural de-
influence brain development, structure, and function throughout
all stages of life (4). Zn2+supplementation can reduce the risk
for certain pregnancy complications (5–7), including congenital
defects, by preventing primary deficiencies caused by diet or by
treating secondary deficiencies, such as acrodermatitis enter-
opathica, a genetic disorder caused by a defect in intestinal Zn2+
transport secondary to a mutation in the Zn2+IRT-like protein
ZIP4 (5). Members of the solute carrier 39 (SLC39) gene family
encoding the Zn2+IRT-like proteins (ZIPs) are important com-
ponents of cellular Zn2+homeostasis and encode proteins that
promote cellular Zn2+uptake in a wide range of species (8). In
vertebrates, mutations in some SLC39 members have been linked
to developmental and metabolic disorders (5, 9–13), generally
leading to pleiotropic phenotypes.
Here, we analyzed published genome-wide microarray data
(14) to determine that slc39a12 (ZIP12) is highly expressed in the
humanbrain relative tootherSLC30andSLC39transporters.We
used a reverse genetics approach to demonstrate that ZIP12 is an
essential Zn2+transporter predominantly expressed in the brain.
pproximately 12% of Americans fail to consume the Esti-
mated Average Requirement for Zn2+and could be at risk
We observed that ZIP12 is important for multiple aspects of
neuronal differentiation, including activation of cAMP response
element-binding protein (CREB), tubulin polymerization, and
neurite extension, in vitro. We show that ZIP12 is required for
neurulation and embryonic viability during Xenopus tropicalis
development. These findings show that the Zn2+transporter
ZIP12 represents a point of regulation that links Zn2+directly to
nervous system development.
ZIP12 Is Highly Expressed in the Brain. We analyzed a previously
published microarray dataset (14) for human genes in the SLC30
and SLC39 families with brain-specific patterns of expression
that are likely to be important for nervous system development
and function (Fig. S1A). Of 32,878 probe sets, 1,130 genes
(3.44%) had a brain expression ratio greater than 5; of these,
1,118 genes (3.40%) were annotated with a gene symbol, Na-
tional Center for Biotechnology Information accession number,
or Celera transcript number (Dataset S1). The gene expression
of slc39a12 is 47-fold higher in the human brain than in other
tissues (Fig. S1A). The top five genes with the highest brain-
expression ratios (Dataset S1) have documented nervous system-
specific expression patterns and functions. The high expression
of ZIP12 in the human brain identified in our analysis is con-
sistent with a previous transcriptome analysis using expressed
sequence tag data (8).
Next, we examined slc39a12 expression in mice to gain insight
into the possible physiological roles of ZIP12. Our gene-
expression analysis revealed high levels of mZIP12 mRNA in
brain (Fig. 1A). We created an mZIP12-specific antibody that
binds to an N-terminal epitope before the first transmembrane
domain (Fig. 1B). mZIP12 protein expression was primarily in
the mouse brain (Fig. 1C) and was detected in the hippocampus,
frontal cortex, striatum, hypothalamus, and cerebellum (Fig. 1D).
Primary mouse neurons have endogenous ZIP12 expression at the
plasma membrane (Fig. 1E). mZIP12 localization to the plasma
membrane also was confirmed via epitope labeling in unpermea-
bilized CHO cells transfected with mZIP12-HA (Fig. S1 B–E).
Sequestration of extracellular Zn2+with the Zn2+chelator N,N,N′,
N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) resulted in
a redistribution of mZIP12 from the perinuclear space to the cy-
toplasm and plasma membrane, as detected by indirect immuno-
fluorescence (Fig. S1D) and cell-surface protein biotinylation (Fig.
S1E). TPEN also increased Zn2+uptake in both control and
mZIP12-transfected cells (Fig. S1F), indicating that mZIP12 is
present at the plasma membrane of transfected CHO cells.
Author contributions: W.C., D.M.G., R.B.R., and M.A.M. designed research; W.C., D.M.G.,
and M.A.M. performed research; C.L.K. contributed new reagents/analytic tools; W.C., D.M.G.,
and M.A.M. analyzed data; and W.C., D.M.G., C.L.K., R.B.R., and M.A.M. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
www.pnas.org/cgi/doi/10.1073/pnas.1222142110 PNAS Early Edition
| 1 of 6
mZIP12 protein also was detected in neurons in the Purkinje
cell layer of the cerebellum (Fig. 1F), medulla oblongata (Fig.
1G), and the frontal cortex (Fig. 1 H and I). No staining was
detected in the granule cells of the cerebellum (Fig. 1F), in-
dicating that not all neurons express ZIP12. The high expression
of ZIP12 in the brain suggests a functional role for ZIP12-
mediated Zn2+transport in the nervous system.
slc39a12 Encodes the Zn2+Transporter, ZIP12. The gene slc39a12
encodes a putative member of the SLC39 gene family of Zn2+
transporters. We characterized the kinetic properties of mZIP12
by transfecting CHO cells and measuring Zn2+uptake using the
radioisotope65Zn in varying concentrations of free Zn2+(Fig. 2).
Zn2+uptake was twofold higher in mZIP12-transfected cells
than in control cells (Fig. 2A). Addition of other metals in excess
did not prevent Zn2+uptake (Fig. 2B), although copper and
cadmium did suppress Zn2+uptake to a slight degree. Excess
cold (unlabeled) Zn2+significantly reduced65Zn uptake in both
control and mZIP12-transfected cells. ZIP12 activity is temper-
ature dependent because we did not observe significant Zn2+
uptake at 4 °C. We estimated the Kmof mZIP12 to be 6.6 nM,
which indicates that mZIP12 has a high affinity for Zn2+, and
a Vmaxof 2.7 pmol Zn2+·min−1·mg protein−1. Using inductively
coupled plasma mass spectrometry (ICP-MS), we observed that
mZIP12 induces the accumulation of Zn2+(Fig. 2C). Over-
expression of ZIP12 increases metal response element (MRE)
activation by metal-regulatory transcription factor 1 (MTF-1),
a sensitive measure of cytoplasmic Zn2+concentrations (Fig. 2D)
(15). Zinc sequestration with TPEN also increased Zn2+uptake in
both control and mZIP12-transfected cells (Fig. S1F). The in-
crease in Zn2+transport by transfection with ZIP12 did not af-
fect cell viability (Fig. 2E). These results indicate that increased
ZIP12 expression results in higher cellular Zn2+uptake, in-
creased cellular Zn2+accumulation, and increased cytoplasmic
Zn2+concentration, consistent with ZIP12 functioning as a Zn2+
ZIP12 Expression and Intracellular Zn2+Increase During Stimulated
Neurite Outgrowth. We conducted studies to determine if neuro-
nal differentiation is accompanied by changes in cellular Zn2+
homeostasis and mZIP12 expression. We used the mouse neuro-
blastoma cell line, Neuro2a (N2a), a well-characterized model for
cript analysis of endogenous mZIP12 in N2a cells revealed an
approximately threefold increase in mZIP12 gene expression in
differentiated cells versus control and reduced serum conditions
(Fig. S2A). A similar increase in mZIP12 mRNA expression was
found in neuronal precursor cells (NPCs) (Fig. S2B) following
differentiation. Endogenous mZIP12 protein expression increased
in N2a cells after differentiation (Fig. S2C). After 48 h differenti-
ated N2a cells displayed significantly increased Zn2+uptake (Fig.
S2D), as measured by65Zn, and increased fluorescence of the zinc
fluorophore Zinpyr-1 in perinuclear compartments, as measured
by microscopy (Fig. S2E) and flow cytometry (Fig. S2 F and G),
neurite extension, an early morphological sign of neuronal differ-
entiation, were observed in N2a cells treated with RA (Fig. S2H).
These changes in Zn2+metabolism induced by differentiation oc-
curred without affecting cell viability (Fig. S2I).
ZIP12 Knockdown Reduces Zn2+Uptake and Neurite Extension During
Differentiation. We used a ZIP12-specific shRNA plasmid to knock
down expression of endogenous mZIP12 in N2a cells. ZIP12
shRNA effectivelyreduced mZIP12 mRNA (Fig.S3A) and protein
(Fig. S3B) expression in differentiated N2a cells. Differentiation of
N2a cells increased Zn2+uptake, which was decreased by ZIP12-
shRNA knockdown (Fig. S3C). No difference in cell viability was
observed between control cells and N2a cells transfected with
ZIP12-shRNA at 48 or 96 h posttransfection (Fig. S3D).
In differentiated N2a cells, knockdown of mZIP12 expression
resulted in fewer and shorter neurites compared with control
mRNA expression detected in various mouse tissues by RT-PCR. (B) Detection
of mouse ZIP12 by immunoblotting in mZIP12-transfected CHO cells. (C and D)
ZIP12 protein expression detected in various mouse tissue lysates (C) and
brain region lysates (D) by immunoblotting. (C) Tissues include (from left to
right): brain, lung, skeletal muscle, liver, small intestine, heart, kidney, and
pancreas. (D) Brain regions include (from left to right): hippocampus (Hipp),
frontal cortex (FC), striatum (Str), hypothalamus (Hyp), and cerebellum (Cerb).
(E) ZIP12 is present at the plasma membrane in primary mouse neurons. (Scale
bar: 10 μm.) (F–I) ZIP12 is present in coronal sections of various regions of the
mouse brain. Brain regions include (F) cerebellum (including Purkinje cell
layer), (G) medulla oblongata, (H) frontal cortex, and (I) corpus callosum (and
cortex). Arrows in F and G indicate neuronal staining. [Scale bar: 50 μm (60×
magnification) in F and G; 200 μm (20× magnification) in H and I.]
ZIP12 is primarily expressed in the human and mouse brain. (A) ZIP12
different concentrations of free external Zn2+by ZIP12 in CHO cells was
measured using65Zn (n = 3, ± SE). The curve marked ZIP12 – Control repre-
sents net Zn2+uptake in ZIP12-transfected cells minus uptake in control-
transfected cells. (B) Zn2+uptake specificity of ZIP12 relative to other metals
determined using65Zn (n = 3, ± SE). Excess Zn2+indicates cold Zn2+added to
uptake buffer during assay. (C) Increased Zn2+content relative to cell number
by ZIP12 was measured by ICP-MS (n = 6, ± SE). (D) ZIP12 increases MRE ac-
tivation, measured by reporter assay (n = 6, ± SE). (E) ZIP12 overexpression
does not affect cell viability at 48 h posttransfection, measured by Trypan
blue exclusion (n = 6, ± SE). **P < 0.01; ***P < 0.001 versus control cells.
ZIP12 is a high-affinity, Zn2+- specific transporter. (A) Zn2+uptake at
2 of 6
| www.pnas.org/cgi/doi/10.1073/pnas.1222142110Chowanadisai et al.
cells (Table S1). Exposure to RA for periods of 48, 72, and 96 h
caused consistently longer neurites in cells treated with control
shRNA than in cells treated with ZIP12-shRNA over the same
time periods (Fig. 3 A and B and Fig. S4 A–C). We observed
a decrease in neurite length when N2a cells were differentiated
with dibutyryl cAMP instead of RA (Fig. S4D). Primary neurons,
either directly dissociated from mouse embryonic brain cortices
(Fig. S4 E and F) or primary mouse NPCs (Fig. S4 G and H), had
shorter neurites after 48 h of transfection with ZIP12 shRNA.
These observations show that the ZIP12-dependent aberrant
neurite phenotype is consistent across many neuronal models of
differentiation and is not limited to a specific cell line or de-
pendent on cell line immortalization or specific inducers of dif-
ferentiation. These data indicate that neurite sprouting and
length are linked to intracellular Zn2+and ZIP12 expression.
We characterized the role of Zn2+on neurite outgrowth by
manipulating mZIP12 expression with ZIP12-shRNA and avail-
ability of extracellular and intracellular Zn2+with the nonper-
meant Zn2+chelator diethylene triamine pentaacetic acid (DTPA)
and the Zn2+carrier, pyrithione (17), respectively. All experiments
reported here were performed on both N2a cells and primary
mouse NPCs differentiated for 48 h. Treatment of N2a cells with
DTPA resulted in fewer cells expressing neurites (Table S1).
DTPA treatment, which reduced available free Zn2+concen-
trations below detection (Fig. S5 A and B) in both N2a cells (Fig.
3C) and differentiated mouse NPCs (Fig. S4I), reduced neurite
length in the cells transfected with control shRNA. This phe-
notype was restored by saturating DTPA with additional Zn2+
(Fig. 3C and Fig. S4I). In comparison, ZIP12 knockdown resulted
in cells with fewer neurites (Table S1) and reduced neurite length
(Fig. 3C and Fig. S4I) compared with control cells without DTPA
treatment. Treatment of ZIP12-knockdown cells with DTPA alone
or Zn2+-saturated DTPA did not have further negative effects on
neurite outgrowth. Similar results were found with Zn2+-deficient,
Chelex-treated medium. The effects of ZIP12 were observed in
N2a cells and differentiated mouse NPCs treated with Zn2+pyr-
ithione. Zn2+pyrithione radiolabeled with65Zn crosses the plasma
membrane (Fig. S5 C and D) in greater amounts compared with
equimolar amounts of added Zn2+alone. In the absence of Zn2+
pyrithione, knockdown of ZIP12 reduced neurite length com-
pared with N2a cells (Fig. 3D) and differentiated mouse NPCs
(Fig. S4J) transfected with control shRNA, whereas neurite
length was recovered with Zn2+pyrithione treatment. Thus,
neurite length could be rescued by increasing intracellular Zn2+
availability with Zn2+pyrithione in ZIP12 knockdown cells. Both
DTPA and Zn2+pyrithione were used at concentrations that do
not affect cell viability (Fig. S5 E–H). These data indicate that
neurite sprouting and length are linked to intracellular Zn2+and
ZIP12 Is Important for CREB Signaling and Tubulin Polymerization.We
investigated the role of ZIP12 on CREB function because we
observed that neurite outgrowth, a CREB signaling-dependent
critical step for neuronal differentiation (18), requires ZIP12.
N2a cells transfected with ZIP12 shRNA showed reduced CREB
activation after differentiation for 48 h (Fig. 4A) and impaired
phosphorylation at serine 133 (Fig. 4B) after incubation in dif-
ferentiating medium for 30 min at 48 h posttransfection. We did
not observe a difference in CREB activation and phosphoryla-
tion in undifferentiated cells transfected with control or ZIP12
shRNA. When we differentiated N2a cells while treating them
with chelator to eliminate extracellular Zn2+availability (Fig. 4C),
we observed that DTPA-treated control-shRNA cells showed
a reduction in CREB activation that was similar to the reduction
induced by ZIP12 shRNA knockdown. This result demonstrates
that chelation of extracellular Zn2+eliminates the effect of
ZIP12 on CREB activation. CREB phosphorylation was reduced
by intracellular Zn2+chelation by TPEN in all conditions tested
(Fig. 4D), including both undifferentiated and differentiated
cells and cells transfected with control or ZIP12 shRNA. These
differences in the interactions of Zn2+chelation and ZIP12 ex-
pression may be caused by the differing characteristics of the cell
impermeant chelator DTPA and the rapid cell permeant che-
lator TPEN and the time courses of the CREB activation and
We used viral protein 16 (VP16)-CREB, a constitutively active
CREB (19), to determine if the effects of Zn2+and ZIP12 on
neurite extension could be rescued by CREB activation without
the induction of CREB phosphorylation. Transfection of VP16-
CREB resulted in an increase in neurite length in N2a cells in-
cubated in control and reduced serum media without RA sup-
plementation after 48 h as compared with cells transfected with
control plasmid (Fig. 4E). The neurite outgrowth induced by
constitutive VP16-CREB activation was not affected by extra-
cellular Zn2+chelation by DTPA (Fig. 4F) or by ZIP12 shRNA
(Fig. 4G). These observations support the concept that CREB
activation is Zn2+-dependent and requires ZIP12 expression
during neuronal differentiation.
We examined tubulin polymerization states in N2a cells to
further our understanding of how neurite length, ZIP12, and
intracellular Zn2+might be connected. Knockdown of ZIP12
resulted in a reduction of polymerized tubulin and an increase in
soluble tubulin (Fig. 4H) compared with the control. Tubulin ex-
pression, as measured by α-tubulin in whole-cell extracts, was not
affected in either treatment (Fig. 4H). The knockdown of ZIP12
did cause an increase in neurite retraction induced by nocodazole,
a microtubule-destabilizing agent. Here, neurite length was mea-
sured before and after nocodazole treatment for differentiated
N2a cells transfected with the control shRNA or the ZIP12
shRNA. Application of 500 nM nocodazole for 30 min resulted in
a decrease in neurite length in ZIP12-knockdown cells but not in
control cells (Fig. 4I). No changes in neurite length were observed
when nocodazole was withheld. Reductions in neurite length were
evident in both control and ZIP12 shRNA cells when a higher
concentration of nocodazole (1.5 μM) was used.
affected by Zn2+availability. (A and B) ZIP12 shRNA reduces neurite length in
N2a cells (n = 50, ± SE). (C) Chelation of extracellular Zn2+with DTPA mimics
the impairment of ZIP12 knockdown on neurite length in N2a cells (n = 50, ±
SE). (D) Zn2+carrier pyrithione (ZP) restores neurite outgrowth impaired
by ZIP12 shRNA knockdown in N2a cells (n = 50, ± SE). (Scale bars: 100 μm.)
**P < 0.0, ***P < 0.001 versus control cells without DTPA or ZP;##P < 0.01,
###P < 0.001 versus control cells with DTPA or ZIP12-shRNA cells without ZP.
Neurite extension is dependent on Zn2+transport by ZIP12 and is
Chowanadisai et al.PNAS Early Edition
| 3 of 6
Differences in tubulin polymerization in N2a cells caused by
ZIP12-shRNA knockdown were visible by staining polymerized
tubulin with a fluorescent paclitaxel. ZIP12 knockdown resulted
in reduced polymerized tubulin in neurites and increased poly-
merized tubulin staining within the cell body at both 8 h and 48 h
differentiation (Fig. S6). Controls displayed staining for polymer-
ized tubulin primarily within neurites. Undifferentiated ZIP12
knockdown cells and control undifferentiated cells displayed no
discernible difference in polymerized tubulin content and orga-
nization. Polymerized tubulin was visibly prominent in neurites
of untransfected cells next to mZIP12-shRNA–transfected cells
in the same field of view. Some mZIP12-shRNA–transfected
cells showed reduced polymerized tubulin staining in neurites or
heavier tubulin staining in the cell body compared with neurites.
Collectively, these results link ZIP12-dependent Zn2+transport
function to polymerized tubulin within extending neurites.
ZIP12 Is Expressed During Neurulation and Embryonic Nervous System
Development in X. tropicalis. The slc39a12 ortholog of X. tropicalis
shares 52% and 50% of its predicted amino acid sequence with
human and mouse ZIP12, respectively, and contains conserved
structural features of ZIP proteins. In X. tropicalis, slc39a12
and neighboring genes (contig NW_003163763) share a syntenic
relationship with human and mouse, indicating that xZIP12 is
the ZIP12 ortholog of this species. Gene expression of xZIP12
in adult X. tropicalis was highest in brain tissue, similar to our
observations for mouse tissues (Fig. 5A). xZIP12 gene expression
during development was first detected during early neural plate
formation (stage 13) and increased at the early tailbud stage
(stage 28). Onset of xZIP12 gene expression seems to be similar
to that of N-tubulin (Fig. 5B), which first occurs during early
neurulation (20). Using quantitative RT-PCR, we observed that
xZIP12 mRNA expression is higher in the neural plate than in
the rest of the embryo (Fig. 5C). In situ hybridization of X. tro-
picalis embryos showed that ZIP12 mRNA expression is highest
around the anterior neuropore in the neural plate (Fig. 5D), and
as embryogenesis proceeds, ZIP12 is present in the forebrain,
midbrain, and presumptive eye of the embryo (Fig. 5D). These
gene-expression patterns suggest that xZIP12 is important during
neurulation and subsequent nervous system development.
ZIP12 Is Required for Neurulation and Tubulin Polymerization. Based
onthedevelopmentalpattern ofZIP12 expression, weinvestigated
the functional role of xZIP12 in neurulation by targeted knock-
prevents the initiation of xZIP12 translation (Fig. S7A), whereas
morpholino slc39a12MO2 causes exon exclusion and premature
termination of xZIP12 translation (Fig. S7 B and C). Embryos
injected with slc39a12MO1 and slc39a12MO2 developed through
gastrulation and neural plate formation but delayed at neural
tube closure and displayed severe neural tube defects. These
developmental arrest after neural tube closure (16/100) for em-
the neural tube (45/109) after 22 h for embryos injected with
slc39a12MO2 (Fig. 5K). All embryos injected with slc39a12MO1
were dead before developmental stage 22 and all embryos injec-
ted with slc39a12MO2 were dead before developmental stage 25
(Fig. 5M). In contrast, embryos injected with a mismatch mor-
pholino to slc39a12MO1 (n = 41) were developmentally normal
and indistinguishable from embryos injected with control mor-
pholino (Fig. 5 H and I). Few embryos injected with control
morpholino (1/153) (Fig. 5 F, H, and J) showed signs of neural
tube defects. Nearly all control-injected embryos (145/153) pro-
ceeded through normal development (stages 32–34). Collectively
these results support the concept that xZIP12 is required for
neurulation during development.
We explored whether xZIP12 affects neurulation through im-
paired tubulin polymerization. N-tubulin is expressed primarily in
the neural tube during closure in Xenopus laevis (21), whereas
α-tubulin is expressed throughout the embryo. Differences in
total N-tubulin and α-tubulin content were not detected in
slc39a12MO1-injected versus control-injected embryo extracts
(Fig. 5N). However, a reduced ratio of polymerized to soluble
N-tubulin was found in slc39a12MO1 compared with control
and B) Differentiation of N2a cells increases cAMP response element (CRE)
activation (n = 6, ± SE) and CREB phosphorylation, both of which are blunted
by ZIP12 shRNA-mediated knockdown. ***P < 0.001 versus control un-
differentiated cells;##P < 0.01 versus control differentiated cells. (C) Extra-
cellular Zn2+chelation by DTPA mimics the blunting of CREB activation in
differentiated cells induced by ZIP12 shRNA knockdown (n = 6, ± SE). ***P <
0.001 versus control undifferentiated cells;##P < 0.01 versus control differ-
entiated cells;+++P < 0.001 versus cells treated with DTPA. (D) Intracellular
Zn2+chelation by TPEN reduces phosphorylation of CREB regardless of ZIP12
shRNA knockdown. (E) Constitutive activation of CREB by VP16-CREB1
transfection increases neurite extension in the absence of RA. ***P < 0.001
versus cells transfected with control plasmid. (F and G) Neurite outgrowth
induced by constitutive activation of CREB is not affected by (F) extracellular
Zn2+chelation or (G) ZIP12 shRNA-mediated knockdown. ***P < 0.001 versus
cells transfected with control plasmid in reduced (Red) serum medium.###P <
0.001 versus cells transfected with control plasmid and differentiating me-
dium. (H) ZIP12 knockdown alters soluble and polymerized tubulin fractions
in N2a cells without affecting total tubulin protein expression. (I) ZIP12
knockdown in differentiated N2a cells increases sensitivity to neurite re-
traction following microtubule destabilization by nocodazole (n = 50, ± SE).
***P < 0.001 versus cells before nocodazole.
ZIP12 is required for CREB signaling and tubulin polymerization. (A
4 of 6
| www.pnas.org/cgi/doi/10.1073/pnas.1222142110 Chowanadisai et al.
extracts (Fig. 5O). Differences in the ratio of polymerized to
soluble α-tubulin were not observed. Taken together, knockdown
of xZIP12 impairs normal neurulation and reduces the amount
of polymerized N-tubulin.
Our data show the Zn2+transporter ZIP12 is critical for neu-
ronal differentiation, neurulation, and embryonic development.
ZIP12 expression in the brain, conserved across human, mouse,
and frog is likely an indicator of evolutionary constraint caused
by the requirement of ZIP12 for nervous system development
and is consistent with other brain-specific genes (22). The
defects in neuronal maturation and neural tube closure caused
by ZIP12 inhibition are consistent with the localization and
expression of mZIP12 and xZIP12. Zn2+transport by ZIP12
may support an increased demand for Zn2+during early nervous
system development (4). Importantly, loss of ZIP12 could not
be offset by other numerous Zn2+uptake pathways, such as
Zn2+-permeable Ca2+channels (23), or other Zn2+transpor-
ters. This finding underscores the importance of ZIP12 in its
nonredundant and critical role in neuronal development and
The Zn2+uptake activity and cellular distribution of ZIP12 are
similar to that of several other members of the SLC39 family.
ZIP12-transfected cells have increased Zn2+uptake, a high cel-
lular Zn2+content, and increased cytoplasmic Zn2+concen-
trations. These changes in cellular Zn2+are similar to those that
can be induced by ZIP1, ZIP3, and ZIP4 of the SLC39 family
(24), which transit between the plasma membrane and peri-
nuclear regions and transport Zn2+from the extracellular medium.
The finding that ZIP12 is detectable at the plasma membrane in
primary mouse neurons and transfected CHO cells is supported
by proteomic profiling (25), which identified ZIP12 (FLJ30499)
by LC-MS using extracted and digested plasma membrane pro-
teins from mouse brain. Similar to the Zn2+transporters ZIP1,
ZIP3, and ZIP4 (24), a significant portion of ZIP12 is present
away from the plasma membrane, and Zn2+chelation by TPEN
results in migration of ZIP12 away from the perinuclear region
and toward the plasma membrane. Interplay between the high,
nanomolar affinity of ZIP12 for Zn2+, regulation of ZIP12 lo-
calization at the plasma membrane, and tissue-specific expres-
sion of ZIP12 in the nervous system likely contributes to the role
of ZIP12 in development.
Neuronal differentiation requires ZIP12, which links Zn2+to
physiological aspects of neuronal development. Previous studies
have shown that low dietary Zn2+intake in rats and mice impairs
neurite outgrowth in the brain (26, 27). CREB activity and neurite
outgrowth are closely linked phases of neuronal differentiation.
RA induces rapid CREB phosphorylation, a critical step that
controls CREB transcriptional activity and neurite outgrowth
(18). Zn2+transport by ZIP12 affects an early stage of neuronal
differentiation through CREB phosphorylation, which is evident
within 30 min of differentiation. Previous studies have shown Zn2+
ionophore PBT2 increases zinc availability, induces CREB phos-
phorylation, and promotes neuroprotection in cellular models
of Alzheimer’s disease (28). Furthermore, the role of Zn2+
transporters in cell signaling pathways (29–31) has been iden-
tified in tissues outside the nervous system. In support of the role
of Zn2+in CREB signaling, the constitutive activation of CREB
signaling by VP16-CREB, which essentially bypasses the re-
quirement of CREB phosphorylation for activation activity, ef-
fectively rescues the impairment in neurite outgrowth caused by
either extracellular Zn2+chelation or ZIP12 knockdown. Given
the wide range of biological functions for Zn2+, neural differ-
entiation may result in an increased need for Zn2+that is met in
part by ZIP12. Although CREB signaling is affected by ZIP12
and Zn2+, it is possible that the impairments in CREB signaling
and other factors such as tubulin polymerization reflect a broad
delay in differentiation, given the diverse roles of Zn2+in cell
metabolism (2). More studies will be needed to determine the
roles of ZIP12 and other Zn2+uptake transporters, such as
ZIP10 (32), which is highly expressed in both brain and liver, at
different stages of neuronal development.
During embryogenesis, knockdown of ZIP12 expression im-
paired neural tube closure and arrested development. A crucial
role of ZIP12 for neurulation is consistent with reports link-
ing dietary Zn2+deficiency in humans to neural tube defects
(7). The location of ZIP12 expression and the developmental
impairments in ZIP12MO embryos are closely linked. High
expression of ZIP12 was detected at the anterior neuropore,
the primary site of neural tube closure impairment. ZIP12 ex-
pression was concentrated in the eye and head region of Xenopus
tailbud-stage embryos, and for ZIP12MO embryos that proceeded
past neurulation, no optic vesicle or lens development could be
and is required for neural tube closure and embryonic viability. (A and B)
ZIP12 and β-actin or GAPDH expression was determined in various adult
tissues and developmental stages by RT-PCR. (C) ZIP12 mRNA expression is
elevated in the neural plate, as determined by quantitative RT-PCR (n = 6, ±
SE). ***P < 0.001 versus whole embryo;###P < 0.001 versus rest of embryo.
(D) ZIP12 mRNA (slc39a12) is expressed during neurulation and early nervous
system development, analyzed by in situ hybridization. (E–M) ZIP12 knock-
down by antisense morpholino microinjection impairs X. tropicalis develop-
ment during neurulation. (N) Microinjection of slc39a12MO1 does not affect
tubulin protein content, analyzed by immunoblotting. (O) Microinjection of
slc39a12MO1 affects the ratio of polymerized to soluble β2-tubulin, as ana-
lyzed by polymerized tubulin fractionation and immunoblotting. (Scale bars:
250 μm in all images.)
ZIP12 is expressed primarily in the neural tube and brain of X. tropicalis
Chowanadisai et al.PNAS Early Edition
| 5 of 6
discerned in the presumptive eye region. Tubulin polymerization
was impaired in the neural plate of slc39a12MO1 morphants.
Disruption of microtubules with different agents slows progression
of neurulation and impairs neural tube closure in vertebrate
embryos (33), similar to the effects seen in ZIP12-knockdown
embryos. Chelation of Zn2+in Xenopus slows development
during neurulation, leading to arrest (34). The correlation be-
tween the temporal and spatial expression of ZIP12 and its
crucial requirement in neurulation all support the hypothesis
that ZIP12 has a specific role in CNS development.
Systematic analyses of tissue-specific patterns of gene coex-
pressionacross differentspecies,asproposedbyAla etal. (35),can
effectively identify genes critical for nervous system function.
McGary et al. (36) have proposed that phenologs, in which dis-
ruptions in orthologous genes result in phenotypes across species,
can identify candidate disease genes in humans. Further in-
vestigation is warranted to determine if ZIP12 is a candidate gene
for nervous system defects during prenatal development with
increased penetrance during low maternal intake of dietary Zn2+.
All procedures were approved by the Institutional Animal Care and Use Com-
Microinjection of X. tropicalis Embryos with Antisense Morpholinos. Embryos
were injected at the one-cell stage with 10 ng morpholino with 0.2% rho-
damine dextran (Invitrogen) as a tracer.
Additional Methods.Detailsofmicroarray dataanalyses,reagents,cellculture,
transfection, Zn2+uptake activity, plasmid construction, luciferase reporter
assays, ICP-MS, cell-surface biotinylation, anti-mouse ZIP12 antibody pro-
duction, indirect immunofluorescence, cell protein isolation, immunoblot-
ting, mouse brain immunohistochemistry, free Zn2+measurements in
solutions, Zinpyr-1 staining, flow cytometry, cell viability, RT-PCR, neurite
length analyses, in situ hybridization, morpholino sequences and validation,
and tubulin organization are described in SI Methods. All statistical analyses
used are detailed in SI Methods.
ACKNOWLEDGMENTS. We thank Maggie Chiu, Carol Oxford, and Joel Com-
misso for their technical expertise; Jonathan Gitlin and Robert Prendergast
for their critical reading of the manuscript; Peter J. S. Smith for his support
provided through the Biocurrents Research Center; and Peter Klein for the
pCS2+FLAG plasmid (Addgene). We are grateful for the expertise of the
National Xenopus Resource, funded by National Institutes of Health (NIH)
P40 OD010997. This work was supported by the University of California,
Davis Center for Health and Nutrition Research (R.B.R.), NIH NCRR Grants
P41 RR001395S1 (to Joshua W. Hamilton and M.A.M.) and P30 GM092374
(to Gary G. Borisy), by the Eugene and Millicent Bell Fellowship Fund in
Tissue Engineering (M.A.M.), and by the Hermann Foundation Research
Development Fund Award (M.A.M.).
1. Song Y, Leonard SW, Traber MG, Ho E (2009) Zinc deficiency affects DNA damage,
oxidative stress, antioxidant defenses, and DNA repair in rats. J Nutr 139(9):1626–
2. Ho E, Ames BN (2002) Low intracellular zinc induces oxidative DNA damage, disrupts
p53, NFkappa B, and AP1 DNA binding, and affects DNA repair in a rat glioma cell
line. Proc Natl Acad Sci USA 99(26):16770–16775.
3. Frederickson CJ, Koh JY, Bush AI (2005) The neurobiology of zinc in health and dis-
ease. Nat Rev Neurosci 6(6):449–462.
4. Ames BN (2006) Low micronutrient intake may accelerate the degenerative diseases
of aging through allocation of scarce micronutrients by triage. Proc Natl Acad Sci USA
5. Dufner-Beattie J, et al. (2007) The mouse acrodermatitis enteropathica gene Slc39a4
(Zip4) is essential for early development and heterozygosity causes hypersensitivity to
zinc deficiency. Hum Mol Genet 16(12):1391–1399.
6. Velie EM, et al. (1999) Maternal supplemental and dietary zinc intake and the oc-
currence of neural tube defects in California. Am J Epidemiol 150(6):605–616.
7. Uriu-Adams JY, Keen CL (2010) Zinc and reproduction: Effects of zinc deficiency on
prenatal and early postnatal development. Birth Defects Res B Dev Reprod Toxicol
8. Eide DJ (2004) The SLC39 family of metal ion transporters. Pflugers Arch 447(5):
9. Giunta C, et al. (2008) Spondylocheiro dysplastic form of the Ehlers-Danlos syndrome—
an autosomal-recessive entity caused by mutations in the zinc transporter gene
SLC39A13. Am J Hum Genet 82(6):1290–1305.
10. Fukada T, et al. (2008) The zinc transporter SLC39A13/ZIP13 is required for connective
tissue development; its involvement in BMP/TGF-beta signaling pathways. PLoS ONE
11. Yamashita S, et al. (2004) Zinc transporter LIVI controls epithelial-mesenchymal
transition in zebrafish gastrula organizer. Nature 429(6989):298–302.
12. Pielage J, Kippert A, Zhu M, Klämbt C (2004) The Drosophila transmembrane protein
Fear-of-intimacy controls glial cell migration. Dev Biol 275(1):245–257.
13. Stathakis DG, et al. (1999) The catecholamines up (Catsup) protein of Drosophila
melanogaster functions as a negative regulator of tyrosine hydroxylase activity.
14. Dezso Z, et al. (2008) A comprehensive functional analysis of tissue specificity of
human gene expression. BMC Biol 6:49.
15. Hara H, Aizenman E (2004) A molecular technique for detecting the liberation of
intracellular zinc in cultured neurons. J Neurosci Methods 137(2):175–180.
16. Tremblay RG, et al. (2010) Differentiation of mouse Neuro 2A cells into dopamine
neurons. J Neurosci Methods 186(1):60–67.
17. Andersson DA, Gentry C, Moss S, Bevan S (2009) Clioquinol and pyrithione activate
TRPA1 by increasing intracellular Zn2+. Proc Natl Acad Sci USA 106(20):8374–8379.
18. Cañón E, Cosgaya JM, Scsucova S, Aranda A (2004) Rapid effects of retinoic acid on
CREB and ERK phosphorylation in neuronal cells. Mol Biol Cell 15(12):5583–5592.
19. Barco A, Alarcon JM, Kandel ER (2002) Expression of constitutively active CREB pro-
tein facilitates the late phase of long-term potentiation by enhancing synaptic cap-
ture. Cell 108(5):689–703.
20. Richter K, Grunz H, Dawid IB (1988) Gene expression in the embryonic nervous system
of Xenopus laevis. Proc Natl Acad Sci USA 85(21):8086–8090.
21. Moody SA, Miller V, Spanos A, Frankfurter A (1996) Developmental expression of
a neuron-specific beta-tubulin in frog (Xenopus laevis): A marker for growing axons
during the embryonic period. J Comp Neurol 364(2):219–230.
22. Khaitovich P, et al. (2005) Parallel patterns of evolution in the genomes and tran-
scriptomes of humans and chimpanzees. Science 309(5742):1850–1854.
23. Sensi SL, et al. (1997) Measurement of intracellular free zinc in living cortical neurons:
Routes of entry. J Neurosci 17(24):9554–9564.
24. Wang F, et al. (2004) Zinc-stimulated endocytosis controls activity of the mouse ZIP1
and ZIP3 zinc uptake transporters. J Biol Chem 279(23):24631–24639.
25. Nielsen PA, et al. (2005) Proteomic mapping of brain plasma membrane proteins. Mol
Cell Proteomics 4(4):402–408.
26. Dvergsten CL, Fosmire GJ, Ollerich DA, Sandstead HH (1984) Alterations in the post-
natal development of the cerebellar cortex due to zinc deficiency. II. Impaired mat-
uration of Purkinje cells. Brain Res 318(1):11–20.
27. Gao HL, et al. (2009) Zinc deficiency reduces neurogenesis accompanied by neuronal
apoptosis through caspase-dependent and -independent signaling pathways. Neu-
rotox Res 16(4):416–425.
28. Crouch PJ, et al. (2011) The Alzheimer’s therapeutic PBT2 promotes amyloid-β deg-
radation and GSK3 phosphorylation via a metal chaperone activity. J Neurochem
29. Haase H, Rink L (2009) Functional significance of zinc-related signaling pathways in
immune cells. Annu Rev Nutr 29:133–152.
30. Aydemir TB, Sitren HS, Cousins RJ (2012) The zinc transporter Zip14 influences c-Met
phosphorylation and hepatocyte proliferation during liver regeneration in mice.
31. Beker Aydemir T, et al. (2012) Zinc transporter ZIP14 functions in hepatic zinc, iron
and glucose homeostasis during the innate immune response (endotoxemia). PLoS
32. Lichten LA, Ryu MS, Guo L, Embury J, Cousins RJ (2011) MTF-1-mediated repression of
the zinc transporter Zip10 is alleviated by zinc restriction. PLoS ONE 6(6):e21526.
33. Smedley MJ, Stanisstreet M (1986) Calcium and neurulation in mammalian embryos.
II. Effects of cytoskeletal inhibitors and calcium antagonists on the neural folds of rat
embryos. J Embryol Exp Morphol 93:167–178.
34. Jörnvall H, Falchuk KH, Geraci G, Vallee BL (1994) 1,10-Phenanthroline and Xenopus
laevis teratology. Biochem Biophys Res Commun 200(3):1398–1406.
35. Ala U, et al. (2008) Prediction of human disease genes by human-mouse conserved
coexpression analysis. PLOS Comput Biol 4(3):e1000043.
36. McGary KL, et al. (2010) Systematic discovery of nonobvious human disease models
through orthologous phenotypes. Proc Natl Acad Sci USA 107(14):6544–6549.
6 of 6
| www.pnas.org/cgi/doi/10.1073/pnas.1222142110 Chowanadisai et al.