MicroRNA-mediated posttranscriptional regulation is required for maintaining undifferentiated properties of blastoderm and primordial germ cells in chickens.
ABSTRACT MicroRNAs (miRNAs) play a critical role in determining the differentiation fate of pluripotent stem cells and germ cells in mammals. However, the mechanism(s) of miRNA-mediated posttranscriptional regulation with regard to lineage specification and differentiation in chick development require further investigation. Therefore, we conducted miRNA expression profiling to explore specific miRNA signatures in undifferentiated blastoderm and primordial germ cells (PGCs). We identified seven miRNAs that are highly expressed in blastoderm and 10 that are highly expressed in PGCs. In this study, miR-302a and miR-456 for blastoderm and miR-181a* for PGCs were analyzed further for their target transcripts and regulatory pathways. Both miR-302a and miR-456 bound directly to the sex-determining region Y box 11 transcript and could act as posttranscriptional coregulators to maintain the undifferentiated state of the chicken blastoderm through the suppression of somatic gene expression and differentiation. Moreover, miR-181a* showed a bifunctional role in PGCs by binding to two different transcripts. miR-181a* inhibited the somatic differentiation of PGCs by silencing homeobox A1 expression. Additionally, miR-181a* prevented PGCs from entering meiosis through the repression of the nuclear receptor subfamily 6, group A, member 1 transcript. Collectively, our data demonstrate that in chickens miRNAs intrinsically regulate the differentiation fate of blastoderms and PGCs and that the specific timing of germ cell meiosis is controlled through miRNA expression.
- The Journal of Poultry Science 01/2012; 49(3):155-162. · 0.79 Impact Factor
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ABSTRACT: The aim of this study was to isolate Embryonic Stem Cells (ESCs) from native chicken and to characterize their pluripotency properties through the cellular and molecular markers. Samples obtained from fertilized eggs from Mazandaran native hens. Cells were isolated from area of pellucida from stage X native hens' blastoderm. Then the cells were cultured on inactivated mouse SNL feeder cells in the presence of LIF, IGF—1, bFGF, CNTF, OSM, SCF, Il—6, and Il—11 growth factors. The native chickens' ESCs colonies were picked up and subsequently passaged. To characterize the cells, they were analyzed for their alkaline phosphatase activity, and also for the expression of SSEA—4, and TRA—1—60 as embryonic—specific markers at the protein level. Furthermore, the expression of pluripotency (cPouV, Sox2, and Nanog) and cell lineage specific (Cvh, Brachyury, and Gata6) gene markers was evaluated at the level of mRNA using quantitative RT—PCR. Isolated cells were passaged repeatedly and successfully up to ten passages. The stemness of embryonic cells has been approved by the activity of the alkaline phosphatase, presence of the SSEA—4, and TRA—1—60 protein, and expression of the molecular marker (cPouV, Nanog, and Sox—2) genes. The spontaneous differentiation of chicken ESCs confirmed the pluripotecy of the cells in differentiation into specialized cell lineages. Our observation showed that ESCs can be isolated successfully from stage X blastoderm of Mazandaran native chickens and these cells maintain their stemness properties during multi—passages in vitro.Cellular and molecular biology (Noisy-le-Grand, France) 01/2014; 60(6):8-15. · 1.46 Impact Factor
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ABSTRACT: Background Genes, RNAs, and proteins play important roles during germline development. However, the functions of non-coding RNAs (ncRNAs) on germline development remain unclear in avian species. Recent high-throughput techniques have identified several classes of ncRNAs, including micro RNAs (miRNAs), small-interfering RNAs (siRNAs), and PIWI-interacting RNAs (piRNAs). These ncRNAs are functionally important in the genome, however, the identification and annotation of ncRNAs in a genome is challenging. The aim of this study was to identify different types of small ncRNAs particularly piRNAs, and the role of piRNA pathway genes in the protection of chicken primordial germ cells (PGCs). Results At first, we performed next-generation sequencing to identify ncRNAs in chicken PGCs, and we performed ab initio predictive analysis to identify putative piRNAs in PGCs. Then, we examined the expression of three repetitive sequence-linked piRNAs and 14 genic-transcript-linked piRNAs along with their linked genes using real-time PCR. All piRNAs and their linked genes were highly expressed in PGCs. Subsequently, we knocked down two known piRNA pathway genes of chicken, PIWI-like protein 1 (CIWI) and 2 (CILI), in PGCs using siRNAs. After knockdown of CIWI and CILI, we examined their effects on the expression of six putative piRNA-linked genes and DNA double-strand breakage in PGCs. The knockdown of CIWI and CILI upregulated chicken repetitive 1 (CR1) element and RAP2B, a member of RAS oncogene family, and increased DNA double-strand breakage in PGCs. Conclusions Our results increase the understanding of PGC-expressed piRNAs and the role of piRNA pathway genes in the protection of germ cells.BMC Genomics 09/2014; 15(1):757. · 4.04 Impact Factor
MicroRNA-mediated posttranscriptional regulation is
required for maintaining undifferentiated properties of
blastoderm and primordial germ cells in chickens
Sang In Leea,1, Bo Ram Leea,1, Young Sun Hwanga, Hyung Chul Leea, Deivendran Rengaraja, Gwonhwa Songa,
Tae Sub Parkb, and Jae Yong Hana,2
aWorld Class University Biomodulation Major, Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Korea; andbAvicore
Biotechnology Institute, Optifarm Solution Inc., Gyeonggi-do 435-050, Korea
Edited by George Seidel, Colorado State University, Fort Collins, CO, and approved May 20, 2011 (received for review April 18, 2011)
MicroRNAs (miRNAs) play a critical role in determining the differ-
entiation fate of pluripotent stem cells and germ cells in mammals.
regulation with regard to lineage specification and differentiation
in chick development require further investigation. Therefore, we
conducted miRNA expression profiling to explore specific miRNA
signatures in undifferentiated blastoderm and primordial germ
cells (PGCs). We identified seven miRNAs that are highly expressed
in blastoderm and 10 that are highly expressed in PGCs. In this
study, miR-302a and miR-456 for blastoderm and miR-181a* for
PGCs were analyzed further for their target transcripts and regula-
tory pathways. Both miR-302a and miR-456 bound directly to the
sex-determining region Y box 11 transcript and could act as post-
transcriptional coregulators to maintain the undifferentiated state
of the chicken blastoderm through the suppression of somatic gene
expression and differentiation. Moreover, miR-181a* showed a
bifunctional role in PGCs by binding to two different transcripts.
miR-181a*inhibitedthe somaticdifferentiationofPGCs bysilencing
homeobox A1 expression. Additionally, miR-181a* prevented PGCs
from entering meiosis through the repression of the nuclear recep-
tor subfamily6, groupA,member1 transcript. Collectively, ourdata
demonstrate that in chickens miRNAs intrinsically regulate the dif-
ferentiation fate of blastoderms and PGCs and that the specific
timing of germcell meiosis is controlledthroughmiRNA expression.
ripotent stem cells through in vitro culture (1). During chicken
germline development, primordial germ cells (PGCs) first appear
from the epiblast in the blastoderm and translocate to the hypo-
blast area of the pellucida (2, 3). During gastrulation, PGCs cir-
culate through the vascular system and settle down in the gonadal
anlagen. Such a differentiation pathway, including germ cell lin-
eage during chicken embryo development, is a systematic process,
governed by the concerted action of multiple unknown regulatory
MicroRNAs (miRNAs) are small, noncoding RNAs ranging
from 18 to 23 nucleotides that posttranscriptionally regulate gene
expression in various tissues and cell types. Typically, miRNAs
act as specific regulators of gene expression and are capable of
controlling the fate of cells in a time- and tissue-specific manner
(7, 8) through regulation of cellular differentiation, in addition to
developmental patterning and morphogenesis (9–11). To date,
several miRNA profiles have been classified as ESC-specific
miRNAs, including miR-290–295 and miR-302–367 clusters (12,
13). However, both the miRNA expression profiling and post-
transcriptional gene regulation for lineage specification, com-
mitment,anddifferentiation duringchicken embryo development
remain largely uninvestigated. It has been shown recently that
miRNA biogenesis and specific expression are required for PGC
and germline development of mouse PGCs (14). Such miRNAs
regulate the gain of lineage-specific differentiation in germ cells,
in addition to the loss of pluripotent potential in stem cells.
t stage X, the chicken blastoderm consists of 40,000–60,000
undifferentiated embryonic cells and is able to develop plu-
However, the intricate posttranscriptional network of miRNA
expression for lineage specification and differentiation during
chicken embryo development has yet to be investigated in detail.
Understanding the cellular and molecular mechanism(s) that
underlie the developmental fateofearly embryos is critical for the
practical use of genetic modifications. In the current study, to
identify miRNA signatures for the maintenance of the undif-
ferentiated state of blastoderms and the germline lineage of
chicken PGCs, global miRNA expression profiles using miRNA
expression microarrays were analyzed. The miRNAs that were
significantly expressed in the undifferentiated blastoderm and
chicken PGCs were examined further to investigate the regulatory
pathways of their expression. We demonstrate that posttran-
scriptional regulation through miRNA expression is important
for the control of differentiation in both undifferentiated blas-
toderm and chicken PGCs.
Preparation of PGCs from Embryonic Gonads. To collect purified
chicken PGCs, FACS was performed using a chicken PGC-
specific marker, anti–stage-specific embryonic antigen 1 (anti–
SSEA-1) antibody, and confirmed by staining with anti–SSEA-1
antibody and the periodic acid-Schiff (PAS) reaction, which are
specific to chicken PGCs (15, 16). The percentage of SSEA-1+
and PAS+cells after FACS analysis were 93 ± 1.4% and 96 ±
0.8, respectively, and the viability of the sorted PGCs was 95.0 ±
0.8% (Fig. S1 and Table S1).
Identification of miRNAs Highly Expressed in Stage X Blastoderm and
Chicken PGCs Based on Customized Chicken miRNA Expression
Microarray. To identify certain miRNAs and miRNA clusters
that specifically appear in undifferentiated blastoderm and
chicken PGCs, we compared the miRNA expression profiles of
stage X blastoderms, SSEA-1+PGCs, and chicken embryonic
fibroblasts (CEFs). The hierarchical clustering showed that the
clustered miRNA expression pattern of the undifferentiated
blastoderm was more closely related to PGCs than to CEFs (Fig.
1A). Based on the comparison with CEF expression profiles, we
identified six miRNAs that were specifically up-regulated in
undifferentiated blastoderm and 18 miRNAs that were specifi-
cally up-regulated in chicken PGCs (Fig. 1B and Tables S2 and
S3; Welch’s t test: P < 0.05). Additionally, five miRNAs that were
significantly expressed in both stage X blastodermal cells and
PGCs were identified and selected (Table S4).
Author contributions: S.I.L., B.R.L., and J.Y.H. designed research; S.I.L., B.R.L., Y.S.H., H.C.L.,
and D.R. performed research; S.I.L., B.R.L., G.S., T.S.P., and J.Y.H. analyzed data; and S.I.L.,
B.R.L., D.R., G.S., T.S.P., and J.Y.H. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1S.I.L. and B.R.L. contributed equally in this work.
2To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| June 28, 2011
| vol. 108
| no. 26www.pnas.org/cgi/doi/10.1073/pnas.1106141108
Validation of Selected miRNAs by Quantitative Real-Time PCR
Analyses. To verify the expression of miRNAs identified by the
microarray, we performed real-time PCR using undifferentiated
blastoderm, PGCs, gonadal stroma cells (GSCs), and CEFs. In
candidate stage X-specific miRNAs, the expression levels of miR-
other tissues (Fig. 2A). In chicken PGCs, 10 of the 18 miRNAs
were confirmed to be predominantly expressed (Fig. 2B). Finally,
when the expression levels of five miRNAs specifically expressed
in both undifferentiated blastoderm and PGCs were examined by
real-time PCR, all were highly expressed in the stage X blasto-
derm and PGCs compared with somatic cells such as CEFs and
GSCs. Interestingly, the expression levels of five miRNAs were
higher in the undifferentiated blastoderm than in the chicken
PGCs (Fig. 2A). We therefore classified these five miRNAs as
miRNAs that were highly expressed in the undifferentiated
blastoderm. Based on the miRNA microarray and real-time PCR
analysis, we identified seven miRNAs that were highly and spe-
cifically expressed in the chicken stage X blastoderm and 10
miRNAs that were highly and specifically expressed in PGCs.
Knockdown of miR-302a and miR-456 in Undifferentiated Chicken
Blastodermal Cells. Among stage X miRNAs, the miR-302 cluster
acts as a critical regulator for pluripotency through regulation of
the cell cycle and methylation of related genes in mammals. miR-
456 has been characterized in chickens but not in mammals. Also,
target binding sites of miR-302a and miR-456 were localized
to the sex-determining region Y box 11 (SOX11) 3′ UTR based
on computational prediction of target miRNA. To examine the
function of the miRNAs in the maintenance of the undifferen-
tiated state in early development, miR-302a and miR-456 were
selected, and their knockdown probes were introduced into un-
differentiated blastodermal cells. Both knockdown probes were
able to silence target miRNA expression significantly. The knock-
down of miR-302a and miR-456 resulted in a significant decrease,
61.0 ± 0.3% (P < 0.001) and 52.0 ± 1.8% (P < 0.01), respectively,
compared with knockdown of control miRNA (Fig. 3A). Second,
we validated changes in the expression levels of pluripotency-
related genes [POU domain class 5 transcription factor 1 (POUV)
and nanog homeobox (NANOG)] and germ cell-associated genes
[deleted azoospermia-like (DAZL) and DEAD box polypeptide 4
(DDX4)]. The knockdown of both miR-302a and miR-456 signifi-
cantly decreased the expression of pluripotency-related genes
POUV (miR-302a, P < 0.05, and miR-456, P < 0.05) and NANOG
(miR-302a, P < 0.05, and miR-456, P < 0.01) but not the germ cell
of somatic genes including brachyury homolog (BRACHYURY),
fibroblast growth factor 8 (FGF8), snail homolog 1 (SNAI1), and
SOX11 were analyzed in the knockdown blastodermal cells. The
expression levels of all somatic genes except SNAI1 were signifi-
than in the control miRNA (Fig. 3C).
We also examined the functional activity of miR-302a and
miR-456in chicken PGCs using the same knockdown probes. The
efficacy of both knockdown probes in silencing target miRNA
was similar to that of blastodermal cells. Interestingly, like blas-
todermal cells, effects of the knockdown of miR-302a and miR-
456 in chickens were similar to those of blastodermal cells (Fig.
S2). These results indicate that silencing both miR-302a and
miR-456 not only induces a decrease in the expression of pluri-
potency-related genes but also increases expression of somatic
genes in both undifferentiated blastodermal cells and PGCs.
Knockdown of miR-181a* in Chicken PGCs. Germ cell specification
involves molecular events that are programmed by somatic cells.
For example, the homeobox (HOX) and mesoderm genes are
repressed by transcriptional regulators such as B-lymphocyte-
array. (A) Hierarchical clustering of the chicken miRNA expression microarray.
Clustering was performed from the normalized expression data of all probes.
Each row represents the relative gene expression of a single miRNA. C, CEF; P,
PGCs; S, Stage X. High expression levels of miRNAs compared with the mean
are indicated in red, low expression levels in blue, and mean expression levels
in yellow. Clustering results are displayed using the TreeView software. (B)
Venn diagram illustrations of miRNA increases in SSEA+chicken PGCs, stage X
blastoderm cells, and CEFs from the customized chicken miRNA expression
microarray. The common genes from the miRNA microarray experiments
were up-regulated at least twofold (P < 0.05) in each cell type.
Data analysis of the customized chicken miRNA expression micro-
expressed in undifferentiated blastoderm cells (A) and PGCs
(B). Real-time PCR analysis was conducted in triplicate and
normalized to the expression levels of small nucleolar RNA
(snoRNA). miRNA expression was compared among stage X
blastodermal cells, PGCs, GSCs, and CEFs. PGCs, GSCs, and
CEFs were retrieved from 6-d-old embryos (stage 29). Error
bars indicate SE of triplicate analyses.
Quantitative expression analysis of miRNAs highly
Lee et al.PNAS
| June 28, 2011
| vol. 108
| no. 26
induced maturation protein 1 (Blimp1). We hypothesized that
miR-181a* was a candidate posttranscriptional regulator because
the binding site of miR-181a* was localized to the homeobox A1
(HOXA1) 3′ UTR, based on target prediction. To investigate the
function of miR-181a* in chicken PGCs, the miR-181a* tran-
script was silenced, and the expression levels of germ cell-related
genes were analyzed (Fig. 3D). In contrast to the knockdown
of miR-302a and miR-456, miR-181a* silencing specifically de-
creased the expression levels of DAZL and DDX4; however, no
change was detected in the expression of POUV or NANOG (Fig.
3E). Additionally, miR-181a* silencing dramatically up-regulated
the expression levels of all the somatic genes (BRACHYRURY,
FGF8, SNAI1, and SOX11) in chicken PGCs (Fig. 3F). These
results indicate that miRNA181a* is involved in the regulation of
germ cells but not pluripotent cells.
Specific Analysis of the Target Gene Transcript for the Regulatory
Roles of miR-302a and miR-456. To examine the posttranscriptional
gene regulation of miR-302a and miR-456, we validated the
binding activity and down-regulation of the miRNA target site
using the 3′ UTRs of the SOX11 transcript, which was predicted
to be a miR-302a and miR-456 target transcript using the com-
putational miRNA target prediction database TargetScan (Fig.
4A; http://www.targetscan.org). We also subcloned the 3′ UTR of
the SOX11 transcript to generate a reporter–target construct in
which the expression of a GFP transcript was coupled with the
miRNA target 3′ UTR (Fig. 4B). To perform a dual fluorescence
reporter assay, coexpression vectors containing Discosoma sp.
red fluorescent protein (DsRed) and miR-302a or miR-456 were
constructed (Fig. 4B). After cotransfection of SOX11 eGFP-3′-
UTR and DsRed-miRNA for miR-302a or miR-456, GFP and
DsRed expression were analyzed by fluorescence microscopy and
FACS (Fig. 4C). The percentage of GFP+cells (55.3% in control
vs. 23.16% in miR-302a and 26.08% and miR-456) and the
density of GFP fluorescence decreased in miR-302a– or miR-
456–transfected cells compared with control samples. These
results indicate that both miR-302a and miR-456 bind directly to
the 3′ UTR of the SOX11 transcript and regulate SOX11 func-
tion during somatic cell differentiation.
Specific Analysis of the Target Gene Transcript for miR181a*. To
analyze the miR-181a* target transcript, we predicted the target
gene(s) of miR-181a* from a computational target prediction
database (TargetScan; Fig. 4D; Gene Expression Omnibus: GSE
15830) (Table S5). From this analysis, both HOXA1 (a somatic
gene involved in somatic cell differentiation) and nuclear re-
miR-456 in undifferentiated blastoderm cells and after knockdown of miR-
181a* in chicken PGCs. (A) Relative miRNA expression in stage X blastoderm
(B) Relative expression of pluripotent genes (POUV and NANOG) and germ
cell-related genes (DAZL and DDX4) in stage X blastoderm cells after trans-
fection of the knockdown probe for miR-302a or miR-456. (C) Relative ex-
pression of somatic genes (BRACHYRURY, FGF8, SNAI1, and SOX11) in stage X
blastoderm cells following transfection of the miR-302a or miR-456 knock-
down probes. (D) Relative miRNA expression in PGCs after miR-181a* si-
lencing. (E) Relative expression of pluripotent genes and germ cell-related
genes in PGCs after miR-181a* knockdown. (F) Relative expression of somatic
genes in PGCs after miR-181a* silencing. Noncomplementary sequences in
the chicken genome were used as a control. real-time PCR was conducted in
triplicate and normalized to control expression of snoRNA (A and D) or
groups are indicated as ***P < 0.001, **P < 0.01, and *P < 0.05. Error bars
indicate the SE of triplicate analyses.
Quantitative expression analysis after knockdown of miR-302a and
vector maps for eGFP with SOX11 3′ UTR and DsRed with each miRNA. The 3′ UTR of the SOX11 transcript was subcloned between the eGFP gene and the
polyA tail to generate the fusion construct of the GFP transcript following the miRNA target 3′ UTR (pcDNAeGFP3′UTR) (Upper), and the miRNA expression
vector was designed to coexpress DsRed and each miRNA (pcDNADsRedmiRNA) (Lower). After cotransfection of pcDNA-eGFP-3′-UTR for the SOX11 transcript
and pcDNA-Ds-Red miRNA for miR-302a or miR-456, the fluorescence signals of GFP and DsRed were detected using fluorescence microscopy (Right) and FACS
(C). (D) Diagram of miR-181a* binding site in the HOXA1 3′ UTR. (E) The expression vector map for eGFP with the HOXA1 3′ UTR, DsRed with miR-181a*, and
the fluorescence signals of GFP and DsRed using fluorescence microscopy. (F) After cotransfection of pcDNA-eGFP-3′-UTR for the HOXA1 transcript and
pcDNA-DsRed-miRNA for miR-181a*, the fluorescence signals of GFP and DsRed were detected using FACS.
In vitro target assay of miR-302a, miR-456, and miR-181a*. (A) Diagram of miR-302a and miR-456 binding sites in the SOX11 3′ UTR. (B) Expression
| www.pnas.org/cgi/doi/10.1073/pnas.1106141108 Lee et al.
ceptor subfamily 6, group A, member 1 (NR6A1 (a gene involved
in germ cell differentiation) were selected and analyzed further.
After the cotransfection of HOXA1-eGFP-3′UTR and DsRed-
miRNA-181a* (Fig. 4E), the percentage of GFP+cells (73.66% in
control vs. 49.17% in miR-181a*) decreased, compared with the
controls (Fig. 4 E and F). To assess NR6A1, which is involved in
retinoicacid(RA)signaling,a criticalregulator formeiosisinearly
germ cells, we analyzed the changes in meiotic gene expression in
chicken PGCs after RA treatment. The mRNA expression of
meiotic genes including synaptonemalcomplexprotein3 (SYCP3),
stimulated by retinoic acid gene 8 homolog (STRA8), and NR6A1
increased gradually as the incubation time with RA increased;
however, miR-181a* levels were down-regulated (Fig. 5A). This
result suggested that miR-181a* could be closely related to the
regulatory pathway of meiotic differentiation in chicken PGCs,
through down-regulation of NR6A1. Interestingly, a binding site
for miR-181a* was located in the 3′ UTR of the NR6A1 transcript
(Fig. 5B). Based on these results, we generated a reporter–target
construct in which the eGFP transcript accompanied the miR-
181a* target 3′ UTR and a coexpression vector containing the
DsRed gene and miR-181a* for dual fluorescence reporter assays
(Fig. 5B). We observed that the percentage of GFP-expressing
cells (75.56% in control vs. 38.70% in miR-181a*) was reduced by
the cotransfection of miR-181a*, compared with controls (Fig. 5 B
and C). Collectively, these results indicate that miR-181a* bound
directly to the 3′ UTR where it regulated NR6A1 function during
cell differentiation or meiosis.
Real-Time PCR Analysis of miR181a* and NR6A1 During Chicken
Embryonic Gonad Development. To confirm the functional rela-
tionship between miR181a* and NR6A1 in vivo, the expression
patterns of miR181a* and NR6A1 transcripts were analyzed by
real-time PCR during chicken embryonic gonad development.
In the female embryo, miR181a* expression was gradually up-
regulated by embryonic day (E) 15.5 during development, and the
NR6A1 transcript levels markedly increased from E13.5 when
chicken PGCs entered meiosis (Fig. 5D). In the male embryonic
gonads, the miR181a* transcript increased between E13.5 and
E17.5,andNR6A1 expression wasunchangedat thesetimepoints.
To confirm the functional relationship between miR181a* and
NR6A1 in vitro, expression patterns were analyzed by NR6A1 si-
lencing and overexpression. In contrast to knockdown of miR-
181a*, NR6A1 silencing specifically decreased the expression
levels of mir-181a* and meiotic genes (SYCP3 and STRA8), al-
though no change was detected in the expression of DAZL or
DDX4 (Fig. S2 D–G and Table S6). NR6A1 overexpression spe-
cifically decreased the expression levels of pluripotency-related,
germ cell-associated, and somatic genes, although increased ex-
pression of meiotic genes was detected (Fig. S2 H–K). These data
indicatethatmiR181a* andNR6A1regulate eachother negatively
during germ cell development.
Functions of miR-181a* in Meiotic Differentiation of Germ Cells. To
confirm the function of miR-181a* during meiotic events, the ex-
pression patterns of meiotic transcripts were analyzed by knocking
down miR-181a* during RA-induced meiotic differentiation of
germ cells. Without RA, miR-181* knockdown increased expres-
sion levels of meiotic transcripts (STRA8, NR6A1) except for
SYCP3, but miR-181a* silencing dramatically induced all meiotic
transcripts including SYCP3 during RA-induced meiotic differen-
interaction between miR-181*and other pathways regulating gene
expression, SYCP3 protein expression was analyzed. SYCP3 pro-
tein was increased by miR-181a* knockdown in chicken PGCs. In
181a* in chicken PGCs. (A) Expression levels of the meiotic genes, including SYCP3, STRA8, and NR6A1, were analyzed in chicken PGCs 0–72 h after RA treatment.
Expression of endogenous miR-181a* in PGCs was down-regulated after RA treatment. (B) Diagram of the miR-181a* binding site in the NR6A1 3′ UTR and the
expressionvector maps foreGFP with NR6A13′ UTRand DsRed withmiR-181a*. Aftercotransfection ofpcDNA-eGFP-3′-UTRfortheNR6A1transcriptandpcDNA-
DsRed-miRNA for miR-181a*, the fluorescence signals of GFP and DsRed were detected using fluorescence microscopy and FACS (C). (D) Quantitative expression
patternofmiR181a*and NR6A1inmale andfemalechicken embryonic gonadsfrom E9.5toE17.5. Errorbarsindicatethe standarderror(SE)oftriplicate analysis.
(E) Relative expression of SYCP3, STRA8, and NR6A1 after silencing with the knockdown probes for miR-181a* at 72 h, followed by RA treatment in chicken
PGCs. *P < 0.05, **P < 0.01, and ***P < 0.001: significant difference compared with control. (F) Expression of SYCP3 protein after silencing with the knockdown
probes for miR-181a* at 72 h, followed by RA treatment in chicken PGCs examined by Western blotting. OE, overexpression, KD, knockdown.
In vitro target assay of miR-181a* on the NR6A1 transcript and a quantitative expression analysis of meiotic genes following the knockdown of miR-
Lee et al. PNAS
| June 28, 2011
| vol. 108
| no. 26
RA-induced meiotic differentiation of germ cells, SYCP3 protein
was increased by miR-181a* silencing and NR6A1 overexpression.
However, SYCP3 protein decreased with miR-181a* overex-
pression and in NR6A1 knockdown cells (Fig. 5F).
miRNAs function as major regulators and pivotal determinants
in numerous critical cellular processes (7, 17, 18). Several studies
and dynamic expression patterns of miRNAs in many organisms
and multiple tissues (19–21). In this study, a chicken miRNA ex-
pression microarray was developed that contained 479 miRNA
sequence probes based on the miRBase Sequence database, ver-
sion 14 (Sanger Institute). The array data obtained were consistent
and highly reproducible and may be useful for global miRNA ex-
pression analyses in a variety of avian species.
Based on the chicken miRNA microarray and qRT-PCR anal-
yses, we identified seven miRNAs and undifferentiated chicken
stage X blastoderm and 10 miRNAs that were highly expressed in
PGCs. Interestingly, five of these miRNAs were selected as can-
didate miRNA signatures specifically expressed in both stage X
blastoderm and chicken PGCs. qRT-PCR analyses also confirmed
that the five miRNAs were highly expressed in the stage X blas-
toderm and PGCs compared with somatic cells, CEFs, and GSCs.
However, the expression levels of the five miRNAs in undiffer-
entiated blastoderm cells were increased compared with their
levels in chicken PGCs (Fig. 2A). Thus, these five miRNAs (miR-
302a, miR-302b, miR-302b*, miR-302c, and miR-302d) were
classified as miRNAs that were highly expressed in the undiffer-
entiated blastoderm (Table S4). The miR-302 cluster is known to
contain critical stem cell-specific miRNAs that maintain an un-
differentiated state in ESCs (13). To evaluate the putative func-
tions of these miRNAs in the undifferentiated state of chicken
embryonic cells, miR-302a and miR-456 were silenced. This si-
but not of the germ cell-associated genes (Fig. 3B). Additionally,
somatic gene expression increased significantly in miR-302a
and miR-456 knockdown cells (Fig. 3C), and the knockdown of
miR302a in chicken PGCs showed similar expression patterns
(Fig. S2). These results indicate that the expression of the miR-
302 cluster and miR-456 are stem cell markers in chicken as well
as in mammals and may prevent stage X blastodermal cells and
PGCs from differentiating into the somatic cell lineages.
In contrast to other somatic cell lineages, PGCs maintain
functional characteristics similar to those of the undifferentiated
germ cells, has been derived and established from PGCs in
mammals and chickens (22–24). Thus, the miR-302 cluster that is
highly expressed in undifferentiated blastodermal cells may be up-
regulated in chicken PGCs, even though it is expressed at lower
levels than in the stage X blastoderm. In this study, the miRNA-
17–92 cluster was expressed predominantly in chicken PGCs (Fig.
miR-17–92 cluster is highly expressed in mouse and human ESCs
and many cancerous tissues and has a seed sequence similar to
ESC-specific cell-cycle miRNAs (25–29). However, the chicken
miRNA-92 cluster was specifically expressed in PGCs but not in
undifferentiated blastoderm cells (Fig. 2 A and B). Recently, it has
been reported that the expression of miR-17–92 affects the bal-
ance between ESC self-renewal and differentiation (30–32). Thus,
specific miRNAs and potential miRNA targets of the miRNA-92
in chicken PGCs but not in undifferentiated blastodermal cells. In
the current study, we also observed that miR-181a* was expressed
specifically in chicken PGCs. In knockdown experiments, we hy-
pothesized that silence of miR-181a* altered the expression net-
works that could cause cellular differentiation, apoptosis, and
proliferation. Interestingly, miR-181a* silencing specifically de-
creased the expression levels of the germ cell-related genes.
However, no change in the expression of the pluripotency-re-
lated genes was observed (Fig. 3E), although miR-181a*
knockdown markedly increased the expression of all somatic
genes in chicken PGCs (Fig. 3F). These results indicate that
miR181a* in chicken PGCs not only represses somatic differen-
tiation through the silencing of somatic gene transcripts but also
promotes germ cell differentiation. miR181a* appears not to be
involved inthe regulationofpluripotency-related geneexpression
in chicken PGCs. However, further investigation is necessary for
miRNA interactions for maintaining the properties of germ cells
and pluripotent cells.
the up-regulation of somatic gene expression in undifferentiated
blastoderm and PGCs. Thus,we investigated further the biological
pathways governed by these chicken miRNAs. For candidate tar-
get somatic genes, SOX11 3′ UTR for miR-302a and miR-456 and
HOXA1 3′ UTR for miR-181a* were predicted using a compre-
hensive database for predicting target miRNAs. In cotransfection
experiments using the eGFP-3′ UTR and miRNA, the number of
GFP+cells and the density of green fluorescence decreased, sug-
gesting that each miRNA bound directly to the target transcript 3′
UTR and silenced its expression. These results indicate that the
miRNAs prevent stage X blastodermal cells and PGCs from dif-
ferentiating into somatic cell lineages with somatic gene silencing.
Collectively, to maintain an undifferentiated state, ESCs, stage X
blastodermal cells, and germ cells not only activate pluripotency-
related genes but also repress somatic genes (Fig. 6).
In mice, the germline is directly specified from the epiblast,
and germ cell specification requires bone morphogenic protein
signaling from the extraembryonic ectoderm and visceral endo-
derm (33–36). As a critical determinant of the germ cell lineage
in mice, Blimp1 interacts with protein arginine methyltransferase
5 (Prmt5), an arginine-specific histone methyltransferase, and the
Blimp1–Prmt5 complex represses the somatic gene expression of
HOX genes and downstream targets during the formation of the
initial PGC population (37). Similar to these reports, we found
that the somatic HOXA1 transcript was a target of miR-181a*,
which down-regulated its expression in chicken PGCs (Fig. 4).
Taken together with knockdown experiments and functional
analyses of the miR181a* target transcript, we propose that these
regulation of miR-302a, miR-456, and miR-181a*. miR181a* prevents chicken
PGCs from entering meiosis by downregulating the NR6A1 transcript that
triggers the meiosis process in chicken PGCs through RA signaling from go-
chickenPGCs by silencing thesomatic gene expression,includingHOXA1. Both
miR-302a and miR-456 regulate the pluripotency in chicken blastodermal cells
and PGCs by silencing the somatic gene expression, including SOX11.
Schematic illustrating the current working hypothesis regarding the
| www.pnas.org/cgi/doi/10.1073/pnas.1106141108Lee et al.
miRNAs play important roles in posttranscriptional gene regu-
lation associated with the suppression of somatic differentiation
during early germ cell development in chickens.
In the current study, we confirmed the function of another
miR-181a* target transcript, NR6A1, which is related to germ cell
development. NR6A1 is involved in RA signaling and is a critical
regulator of meiosis in developing germ cells (38). Interestingly,
after the treatment of chicken PGCs with RA, we found that
the expression levels of meiotic genes as well as NR6A1 were
increased dramatically in accordance with the incubation time
with RA (Fig. 5A). Additionally, the in vivo expression profiles of
miR-181a* and NR6A1 transcripts showed an inverse relation-
ship during chicken embryonic gonad development (Fig. 5D). In
the female embryo, miR181a* expression was down-regulated
gradually as female embryonic gonads developed; however, the
NR6A1 transcript was increased dramatically, particularly around
day 13.5. Smith et al. (39) reported that germ cells in the chicken
female gonad initiate meiosis at around E12.5, at which time
STRA8 is up-regulated in the female left gonad. In contrast to the
female embryo, miR181a* was highly expressed in male embry-
onic gonads; however, NR6A1 expression was suppressed after
E13.5 (39). Thus, we propose that miR-181a* and NR6A1 could
be closely related to the regulatory pathways of meiotic differ-
entiation in chicken PGCs. The meiotic pathway was suppressed
by silencing the NR6A1 transcript (Fig. 6). In addition to the
repression of the NR6A1 meiotic gene and HOXA1 somatic gene
transcript, miR-181* may modulate the different regulatory
pathway(s) for meiosis in chicken early germ cells. The mecha-
nisms by which miR-181a* and NR6A1 regulate germ cell dif-
ferentiation in chicken thus require further investigation (Fig. 6).
Here we report miRNA expression profiling and miRNA
functional analysis in the stage X blastoderm and PGCs in
chickens. Furthermore, we demonstrated that the germ cell
differentiation pathway of meiosis initiation is controlled in a
time-dependent manner by these miRNAs. Collectively, our data
indicate that miRNAs are key posttranscriptional regulators for
the control of differentiation of both undifferentiated blasto-
dermal cells and germ cells in chickens.
Materials and Methods
The preparation of PGCs and additional methods are described in SI Material
Design of Chicken miRNA Expression Microarray and Expression Profile
Analysis. We developed chicken miRNA expression chips (AMDID 027206;
Agilent) that contained 479 miRNA sequence probes, based on the miRBase
database, version 14 (Sanger Institute). For miRNA microarray analysis, total
Labeling and Hyb Kit (Agilent Technologies) and then was placed on the
chicken miRNA v.14 chips (AMDID 027206; Agilent). Slides were hybridized
for 16 h at 42 °C in the Agilent hybridization system and washed with
0.0005% Triton X-102 for 5 min followed by a second washing for 5 min. The
slides then were centrifuged and dried at room temperature.
miRNA expression profiles were analyzed using the GeneSpring GX v.11
(Agilent Technologies). All data were analyzed with the standard normali-
zation method for one-channel microarrays, namely, percentile median
normalization. Fold-change values were calculated for unpaired comparisons
between normal and treated groups. The fold-change filters included the
requirement that miRNAs should be increased at least 150% compared with
the controls for the up-regulated miRNAs and decreased by 66.67% com-
pared with the controls for the down-regulated miRNAs. Welch’s t test was
performed to identify significant (P < 0.05) changes in expression.
ACKNOWLEDGMENTS. This work was supported by a grant from the The
Next Generation BioGreen 21 Program (No. PJ008142), Rural Development
Administration, and by Grant R31-10056 from the World Class University
Program through the National Research Foundation of Korea funded by the
Ministry of Education, Science and Technology, Republic of Korea.
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| June 28, 2011
| vol. 108
| no. 26