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: firstname.lastname@example.org.
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