Biochemical and Biophysical Research Communications 306 (2003) 1008–1013

www.elsevier.com/locate/ybbrc

BBRC

Yin Yang 1, a vertebrate Polycomb group gene, regulatesantero-posterior neural patterning

Hye-Joo Kwon and Hae-Moon Chung*

School of Biological Sciences, Seoul National University, San 56-1, Shillim-dong, Kwanak-gu, Seoul 151-747, Republic of Korea

Received 20 May 2003

Abstract

Polycomb group (PcG) genes are required for the stable repression of the homeotic genes and other developmentally regulated

genes. Yin Yang 1 (YY1), a vertebrate homolog of the Drosophila PcG pleiohomeotic (Pho), is a multifunctional protein that can act

as a repressor or activator of transcription. Xenopus YY1 (XYY1) protein was localized in the central nervous system (CNS),

particularly anterior neural tube of tailbud stage embryos. To elucidate the role of endogenous XYY1, loss-of-function studies were

performed using XYY1 antisense morpholino oligonucleotide (XYY1 MO). Inhibition of XYY1 function resulted in embryos with

antero-posterior axial patterning defects and reduction of head structures. XYY1 MO also reduced the expression of En2, a mid-

brain/hindbrain junction marker, which was rescued by co-injection of XYY1 mRNA. However, XYY1MO-injection did not affect

the expression of HoxB9, a spinal cord marker. These results suggest that YY1 controls antero-posterior patterning of the CNS

during Xenopus embryonic development.

� 2003 Elsevier Science (USA). All rights reserved.

Keywords: Yin Yang 1; Xenopus; Polycomb group genes; Antisense morpholino oligonucleotide; Antero-posterior patterning; CNS

The Polycomb group (PcG) is an important, widely

conserved group of transcriptional repressors best

known for their function in stably maintaining the in-

active expression patterns of key developmental regu-

lators [1]. They were first identified in Drosophila as a

group of genes required for maintenance of correct ex-

pression patterns of homeotic genes [2,3]. In recentyears, PcG homologs have also been found in verte-

brates [4], such as mammals, chick, and Xenopus [5–14]

and the developmental functions of the PcG appear to

be conserved between Drosophila and vertebrates [15].

Yin Yang 1 (YY1), a homolog of the Drosophila

PcG gene pleiohomeotic (pho) product, is a zinc finger-

containing transcription factor highly conserved among

animal species. Unlike other PcG genes, YY1 is amultifunctional protein that can act as a transcriptional

repressor, an activator, or an initiator as its name re-

flects [16,17]. Little is known about the role of YY1 in

* Corresponding author. Fax: +82-2-872-1993.

E-mail address: hmchung@snu.ac.kr (H.-M. Chung).

0006-291X/03/$ - see front matter � 2003 Elsevier Science (USA). All rightsdoi:10.1016/S0006-291X(03)01071-4

vertebrate embryonic development. Targeted disruption

of mouse YY1 results in embryonic lethality, suggest-

ing that it is essential for mammalian embryonic de-

velopment [18]. The Xenopus homolog of YY1 (XYY1)

has been isolated from oocyte cDNA library [7].

Abundant levels of XYY1 mRNA and protein are de-

tected in all stages of oocyte and in the subsequentstages of embryonic development through the swim-

ming larval stages [19], and XYY1 is a component of

mRNP particles in the cytoplasm of oocytes [20].

XYY1 interacts with EED PcG protein and overex-

pression of XYY1 mRNA in early embryo directly

induces neural tissue but is unable to induce meso-

dermal tissues [13].

In this paper, we used a different experimental ap-proach based on antisense morpholino oligonucleotides

(MOs) to gain new insights into the precise roles of

XYY1 during Xenopus embryogenesis. Our results pro-

vide evidences to suggest that vertebrate YY1 is re-

quired for antero-posterior neural patterning in early

embryonic development.

reserved.

Fig. 1. Spatial distribution of YY1 protein in Xenopus embryo. XYY1

proteins were analyzed by immunostaining. (A,C) Lateral views of late

tailbud stage embryos (stage 32 (A) and 36 (C)). (B) Neural tube (top)

and notochord (bottom) isolated from (A). Both are oriented anterior

to posterior, left to right. There is strong staining in the anterior region

of the neural tube and eye. (D) Tailbud stage embryo stained with

MZ15, notochord specific antibody. (E) Neural tube and notochord

isolated from (D). (F) No primary antibody as a negative control. nt,

neural tube; nc, notochord; ev, eye vesicle.

H.-J. Kwon, H.-M. Chung / Biochemical and Biophysical Research Communications 306 (2003) 1008–1013 1009

Materials and methods

Embryonic manipulations. Xenopus laevis embryos were obtained

after artificial induction of mating by injecting 150 and 350 IU of

human chorionic gonadotropin (LG PhD) to the male and female,

respectively. Fertilized eggs were dejellied by swirling gently in 2%

(w/v) cysteine solution (pH 8.0) and reared in 0.1� Marc�s modifiedRinger�s solution (MMR). Staging was done according to Nieuwkoopand Faber [21].

Whole-mount immunostaining. Embryos were fixed with MEMFA

(0.5M Mops, pH 7.4, 100mM EGTA, 1mM MgSO4, and 4% form-

aldehyde), rehydrated through a graded series of alcohols, and trans-

ferred to bleaching solution (15% H2O2 in PBS). Bleached embryos

were washed in PBS and incubated in PBT (PBS with 2mg/ml BSA

and 0.1% Triton X-100) with continuous rotation. Primary antibodies

were rabbit anti-human YY1 polyclonal. (Santa Cruz, sc-281. The

amino acid sequence alignment with peptide mapping at the carboxy

terminus of YY1 used for the antibody is 100% homologous between

the human and Xenopus YY1. Its cross-reactivity and specificity were

confirmed by Western blot analysis of Xenopus embryonic lysates.) The

YY1 antibody was used at 1:500 dilution. Incubation with the sec-

ondary antibody for detection of YY1, alkaline phosphatase-conju-

gated goat anti-rabbit IgG (Fc), was performed at 1:500 dilution. As a

substrate for color development, NBT and BCIP were used. MZ15

antibody was a generous gift from F.M. Watt [22].

Microinjection of XYY1 MO and mRNA. Antisense morpho-

lino oligonucleotides (50-CCAGCTCAGTTTCCCCCCTCAGTTT-30)

complementing the 50 UTR region of the XYY1 mRNA were designed

to block translation. The control morpholino was the standard control

morpholino (50-CCTCTTACCTCAGTTACAATTTATA-30) supplied

by Gene Tools, LLC. MOs were solubilized in CMFM [23] at the

concentration of 1mM (�8.3mg/ml) and the resulting stock was di-luted to working concentrations of 0.05–0.5mM. XYY1 mRNA was

synthesized from pT7TsYY1 plasmid (a gift of E. Beccari) containing

the entire translated region of the Xenopus YY1 cDNA, linearized with

BamHI, using T7 RNA polymerase. All mRNAs used for microin-

jection were capped with 50 7MeGpppG 50 cap analog (Stratagene) by

in vitro transcription. The injection volume of XYY1 mRNA or XYY1

MO was 4.6 nl into each blastomere at the concentration indicated.

Embryos for microinjection were transferred to 0.5�MMR containing5% Ficoll and injected at the two-cell stage. After injection, embryos

were kept in the same solution for 3 h and then cultured in 0.1�MMRuntil they reached appropriate stages.

Quantitative RT-PCR analysis. Total RNA (1lg) extracted fromwhole embryos was used for each RT-PCR. XYY1 primers were 50-

CTCAGACGACTTGGTCCATCC-30 (forward) and 50-CCACTC

AGGTAGCTTTTCTTGC-30 (reverse). Other primers were as de-

scribed previously [24] or obtained from the Xenopus Molecular

Marker resource/Xenbase Web sites. Cycle number and the template

input were determined empirically in each case, within the linear range

of amplification. PCR products were electrophoresed on 1.7% agarose

gels, post-stained with Vistra Green (Amersham), directly scanned

using FLA 2000 FluoroImager (FujiFilm), and quantified by Image

Gauge v.3.12 software (FujiFilm). All RT-PCRs were repeated at least

three times for every set of primers.

Results and discussion

Spatial distribution of XYY1 protein in Xenopus embryos

The spatial distribution of XYY1 protein was deter-

mined by whole-mount immunostaining with YY1 an-

tibody (Fig. 1) In early embryos, XYY1 protein was

present in the whole embryo ubiquitously; it was hard to

find out localized expression of XYY1 (data not shown).

The expression became restricted to the dorsal region as

neurulation proceeded and subsequently localized to the

neural tube and anterior structures at tailbud stages(Fig. 1A). At a more advanced stage, it was highly

maintained in the central nervous system (CNS) and

faint staining was also observed in the somites (Fig. 1C).

To define the regional expression pattern of XYY1

protein in the central nervous system, neural tube was

isolated from the immunostained embryo. As seen in

Fig. 1B, XYY1 was predominantly expressed in the

antero-dorsal neural tube, developing brain includingeye vesicle. In contrast, there was relatively weak signal

in the posterior neural tube, spinal cord and no signal in

the notochord. In mouse embryo, YY1 protein is ex-

pressed in the developing midbrain, hindbrain, and

cerebellar primordia [18]. These results suggest that

vertebrate YY1 may have a role in specific region of

central nervous system during embryonic development.

Phenotype of X. laevis embryos after knockdown of

XYY1 function by antisense morpholino oligonucleotide

To determine whether the XYY1 is essential for early

development in Xenopus, we used XYY1 antisense

morpholino oligonucleotide (XYY1 MO) to knock

down endogenous XYY1. Antisense morpholino oligos

have proven an effective means of specifically reducing

or eliminating gene function in Xenopus and zebrafish[25,26]. These highly stable modified oligos act by

blocking translation of targeted mRNAs [27]. Various

concentrations of XYY1 MOs designed against 50 UTR

Fig. 2. Phenotypic effects of loss-of-XYY1 function. Embryos were injected at the two-cell stage into each blastomere with the control morpholino

(Control MO; A, C, E) or XYY1-specific antisense morpholino oligonucleotide (YY1 MO; B, D, F) Injected embryos were fixed when sibling

embryos reached stage 12.5 (A,B), stage 32 (C,D), and stage 41 (E,F). At 14.5 h post-fertilization, control MO-injected embryo was at stage 12.5,

small yolk plug stage (A), while XYY1 MO-injected embryo resembled normal embryos at stage 12, medium yolk plug stage (B). (D,F) XYY1 MO

injection severely affects head structures and A-P axis patterning.

1010 H.-J. Kwon, H.-M. Chung / Biochemical and Biophysical Research Communications 306 (2003) 1008–1013

of the Xenopus YY1 gene were injected into each blas-

tomere at the two-cell stage embryo. In the early em-

bryonic stage, most of the XYY1 MO-injected embryos

Fig. 3. Effects of the XYY1MO-injection on the expression of neural marker

embryos (lane 2), and uninjected embryos (lane 4) by quantitative RT-PCR as

blastomeres at the two-cell stage (3.8 ng/cell). Minus RT ()RT) indicates PCRconstitutively expressed ornithine decarboxylase (ODC) transcript was used fo

had no noticeable effect on survival or morphology even

with high-dose (76 ng) injection. During gastrulation,

these XYY1 MO-injected embryos involuted properly

s. (A,B) Molecular analysis of control MO (lane 1), XYY1MO-injected

say at stage 13.5 (A) and stage 19 (B). XYY1MOwas injected into both

amplification without reverse transcriptase as a negative control. The

r normalizing results (n ¼ 3 independent experiments; error bars, SEM).

Fig. 4. Rescue of XYY1-MO induced effects by the injection of XYY1

mRNA. (A,B) XYY1 MO (38ng) was rescued by coinjection with

XYY1 mRNA (1 ng). Lateral views of standard control MO-, XYY1

MO+XYY1mRNA-injected embryos at stage 32 (A) and stage 41 (B).

(C) RT-PCR analysis of stage 13.5 embryo samples. Note the restored

expression of En2 gene in embryos injected with 7.6 ng XYY1MO and

500 pg XYY1 mRNA (lane 3) compared with XYY1 MO-injected

embryos (lane 2).

H.-J. Kwon, H.-M. Chung / Biochemical and Biophysical Research Communications 306 (2003) 1008–1013 1011

with only a slight developmental delay compared withcontrol embryos (Figs. 2A and B). Western blots re-

vealed significant reduction of XYY1 protein in XYY1

MO-injected embryos as compared to controls (data not

shown). There is increasing evidence that PcG proteins

regulate the cell cycle [28]. It is possible that the decrease

in XYY1 protein affected cell cycle length, thus delaying

embryonic development. Developmental defects after

XYY1 knockdown became visible after tailbud stages.XYY1 MO-injected embryos showed defects in antero-

posterior (A-P) axis pattern formation (Figs. 2D and F).

Despite comparatively normal development of trunk

structures, head structures and eyes were diminished.

The rate of embryos exhibiting this phenotype was dose-

dependent, with 6.3%, 34.7%, and 62.1% of embryos

(n ¼ 95) exhibiting abnormality at 15, 38, and 76 nginjected XYY1 MO, respectively. The most severely af-fected embryos did not undergo correct extension and

displayed short A-P axis (Fig. 2F, bottom). This phe-

notype was specific to the XYY1 MO injection and was

not seen in embryos injected with control MO (Figs. 2A,

C, and E). Thus morpholino-mediated translational

knockdown of XYY1 results in embryos that display

abnormal A-P axis patterning, supporting a role for this

gene in embryonic A-P axis development.

XYY1 is required for the expression of anterior neural

marker genes

XYY1 protein was expressed in the neural tube and

XYY1MO-injected embryos showed abnormal A-P axis

development. Thus, we questioned whether XYY1 is

involved in A-P neural patterning by analyzing the effect

of XYY1 MO-injection in more detail. Embryos were

analyzed at neurula stages for neural markers usingquantitative RT-PCR assay. Although no obvious gross

phenotype alteration was noted in XYY1 MO-injected

embryos during neurulation, the expression of NCAM, a

pan-neural marker [29], was suppressed in the XYY1

MO-injected embryos (Fig. 3). Previous study has

shown that overexpression of XYY1 mRNA can directly

induce neural tissue in the whole embryo and ectopic

NCAM expression in animal cap ectoderm explants [13].These results reinforce one another and both point to-

wards an early role of XYY1 in neural development:

induction or maintenance of the neural tissue.

As shown in Figs. 3A and B, injection of the XYY1

MO led to significant reduction of En2, a marker of the

midbrain–hindbrain boundary [30], and Krox20, a

marker of rhombomeres 3 and 5 in the hindbrain [31].

At early neurula (stage 13.5, the initial neural platestage), the expression of Otx2, a marker of the forebrain

[32], and HoxB9, a marker of the spinal cord [33],

showed relatively small change. When those embryos

reached the late neurula stage (stage 19), Otx2 tran-

scripts were also markedly decreased (Fig. 3B). How-

ever, no obvious effect on the expression of HoxB9,posterior neural marker, was observed as in the earlier

stage. These results indicate that XYY1 is necessary for

the expression of anterior neural markers specifically.

This result may be related to the localization of XYY1

protein in the anterior neural tube and the head defects

observed in the XYY1 MO-injected embryos.

To assess the specificity of the XYY1 MO-mediated

phenotypic alterations, XYY1 mRNA (lacking theXYY1 MO target sequence) was injected together

with XYY1 MO. Embryos co-injected with this

mRNA and XYY1 MO developed almost normally,

demonstrating an efficient rescue of the XYY1 MO-

induced phenotype (82.7%, n ¼ 98). Typical imagesare shown (Figs. 4A and B). Furthermore, expression

of En2, almost reduced to the baseline by XYY1 MO,

at early neurula stage, was indeed rescued by co-in-jection of XYY1 mRNA with XYY1 MO (Fig. 4C,

compare lane 3 with lane 2). We conclude that the

abnormal A-P patterning and the reduction of ante-

rior neural marker gene expression resulting from

XYY1 MO-injection are due specifically to a decrease

in XYY1 function.

The results presented here strongly support the con-

clusion that the Polycomb group gene YY1 is essentialfor A-P patterning of the CNS in Xenopus. We have

shown that XYY1 protein was localized to the CNS at

tailbud stages and highly expressed in anterior neural

1012 H.-J. Kwon, H.-M. Chung / Biochemical and Biophysical Research Communications 306 (2003) 1008–1013

tube. Decreased XYY1 function using XYY1 antisensemorpholino oligonucleotides caused abnormal A-P axis

patterning and deficiency of anterior neural develop-

ment. We have shown that the midbrain–hindbrain

junction marker En2, and hindbrain marker Krox20, are

target genes of XYY1 and that XYY1 functions to ac-

tivate or maintain these anterior marker genes. Con-

versely, XYY1 does not repress the spinal cord marker

HoxB9. These results implicate an important role forXYY1 in the patterning of the nervous system. In-

triguingly, similar effects were observed in the overex-

pression of other Xenopus PcG genes, XPcl1, Xbmi1,

and XEZ [10,11]. The reason why the similar results

were obtained in exact opposite condition (gain-of-

function vs. loss-of-function) is uncertain. YY1 can act

either as a repressor or an activator of transcription by

distinct functional domains. Although there was a viewthat XYY1 operates as a transcriptional repressor in

inducing neural tube [13], YY1 can activate some pro-

moters but repress others in the same cell [17]. There-

fore, the question which activity of XYY1 is involved in

the regulation of anterior neural markers remains to be

elucidated. In Drosophila, YY1 homolog, pleiohomeotic

(Pho) mutants show abnormal development of the cen-

tral nervous system [34]. Also, a subset of heterozygoteYY1)/+ mice embryos displays neurulation defects [18].Taken together, our loss-of-function data in Xenopus

provide strong evidence that YY1 acts as a regulator of

neural development in embryogenesis.

In conclusion, we provide evidences to suggest that

YY1, a vertebrate PcG gene, is an important regulator

during early development of Xenopus. Based on these

results, we propose that the role for YY1 in vertebrateembryonic development is regulation of A-P neural

patterning.

Acknowledgments

We thank Dr. H.K. Chung for critical reading of the manuscript.

We acknowledge Dr. F.M. Watt for providing the MZ15 antibody. We

are grateful to Dr. E. Beccari for the pT7TsYY1 plasmid. This work

was supported by grants from the Basic Research Program of the

KOSEF (Korea Science and Engineering Foundation). H.J. Kwon was

supported by BK21 Research Fellowship from the Ministry of Edu-

cation and Human Resources Development.

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