Download Expression patterns of PRDM10 during mouse embryonic

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reports
Expression patterns of PRDM10 during mouse embryonic
development
Jin-Ah Park & Keun-Cheol Kim*
Medical & Bio-Material Research Center and Division of Life Sciences, College of Natural Sciences, Kangwon National University,
Chuncheon 200-701, Korea
It is well known that PR/SET family members participate in
transcriptional regulation via chromatin remodeling. PRDM10
might play an essential role in gene expression, but no such
evidence has been observed so far. To assess PRDM10 expression
at various stages of mouse development, we performed immunohistochemistry using available PRDM10 antibody. Embryos
were obtained from three distinct developmental stages. At E8.5,
PRDM10 expression was concentrated in the mesodermal and
neural crest populations. As embryogenesis proceeded further to
E13.5, PRMD10 expression was mainly in mesoderm-derived
tissues such as somites and neural crest-derived populations such
as the facial skeleton. This expression pattern was consistently
maintained to the fetal growth period E16.5 and adult mouse,
suggesting that PRDM10 may function in tissue differentiation.
Our study revealed that PRDM10 might be a transcriptional
regulator for normal tissue differentiation during mouse embryonic
development. [BMB reports 2010; 43(1): 29-33]
INTRODUCTION
PRDM contains a PR (PRDI-BF1 and RIZ homology) domain
and is a subgroup of PR/SET transcription factor family, because its PR domain shows similarity to the catalytic motif of
the SET domain of histone methyltransferase (HMTase) (1, 2).
There are approximately 20 PR and 30 SET domain-containing
genes in the human genome, a growing number of which have
been found to possess intrinsic HMT activity towards specific
lysine residues along the N-terminal tails of histones (2-5).
These histone-modifying proteins participate in the regulation
of genome expression and chromatin remodeling.
Most of the PRDM subfamily has been implicated in cell proliferation and gene repression in cancer and normal mammalian development (6-8). Human PRDM2 (also known as RIZ1) is
a strong tumor suppressor that induces G2/M cell cycle arrest
*Corresponding author. Tel: 82-33-250-8532; Fax: 82-33-251-3990;
E-mail: [email protected]
Received 9 July 2009, Accepted 3 September 2009
Keywords: Gene repression, Mesoderm, Mouse development, Neural
crest, PRDM10, PR/SET, Tissue differentiation
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and/or apoptosis in various tumor cells (6, 9, 10). Its PR domain
can methylate histone H3 lysine 9 (H3K9) and is necessary in
the repression of estrogen signaling genes (3, 11). Deletion of
mouse PRDM2 was closely associated with B cell lymphoma in
a knockout mouse experiment (10). Mouse PRDM1 (also
known as Blimp1) has been identified as a key factor for PGC
(primordial germ cell) specification, which is the major source
for oocytes and spermatozoa (12). Interestingly, PGC specification
requires the concerted orchestration of two PR domain-containing
transcriptional regulators, PRDM1 and PRDM14 (13). PRDM14
is a key protein in the launching of the germ cell lineage in
mice, suggesting that this event is accomplished by the sequential
expression of multiple genes (13). PRDM14 also maintains the
self-renewal of human ES cells by suppressing gene expression
in embryoid bodies (14). The expression of multiple PRDM family
genes (PRDM6, 8, 12, 13 and 16) in the developing mouse central
nerve system occurs in a spatially and temporally restricted
manner, thereby generating a great deal of interest. PRDM8 and
12 are found in the neuronal progenitor zones of developing
spinal cord, where as PRDM12 and 16 occur in the ventricular
zone of developing telencephalon in a lateral to medial graded
manner; PRDM8 also is present in postmitotic neurons. This
suggests that dynamic changes in PRDM family genes occur in
the developing embryo (15).
In this study, we examined the expression of PRDM10 during
mouse embryogenesis. Immunohistochemical analysis was performed on sections obtained from distinct developmental stages
of the embryo during embryo to fetus or tissue differentiation.
RESULTS
Expression of PRMD10 at the embryo to fetus differentiation
(E8.5)
Immunohistochemistry (IHC) with antibodies specific to
PRDM10 was used to examine. PRDM10 expression during
various stages of mouse embryogenesis 8.5 to 16.5 days
post-coitus. Hematoxylin counterstaining was used as a
control. During early stages (E8.5), PRDM10 was strongly expressed in the middle embryo region, suggesting a cell population in the paraxial mesodermal or migrating neural crest
regions as shown in Fig. 1. All mesenchymal cell populations
can differentiate after neural tube closure according to proBMB reports
29
PRDM10 expression during mouse development
Jin-Ah Park and Keun-Cheol Kim
gramming (16, 17). Therefore, PRDM10 is likely expressed in
robust mesenchymal cells derived from the mesoderm and
neural crest during early mouse development.
PRMD10 expression in somital and craniofacial tissues
during organogenesis
To assess PRDM10 expression during late mouse development,
sagittal sections from E13.5 were analyzed. Compared to the
IgG negative control, PRDM10 was mainly expressed in the cra-
niofacial, somital and notochord regions (Fig. 2). PRDM10 expression was also apparent in the ventricles at this stage, but not
in the intestines or liver. Although experiments were not quantitative, the relative levels of PRDM10 expression are apparent. A
more detailed examination was performed using sagittal sections
of the embryo obtained from E16.5. As shown in Fig. 3, IHC
demonstrated that PRDM10 is ubiquitously expressed patterns
in various tissues. Specifically, PRDM10 was mainly visible in
the developing reticular dermis layer of differentiated head tissues
(Fig. 3A), mandible regions (Fig. 3B), and the adrenal medulla
(Fig. 3C), but was concentrated in the skeletal cartilage (Fig.
3D). Since these PRDM10-expressing tissues were mainly derived from mesodermal or neural crest cell populations, the results
suggest that PRDM10 is consistently expressed for mesodermal
differentiation during mouse development.
PRMD10 expression in adult mouse tissues
Fig. 1. Expression of PRDM10 protein in the embryonic head region
8.5 days after fertilization (E8.5). Transverse sections of the embryonic
head were prepared and stained by IHC using PRDM10 antibody.
Immunoreactivity was found in the mesodermal and neural crest
regions. Arrowhead: neural tube, Brown: PRDM10, Purple: hematoxylin counterstaining.
Adult mice were sacrificed in order to examine PRDM10 expression in various tissues by IHC and RT-PCR analysis. IHC detected PRDM10 expression in cartilage and kidney, in agreement with PRDM10 expression during embryonic development
(Fig. 4A). However, RT-PCR analysis found that the patterns of
PRDM10 expression in certain tissues, such as liver, spleen and
rectum, were slightly different compared to embryonic stages,
suggesting that PRDM10 is distinctly expressed in adult mice
(Fig. 4B). This discrepancy could be explained by the fact that
the embryonic tissues were too small. Therefore, these results
suggest that PRDM10 plays a role in tissue differentiation during
mouse embryogenesis and adult development.
DISCUSSION
Although PRDM10 shares characteristics with the PR/SET fam-
Fig. 2. PRDM10 expression in the developmental stage (E13.5). Sagittal sections of
mouse embryos were prepared 13 days after
fertilization (E13.5). PRDM10 proteins were
detected mainly in the craniofacial, somital
and notochord regions. Immunoreactivity
with brown color was determined by incubation with PRDM10 antibody (B), whereas there was no reaction in the negative control (A). NC: notochord, TG: tongue, VT:
ventricle, S: somite, L: liver.
30 BMB reports
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PRDM10 expression during mouse development
Jin-Ah Park and Keun-Cheol Kim
Fig. 3. PRDM10 expression in various embryonic
tissues obtained from E16.5. (A) head, (B) mandible, (C) adrenal medulla, (D) skeletal cartilages.
The scale bar is identical in each image. Brown:
PRDM10, Purple: hematoxylin counterstaining.
Fig. 4. PRDM10 expression in adult mouse
tissues. (A) IHC analysis of PRDM10 expression in adult mouse. Left, cartilage;
Right, kidney; Brown: PRDM10, Purple:
hematoxylin counterstaining. (B) RT-PCR
analysis of PRDM10 expression in various
mouse tissues. cDNA was prepared from
various tissues and used as a DNA template for PCR amplification using PRDM10
specific primers. A single band of PRDM10
was detected at 401 bp. GAPDH was used
as a loading control to confirm correct
cDNA synthesis.
ily with the PR/SET domain and clusters of krüppel-like zinc fingers DNA binding motifs, the functions of PRDM10 are only beginning to be determined (18). It is known that most of the
PR/SET gene family modulates gene expression, specifically the
spatial and temporal regulation of tissue differentiation during
mouse embryonic development (5, 15, 19, 20). Functionally,
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PR/SET gene family members directly or indirectly interact with
histone methylation proteins that possess HMTase activity as
well as components of the transcription machinery (1, 8, 11).
Our present study suggests that PRDM10 expression stimulates
the sophisticated morphogenesis of tissue differentiation, including
somites and craniofacial tissue formation. Therefore, we postulate
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PRDM10 expression during mouse development
Jin-Ah Park and Keun-Cheol Kim
that PRDM10 has an essential role in regulating gene expression by binding target gene promoters and regulating tissue differentiation genes. PRDM10 could be recruited to its
target promoters, depending on the activity derived from its
conserved PR/SET domain. Even though the intrinsic activity of
PRDM10 remains unknown, we have consistently obtained
data showing that PRDM10 is a tri-H3K9 HMTase-related protein (unpublished data). In general, silenced genes are associated with tri-methylated H3K9, which represses transcription
(11). This suggests that PRDM10 expression during mouse embryonic development may function as a gene repressor for normal somite and craniofacial formation.
Previous reports have suggested that PRDM10 may be involved in the pathogenesis of gangliosidosis (GM2), a neuronal
storage disease (18). Based on its high expression in the mesodermal region, PRDM10 might be associated with the pathogenesis of the other mesoderm-derived diseases as well.
Therefore, further studies are needed to explore the relationship between PRDM10 expression and diseases associated
with cartilage defects such as chondroma or inflammatory
arthritis. Although there is no expression data for craniofacial
syndromes, these diseases have genetic backgrounds containing mutated candidate target genes (21). PRDM10 might also
be an important target protein for the growth and development
of the craniofacial complex based on its expression in craniofacial tissues.
In summary, our study suggests that PRDM10 was expressed
in somites and craniofacial tissues during mouse embryonic
development. PRDM10 may be a target gene for tissue differentiation and bone formation defects.
the maternal uterus and fixed in neutral formalin for 2 days, after
which they were cut in half and fixed in neutral formalin for an
additional 3 days. The fixed samples were processed in gradient
alcohols, xylene and embedded in paraffin prior to being sectioned at 4 μm for IHC analysis. Paraffin was removed from
sections using a citrus clearing solvent for 20 min. To block endogenous peroxidase activity, sections were incubated in a 3%
hydrogen peroxidase-methanol mixture for 15 min. Primary
PRDM10 antibody was diluted 1 : 100, incubated for 2 hours,
and treated with secondary antibody. Chromogenic DAB was
used as a substrate and was couterstained with hematoxylin.
Finally, the slides were hydrated through alcohol, cleared with
citrus clearing solvent and mounted with Permount mounting
medium (Fisher Scientific Inc., NJ, USA).
RT-PCR
MATERIALS AND METHODS
Adult ICR mouse tissues were collected and examined by
RT-PCR. Total cellular RNA was extracted from homogenized
tissues using trizol reagent (Promega Co., WI, USA). cDNA
was synthesized using reverse-transcriptase according to the
manufacturer’s instructions and was used as template for PCR
amplification along with PRDM10-specific primers. The primer
sequences are as follows: PRDM10 (sense), 5’-ATC TAC CAG
TGT ACA GAG TGT GA-3’, PRDM10 (antisense), 5’-ATA CTG
ACA GAA GTA ATC TCG AT-3’, GAPDH (sense), 5'-ATG ACA
ACT TTG GCA TTG TGG AA-3’, GAPDH (antisense), 5’-CTG
TTG CTG TAG CCG TAT TCA TT-3’. GAPDH was used as a
o
loading control. Samples were incubated at 95 C for 20 sec,
o
o
60 C for 25 sec and 72 C for 30 seconds for 35 cycles.
Reaction samples were then incubated for an additional 7 min
o
o
at 72 C and cooled to 4 C. PCR products were resolved on a
1% agarose gel.
Antibody and reagents
Acknowledgements
Animals and mating
REFERENCES
Polyclonal PRDM10 antibody was kindly provided from
Abgents Inc. (CA, USA). Secondary antibody was purchased
from Santa Cruz Inc. (CA, USA). Reagents for IHC analysis
were obtained from Vector Laboratories, Inc. (MI, USA). 3,3’diaminobenzidine (DAB) was used as a chromogenic substrate
and was counterstained with hematoxylin.
Eight week-old ICR mice were obtained from Orient Bio Inc.
(Korea) and either used for mating or sacrificed for further
analysis. Mating was performed according to a 3 : 1 female to
male ratio and was verified by vaginal plug formation. The embryonic stage was designated embryonic day 0.5 (E0.5) on the
day of vaginal plug formation. Embryos or fetuses were prepared on E8.5, E13.5 and E16.5. Animals were handled according to the Guide for the Care and Use of Laboratory Animals at
Kangwon National University, Chuncheon, S. Korea.
Immunohistochemistry (IHC)
Following Forane anaesthesia, embryos were removed from
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The authors appreciate the Korea Basic Science Institute (KBSI)
for technical assistance regarding microscopic image analysis.
This work was supported by grants from the Korean Research
Foundation (KRF C00141) and the Ministry of Education,
Science and Technology (the Regional Research Universities
Program/Medical & Bio-Materials Research Center), S. Korea.
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