Connective Tissue Research, 43: 148–152, 2002

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c 2002 Taylor and Francis
0300-8207/02 $12.00 + .00
DOI: 10.1080/03008200290000970

Msx1 Homeogene Antisense mRNA in Mouse Dental

and Bone Cells

A. Berdal,1 F. Lezot,1 L. Pibouin,1 D. Hotton,1 S. Ghoul-Mazgar,1 C. Teillaud,1

B. Robert,2 M. MacDougall,3 and C. Blin1
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1
Laboratoire Biologie-Orofaciale et Pathologie, INSERM EMI-U 0110, Université Paris 7, IFR-58,
Institut Biomédical des Cordeliers, 7, Paris, France
2
Département Biologie moléculaire, Génétique moléculaire de la morphogenèse, CNRS URA 1947,
Institut Pasteur, Paris, France
3
Department Pediatric Dentistry, University Texas Health Science Center San Antonio, San Antonio,
Texas, USA

INTRODUCTION
Msx1 plays a key role in early dental and cranio-facial pat- In mammals, three Msx (muscle segment homeobox gene)
For personal use only.

terning. A systematic screening of Msx1 transcripts during late
postnatal stages of development evidenced not only sense mRNA
but also antisense mRNA in the skeleton. Natural antisenses are
able to bind their corresponding sense RNAs and block protein ex-
pression. Specific reverse-transcription polymerase chain reaction
(RT-PCR) Northern-blotting using riboprobes and primer exten-
sion analysis allowed to identify and sequence a mouse 2184-base
Msx1 antisense transcript. The transcription start site was located
in a region including a consensus TATA box. In situ hybridization
evidenced an increase in antisense mRNA expression during den-
tal and bone cell differentiation in prenatal (Theiler stages E15.5–
18.5) and newborn mice. This upregulation was related to Msx1
protein downregulation in cells expressing Msx1 sense mRNA. In
vitro, transient Msx1 sense and antisense mRNA overexpression
was performed in MO6-G3 cells, which pertain to the odontoblast
lineage (polarization and dentin sialoprotein and phosphoprotein
synthesis). The balance between antisense and sense Msx1 mRNAs
appeared to control Msx1 protein levels. These data suggest that a
bidirectional transcription of Msx1 homeogene may control Msx1
protein levels, and therefore may be critical in cell communication
and differentiation during dental and cranio-facial development
and mineralization.
Keywords Homeogene, Msx, Antisense, Development, Biomineral-
ization.
Received 19 June 2001; accepted 28 November 2001.
Address correspondence to Dr. Ariane Berdal, Laboratoire
Biologie-Orofaciale et Pathologie, INSERM EMI-U 0110, Université
Paris 7, IFR-58, Institut Biomédical des Cordeliers, Esc. E-2è ét.,
15-21 rue de l’Ecole de Médecine, 75270 Paris Cedex 06, France.
E-mail: biol odonto fr@yahoo.com
genes have been identified and related to the Drosophila Msh
gene [1, 2]. This study is devoted to one member of this home-
ogene family, Msx1, and the regulation of its expression in den-
tal and cranio-facial skeleton. Several mutations in mice [3, 4]
and humans [5–7] have established the important role of Msx1
in cranio-facial development. The observed developmental de-
fects include hypodontia and palatal clefting and support that
this homeogene controls early patterning of neural crest-derived
skeleton. On the other hand, an overall screening of Msx1 ex-
pression from early antenatal [2, 8, 9] until postnatal stages
[10] and even during late adulthood [11] has established that
the expression of this homeoprotein is maintained in mammals
throughout their lifetime. The cells committed in the Msx1 sig-
naling pathway include dental and the heterogenous osteoblast
and osteoclast bone cells [10]. The reported pattern suggests
that Msx1 protein expression is not only important during early
patterning but also in the adult skeleton by maintaining stem
cells inside specific compartments. Concerning the potential tar-
get genes of Msx homeoproteins, both Msx1 and Msx2 inhibit
the transcription of the phenotypic marker of osteoblast and
odontoblast differentiation, the osteocalcin gene [12]. Further-
more, tissue-specific master-gene expression may be repressed
by Msx1 and therefore control cell committent. Such a concept is
based on myoblast transformation induced by Msx1 overexpres-
sion while MyoD gene expression is repressed [13]. Msx1 over-
expression was also shown to decrease the steady-state levels of
Cbfa1/Osf2 [14], which targets specific transcription of osteocal-
cin gene in bone. In conclusion, Msx1 downregulation appears
to be a prerequisite to the terminal differentiation, presumably

148
 Msx1 ANTISENSE mRNA IN THE SKELETON 149

via the derepression of tissue-specific master-gene expression
and consequently triggering of phenotypic markers synthesis.
A systematic screening of Msx homeogene expression by
Northern blot analysis identified Msx1 transcripts in the in-
cisor of postnatal rat and mice. Conversely, the absence of Msx1
homeoprotein expression was previously reported in the same
experimental model at the same developmental stage [15]. These
apparent conflicting data led us to more precisely analyze Msx1
mRNAs in dental and bone cells in vivo and in vitro. This study
reports the bidirectional transcription of Msx1 homeogene and
overviews the potential role of antisense mRNA in the regulation
of protein expression.
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initiation site was located in an highly conserved region of Msx1
homeoprotein from 5 species (mouse, human, rat, bovine and
chicken) showing a 66-bp identical region including the presence
of a consensus TATA box. Finally, in MO6-G3 cells, which ex-
press the tissue-specific dentin sialoprotein and phosphoprotein
as well as osteocalcin genes, Western blotting and immunoflu-
orescence (Figure 1) did not provide evidence of Msx1 homeo-
protein, while Msx1 sense mRNA was detected. In contrast, after
lipotransfection of Msx1 sense vector (kindly provided by Cory
Abate-Shen, Piscataway, NJ), Msx1 homeoprotein could be de-
tected (Figure 1). Cotransfection of Msx1 expression vector (S)
and an antisense expression vector (A) with an increasing ratio
A/S = 1/1, 5/1, 10/1, and 20/1 resulted in a decreasing signal

for Msx1 homeoprotein (Figure 1).

METHODS AND RESULTS
Structure and Potential Role of Msx1 Sense
and Antisense Transcripts
The first evidence for Msx1 antisense mRNA was obtained
by using Msx1 sense riboprobes on Northern blotting from mice
dental cells and immortalized MO6-G3 odontoblasts (data not
shown). In order to determine the sequence of Msx1 antisense
transcript, a sequential reverse-transcription polymerase chain
reaction (RT-PCR) was performed with a number of primer sets
selected in the mouse Msx1 homeogene sequence (Genbank AC
number S73812). The antisense transcription start sites were
For personal use only.
Bidirectional Transcription and Cell Differentiation
in the Cranio-Facial Skeleton
In order to validate the concept that Msx1 antisense tran-
script may inhibit in vivo the expression of Msx1 homeopro-
tein, in situ hybridization was performed with specific sense
and antisense riboprobes (exon 2). These data were compared to
Msx1 homeoprotein expression in a knock-in transgenic mice
model where the reporter lacZ gene is inserted inside exon 2. In
heterozygous Msx1+/− mice, β-galactosidase labeling allowed
us to delineate the cell expression pattern of Msx1 homeopro-

identified by primer extension in mouse MOG-63 cells and hu- tein as illustrated in a joint paper [10]. More specifically, in the
man cranio-facial samples. By using a 50 -TTCTATTTAACAGT mandible periosteum, osteoblast progenitors appeared to over-
ACATT-30 primer, extended fragments of 75 bp and 72 bp were express the protein, while in differentiated osteoblasts and os-
obtained for murine and human RNA, respectively. This teocytes the protein was not detected. In this area, it was possible

Figure 1. Immunolabelling of Msx1 homeoprotein in MO6-G3 odontoblasts. (A) Basal immunoreactive levels of Msx1 homeoprotein appears to be negligible
in MO6-G3 cells. (B) In contrast, overexpression of cDNA using CMV promoter and lipotransfection induces an increase in immunoreactive Msx1 homeoprotein.
(C) The cotransfection of Msx1 sense cDNA and a 20-fold ratio of Msx1 antisense partial cDNA (exon 2) result in a clear downregulation of immunolabeling
intensity for Msx1 homeoprotein. Scale bar = 3.5 µm.
 150
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Figure 2. In situ hybridization in basal bone of neonate mouse mandible using sense exon 2 riboprobe. (A) The labeling distribution appears to be cytoplasmic
in most osteocytes. (B) However, the transcripts may also be located in the nuclear compartment in some osteocytes. Scale bar = 10 µm.
that Msx1 sense transcript levels were progressively downregu-
lated during osteoblast differentiation while, conversely, Msx1
antisense transcript levels were progressively upregulated in the
differentiated osteoblasts and osteocytes (Figure 2). The sub-
cellular distribution of Msx1 antisense transcripts was dual:
cytoplasmic in the majority of the cells and nuclear in some
osteocytes.
DISCUSSION
This study showing a naturally occuring antisense Msx1
mRNA is not an isolated report. Naturally occuring antisense
RNAs have the potential to form a duplex with their complemen-
tary sense mRNA, thereby inhibiting protein expression [16].
Such a situation has been shown in a virus [17]. In prokaryotes,
the regulatory mechanism is very basic and may be summarized
as the hybridization of sense transcript with the complementary
antisense transcript, blocking the translation process and inhibit-
ing protein synthesis. In eukaryotes, natural antisense mRNAs
have been reported [18] and shown to control N-myc transcrip-
tion [19] and decrease the mRNA half-life of fibroblast growth
factor (FGF) [20]. However, cell compartmentalization is com-
plex in eukaryotes, implying different mechanisms of antisense
actions [18] (Figure 3). In the vast majority [21], the regula-
tory step is the translation process and occurs within cytoplasm.
A. BERDAL ET AL.
But some studies have also described alterations in the nuclear
compartment, such as inhibition of sense RNA transcription (for
review, see ref. 21), RNA splicing, and nucleocytoplasmic trans-
port and upregulation of enzymatic activity (double-stranded
RNA adenosine deaminases–ADAR, RNA helicase/RNA du-
plex unwindase). Finally, ADAR may edit RNA [22] by muta-
tion of their coding capacity. Such is the case for glu-R5 and
glu-R6 glutamate receptor subunit transcripts. In this last situa-
tion, natural antisense plays a role in creating protein biodiver-
sity. The underlying mechanisms of protein synthesis inhibition
are based on the distinct location of antisense transcripts in cy-
toplasmic and nuclear compartments as shown here by in situ
hybridization.
In this report, as previously proposed [14], Msx1 antisense
transcript is shown to be expressed in several systems where the
existence and morphogenetic role of Msx1 sense transcripts have
been previously shown, such as teeth, bone, and hair follicle.
The control of Msx1 sense transcription has been extensively
studied. Epithelial–mesenchymal interactions trigger the expres-
sion of Msx1 within the dental mesenchyme, which then upreg-
ulates BMP4 expression [23]. Consistently, the null mutation of
Msx1 homeogene is associated with the arrest of tooth develop-
ment at the bud stage secondary to the interrupted BMP4-Msx1
signaling cascade [3, 4]. Epithelial–mesenchymal dissociation
and reassociation experiments, combined with the addition of
 Msx1 ANTISENSE mRNA IN THE SKELETON
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Figure 3. Presumed regulatory pathways for the Msx1 antisense-induced
downregulation of Msx1 homeoprotein synthesis. The sense pre-mRNA con-
tains two exons (1 and 2) and one intron. The antisense transcript includes exon
2 and part of the intron and does not appear to be spliced. Its sequence pre-
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sumably allow duplex formation with both the premessenger and messenger
Msx1 sense RNA. In the nucleus, double-stranded RNA (ds RNA) may inhibit
mRNA maturation (notably splicing) and target RNAase degradation of sense
transcript. In the cytoplasm, the translocation may be located at each step and
unwindase activity may accelerate transcript turnover. In both compartments,
DRADA have been shown to edit sense RNA in other exemplary models.
several growth and differentiation factors, notably BMP4,
segregated into agarose beads onto dental mesenchyme, have
delineated these molecular interactions. In the other hand, anal-
ysis of Msx1 sense promotor has identified the autoregulatory ca-
pacity of Msx1 homeoprotein in the proper expression of Msx1
sense transcript [24]. Transgenic mice where selected regions
of Msx1 homeogene promoter were inserted upstream to a re-
porter gene showed a site-specific pattern of promoter activity:
limb buds for a 78-bp proximal element located −2198 to −2276
and cranio-facial primordia for a 240 bp distal element located
−4006 to −4252. Therefore, Msx1 promoter contains respon-
sive elements that control site-specific Msx1 sense transcription.
The present study focuses on the transcription of the Msx1 re-
verse strand. It shows that: (a) In vivo, the developmental pattern
of Msx1 sense and antisense transcripts are complementary and
related to Msx1 homeoprotein expression; and (b) in vitro, the
effective levels of Msx1 homeoprotein are downregulated by
the overexpression of Msx1 antisense transcripts in immortal-
ized MO6-G3 odontoblast cell. Based on these data, it is possi-
ble to propose that Msx1 homeoprotein expression is controlled
via two pathways: the control of Msx1 sense transcription, and
in the corresponding 30 region, the control of Msx1 antisense
transcription (Figure 3). Such a complex regulatory pathway
151
may help to interpret the phenotype variability in Msx1 homeo-
gene mutations through several combinations of mutated/wild-
type sense and antisense mRNAs inside the potential duplexes.
The organization of the suspected promoter of Msx1 antisense
transcription is highly conserved [14] among five species (hu-
man, bovine, mouse, rat, and chicken). The promoter region
contains an identical 66-bp sequence where the consensus se-
quence for a TATA box is found. Furthermore, primer extension
analysis identified the transcription start site in a reliable
position.
The molecular mechanism(s) that lead the decreased expres-
sion of Msx1 homeoprotein by antisense overexpression illus-
trated here and shown by immunoblotting experiments [14] are
not identified. The sequence of Msx1 antisense transcript may
allow the formation of RNA duplex with the Msx1 sense mRNA,
as the entire exon 2 is shared by sense and antisense transcripts
(Figure 3). Furthermore, part of the intron constitutes the 30 end
of Msx1 antisense transcript. This complementary sequence of
antisense mRNA and sense pre-mRNA may therefore allow the
physical interaction and affect sense pre-mRNA processing. The
potential duplex (sense mRNA–antisense mRNA and sense pre-
mRNA–antisense mRNA) are not exclusive. Their formation is
presently supported by the dual nuclear and cytoplasmic distri-
bution of Msx1 antisense mRNA (Figure 2). However, it must be
taken in account that the probe used in the present study is spe-
cific to exon 2, the region potentially involved in the formation
of sense/antisense duplex mRNA. In the duplexed state, the hy-
bridized sense mRNA would not be available for the labeled an-
tisense riboprobe. Therefore, this labeled probe would hybridize
only with single-stranded Msx1 antisense mRNA present in ex-
cess. Consequently, in situ hybridization would underestimate
the effective ratios of sense and antisense transcripts when they
are hybridized inside the duplex.
Finally, Msx1 homeogene may be important in mineralized
tissues by a mechanism shared with myoblasts [13], the repres-
sion of tissue-specific master gene expression. Indeed, Cbfa1,
which controls osteocalcin expression in osteoblasts and is ex-
pressed in odontoblasts, is downregulated by Msx1 homeo-
protein [14]. Preliminary studies have shown that Msx1 sense
transcripts are expressed in murine odontoblasts in vivo [25]
(Ghoul-Mazgar et al., unpublished data). However, Msx1 home-
oprotein, using antibodies, immunolabeling, and Western blot-
ting, as well as β-galactosidase histoenzymology, on Msx1+/−
knock-in mice failed to detect Msx1 homeoprotein in vivo on
wild-type mice extracts [15]. Consequently, in order to identify
the effective function of Msx1 in vivo, a systematic screening
of the protein expression is critically required, in addition to the
mapping of Msx1 sense mRNA.
In conclusion, the present study illustrates an opening field
for developmental biology, the posttranscriptional regulation of
protein expression, which involves natural antisense strategy,
differential translation start sites (IRES) and constitutes a path-
way complementary to the regulation of gene transcription at
the promoter level.
 152 A. BERDAL ET AL.
ACKNOWLEDGMENTS
We thank Dr. Cory Abate-Shen for providing Msx1 expres-
sion vectors. This study was realized with INSERM EMI-U 0110
grants and IFRO (Institut Français pour la Recherche Odon-
tologique) funding (Laurence Pibouin).
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