Basonuclin 2 has a function in the multiplication
of embryonic craniofacial mesenchymal cells
and is orthologous to disco proteins
Amandine Vanhoutteghema, Anna Maciejewski-Duvala, Cyril Bouchea, Brigitte Delhommea, Franc ¸oise Herve ´a,
Fabrice Daubigneya, Guillaume Soubigoub, Masatake Arakic, Kimi Arakic, Ken-ichi Yamamurac, and Philippe Djiana,1
aUnite ´ Propre de Recherche 2228 du Centre National de la Recherche Scientifique, Universite ´ Paris Descartes, 45 rue des Saints-Pe `res, 75006 Paris, France;
bInstitut Pasteur, Ba ˆtiment 14, 28 rue du Dr. Roux, 75015 Paris, France; andcInstitute of Resource Development and Analysis, Kumamoto University, 2-2-1,
Honjo, Kumamoto 860-0811, Japan
Communicated by Howard Green, Harvard Medical School, Boston, MA, June 26, 2009 (received for review January 29, 2009)
Basonuclin 2 is a recently discovered zinc finger protein of unknown
function. Its paralog, basonuclin 1, is associated with the ability of
keratinocytes to multiply. The basonuclin zinc fingers are closely
the relation between disco proteins and basonuclins has remained
elusive because the function of the disco proteins in larval head
development seems to have no relation to that of basonuclin 1 and
also has no similarity to that of the basonuclins. We have generated
mice lacking basonuclin 2. These mice die within 24 h of birth with a
embryonic head, expression of the basonuclin 2 gene is restricted to
mesenchymal cells in the palate, at the periphery of the tongue, and
in the mesenchymal sheaths that surround the brain and the osteo-
these mesenchymal cells is greatly diminished. Therefore, basonuclin
2 is essential for the multiplication of craniofacial mesenchymal cells
during embryogenesis. Non-Drosophila insect databases available
since 2008 reveal that the basonuclins and the disco proteins share
much more extensive sequence and gene structure similarity than
that basonuclin 2 is both structurally and functionally the vertebrate
ortholog of the disco proteins. We also note the possibility that some
human craniofacial abnormalities are due to a lack of basonuclin 2.
cell multiplication ? cleft palate
general structure: they are about 1,000 residues in length and
contain 3 separate pairs of zinc fingers and a nuclear localization
signal (NLS). The deduced amino acid sequences of bnc1 and bnc2
are only slightly more than 40% identical, but the sequence identity
is much higher in the N-terminal region, the zinc fingers, and the
region of the NLS than in the rest of the molecule (1, 2). The
basonuclin zinc fingers are related to those of the Drosophila
proteins disconnected (3) and discorelated (the disco proteins),
which have an essential function in larval head development (4).
in basal keratinocytes of stratified squamous epithelium and in
reproductive germ cells (5, 6). Although the evidence is by no
potential for cell proliferation (7, 8). The only known function of
bnc1 is that of a transcription factor in the synthesis of ribosomal
RNA (9, 10), but it is possible that bnc1 possesses a nucleoplasmic
function in the regulation of expression of genes transcribed by
RNA polymerase II (11, 12). Knock-down of the mouse bnc1 gene
in oocytes shows that bnc1 is required for oogenesis and possibly
early embryogenesis (13).
The gene encoding bnc2 was discovered in 2004 (1, 2). The bnc2
mRNA is abundant in cell types that possess bnc1, but it is also
found only in vertebrates. Bnc1 and bnc2 share the same
The genes for bnc1 and bnc2 differ greatly in size and are located
on different chromosomes, but it is clear that they have a common
evolutionary origin. Bnc1 and bnc2 are thought to possess different
functions, since bnc2 but not bnc1 localizes to nuclear speckles
and therefore is likely to have a function in nuclear processing
of mRNA (14).
The extreme evolutionary stability of the bnc2 sequence suggests
that the protein possesses an important function (2). To elucidate
this function, we have generated mice lacking bnc2. These mice die
within 24 h of birth with a cleft palate and abnormalities of
craniofacial bones and tongue. We show here that this phenotype
results from a direct effect of bnc2 on the multiplication of
craniofacial mesenchymal cells. From the study of GenBank data,
the insect disco proteins. We note the similarity between the
function of bnc2 in the mouse and that of the disco proteins in
Drosophila. We also note that some human craniofacial abnormal-
ities such as cleft palate might be due to lack of bnc2.
Ayu21–18, an ES Cell Line with a Gene-Trap Insertion in the bnc2 Gene.
We searched gene-trap ES cell repositories for bnc2 insertions. A
BLAST search using the mouse cDNA sequence identified line
Ayu21–18 (15). The description of the generation of Ayu21–18 can
sequencing located the vector insertion site of Ayu21–18 in the
intron that separates bnc2 exons 2a and 3. This allowed us to
generate a probe and to design primers for genotyping by Southern
analysis (Fig. 1A) and multiplex PCR (Fig. 1B). Probing of BamHI,
PstI, ScaI and BglII digests of bnc2?/?genomic DNA with a
fragment of the vector demonstrated that Ayu21–18 mice con-
tained no other copy of the transgene inserted elsewhere in the
To determine whether any bnc2 mRNA remained in bnc2?/?
mice, we carried out northern analysis and RT-PCR on RNA
of 2, 4, 6, and 9 kb previously identified (1) were detected by
northern analysis in wild-type (wt) and heterozygotes. The 2-kb
RNA produced the most intense band; this RNA is too small to
RNAs of 4, 6, and 9 kb were not detected, but a small amount of
the 2-kb RNA remained (Fig. 1C). No bnc2 mRNA was detected
in a total of 8 bnc2?/?embryos examined either by RT-PCR with
Author contributions: A.V., A.M.-D., C.B., B.D., F.H., G.S., and P.D. designed research; A.V.,
A.M.-D., C.B., B.D., F.H., F.D., and G.S. performed research; A.V., M.A., K.A., and K.-i.Y.
contributed new reagents/analytic tools; A.V., A.M.-D., C.B., B.D., F.H., G.S., and P.D.
analyzed data; and A.V. and P.D. wrote the paper.
The authors declare no conflict of interest.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
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primers in exons 2a and 3.
Since the gene-trap vector was inserted in a large bnc2 intron, it
a gene other than bnc2, and whose altered expression could then
contribute to any observed phenotype. To eliminate such a possi-
bility, we examined the expression of all genes located within 2 Mb
of the vector insertion site in 3 bnc2?/?and 3 bnc2?/?embryos by
RT-PCR. No alteration of expression was detected in homozygous
mutants for any of the 12 genes examined. Microarray analyses
confirmed that among the genes located within 23 Mb of the
gene-trap insertion site, only bnc2 showed altered expression in
Neonatal Lethality, Reduced Size of the Head, Abnormalities of the
bnc2?/?females produced litters of normal size, in which about 1/4
with aerial distention of their digestive tract. All dead newborns
were bnc2?/?, whereas surviving ones were either bnc2?/?or
bnc2?/?. A cause of aerial distention of the digestive tract in mice
is cleft palate. All bnc2?/?mice had a cleft of the secondary palate,
which was complete in about half the newborns and posterior-only
in the other half.
To determine whether lack of bnc2 impaired either elevation or
growth of the palatal processes, heads of embryos and neonates
were embedded in paraffin and sectioned in the coronal plane. No
obvious difference was observed between bnc2?/?and wt mice
either at E14.5, when palatal processes were still in a vertical
position (Fig. 2A), or at E15.5, when the anterior part of the
processes had begun to elevate (Fig. 2B). At E16.5, the palatal
mutants, although the processes had elevated, they failed to estab-
lish contact because of insufficient growth after their elevation.
(Fig. 2C). At birth, a wide palatal cleft was obvious and the heads
and tongues were clearly smaller than those of their wt littermates
Developmental Abnormalities of Craniofacial Bones in bnc2?/?Mice.
of bnc2?/?newborns, we stained bone and cartilage with alizarin
red and alcian blue. This showed that the maxillary processes had
failed to grow toward the midline and to extend cranially. The
had not extended caudally. The size of the alisphenoid was reduced
an excessively wide sagittal suture and an abnormally large poste-
rior fontanel was clearly visible (Fig. 3B). The rest of the skeleton
appeared normal (Fig. 3C). We conclude that lack of bnc2 specif-
ically affects the growth of craniofacial bones.
High Expression of the bnc2lacZGene Around the Palatal Cleft. Al-
within the palate itself, it could also be secondary to other cranio-
facial bone defects (17). To investigate these alternatives, we
analyzed the expression of the bnc2 gene in developing palatal
processes. Both bnc2?/?and bnc2?/?mice express a fused bnc2-
lacZ gene (bnc2lacz) whose transcription is under the control of the
bnc2 promoter. Therefore, cell types expressing bnc2 could be
lacZ. RT-PCR in various tissues showed that the relative levels of
the mRNAs for ?-gal and bnc2 were correlated in heterozygotes,
thus demonstrating that insertion of the vector did not affect the
tissue-specific regulation of the bnc2 promoter. Whole-mount
staining of bnc2?/?newborns for ?-gal activity using X-gal as a
chromogenic substrate demonstrated that enzyme activity was
strongest in the skull, particularly sutures and fontanels, and in the
areas surrounding the palatal cleft (Fig. S1). Since the bnc2 gene is
strongly expressed at the sites affected by lack of bnc2, the protein
Expression of the bnc2lacZGene in Embryonic Head Is Specific to
Mesenchymal Cells in the Developing Palatal Processes, the Tongue,
or epithelial cells of the palate expressed bnc2lacZ, we double-
stained coronal sections of bnc2?/?embryonic heads with antibod-
ies to ?-gal and keratins (Fig. 4). At E14.5, a moderate ?-gal
staining was virtually confined to the anterior part of the palatal
processes; little staining was detected in the rest of the head (Fig.
4 A–C). At E16.5, a much stronger ?-gal staining was present along
the entire anteroposterior axis of the palate and in sheaths of cells
around the brain, the nasal cartilage, and the molar tooth buds, as
well as at the base and the periphery of the tongue (Fig. 4 D–F).
Examination of the double-stained sections at high magnification
PCR, northern blotting, and RT-PCR. (A) Genotyping by
Southern blot analysis, using BamHI. (B) Genotyping by
multiplex PCR. (C) Northern analysis demonstrating the
virtual absence of bnc2 mRNA in bnc2?/?embryos at
absence of bnc2 mRNA in bnc2?/?embryos.
Vanhoutteghem et al.PNAS ?
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cells and was absent from epithelial cells (Fig. 4 G and H).
To confirm the specific expression of the bnc2 gene in mesen-
chymal cells, heads of bnc2?/?embryos were fixed at E16.5,
skinned, and incubated in the presence of X-gal. Heads were then
embedded in paraffin and sectioned. ?-gal activity was found to be
restricted to the mesenchymal cells in the palate, and to the sheaths
of mesenchymal cells surrounding cartilage, bone (Fig. 4 I and J)
and brain. No ?-gal activity was detected in other mesenchymal
cells, in the differentiated progeny of mesenchymal cells, such as
cartilage and bone, or in non-mesenchymal cell types such as
epithelial and brain cells.
Bnc2 Controls the Multiplication of Mesenchymal Cells in the Late
Embryo. The diminished size of mesenchyme-derived structures in
the bnc2?/?mice suggests that bnc2 might control the multiplica-
processes have elevated but have failed to fuse because of insufficient growth toward the midline, thus causing cleft palate. (D) At P0, the wt mouse shows normal
fused palate, whereas a cleft palate is evident in the mutant. Head and tongue of the homozygous mutant are smaller than those of the wt. Asterisks indicate cleft
Small size of the head, cleft palate and abnormalities of the tongue in bnc2?/?neonates. Mouse heads were embedded in paraffin, sectioned and stained
have converged toward the midline. In the bnc2?/?mouse, both processes are absent thus exposing the vomer and presphenoid. The internal pterygoid processes of
the bnc2?/?mouse have failed to develop caudally. The lower 2 circles are schematic representations. Bone processes that have failed to grow in the bnc2?/?mouse
are outlined in red. Arrows indicate direction of axial and rostral growths that did not occur. AL: alisphenoid, BS: basisphenoid, ePp: external pterygoid process, iPp:
internal pterygoid process, M: maxillary, Mp: maxillary process, PL: palatine bone, PM: premaxillary, PS: presphenoid, S: sphenoid, V: vomer. (B) Superior view of skull
showing that sutures and posterior fontanel are abnormally large in the homozygous mutants. (C) Shorter stature of mutant and absence of gross skeletal
abnormalities. Tails have been cut for genotyping.
Abnormalities of craniofacial bones in bnc2?/?newborn mice. Newborn mice were stained with alcian blue/alizarin red and cleared with KOH. (A) Inferior
www.pnas.org?cgi?doi?10.1073?pnas.0905840106 Vanhoutteghem et al.
tion of mesenchymal cells. The proportion of cells in mitosis can be
phosphorylation of histone H3 is strictly associated with the chro-
of embryonic heads for histone H3 phosphorylated on serine 10
revealed that bnc2?/?embryos possessed 30–50% fewer phospho-
histone-containing cells (Fig. 5 A–F). Double staining with the
?-gal-containing mesenchymal cells, particularly the palatal pro-
cesses at E14.5 (Fig. 5 G–J). These results were confirmed by
measuring the mitotic index in paraffin-embedded sections stained
with hematoxylin/eosin. The proportion of cells in metaphase,
anaphase or telophase in the palatal processes was decreased by
nearly 50% at E14.5 and E16.5 in homozygous mutants.
To determine whether lack of bnc2 increased cell death, DNA
strand breaks characteristic of apoptotic cells were labeled with
biotin-dUTP (see SI Text). We found no evidence of increased
apoptosis in bnc2?/?mice examined at E16.5 and E18.5. We
conclude that lack of bnc2 does not decrease the number of mitotic
cells by causing cell death.
Bnc2 Is Orthologous to the Disco Proteins.Thesimilaritybetweenthe
and discorelated has long been recognized (1, 2, 5, 12), but the
significance of this similarity has remained unclear because the
sequence of the Drosophila disco proteins has no similarity to that
of basonuclin, apart from the zinc fingers and the function of the
disco proteins in larval head development also seems to have no
relation to that of bnc1. In view of the similarity between the
function of bnc2 in mouse head development and that of the
Drosophila disco proteins in larval head development (4), we
The recent sequencing of a number of insect genomes has shown
that Drosophila and other dipterans have undergone accelerated
evolution and have diverged considerably more from vertebrates
than other arthropods (20). We reasoned that comparison of the
basonuclin sequences with the nondipteran disco sequences could
shed light on the relatedness of the 2 groups of proteins.
double-stained with anti-?-gal (red) and anti-pankeratin (green) antibodies. (A–C) Embryo at E14.5. (A) View of the whole section showing ?-gal staining virtually
limited to palatal processes. Staining is present in the anterior region of the palatal processes (B), but not in the posterior part (C). (D–H) Embryo at E16.5. (D) shows
parts of the processes. (G and H) Enlargement of the palatal processes boxed in D, counterstained with DAPI (blue) for DNA. ?-gal is found in mesenchymal cells and
is absent from the epithelia lining the palate and the tongue (G), stained green by the anti-keratin antibody (H). (I and J) Paraffin-embedded sections of nasal region
at E16.5, after whole-mount staining with X-gal and counterstaining with eosin. Regions stained with X-gal appear blue. ?-gal is specific to the mesenchymal cells
surrounding cartilage. b: brain, e: eye, m: molar tooth, n: nasal region, pp: palatal processes, t: tongue.
Detection of ?-gal and keratins in coronal sections of embryonic heads. (A–H) Frozen sections of bnc2?/?embryonic heads fixed in methanol/acetone and
At E14.5 (A and B), E16.5 (C and D), and E18.5 (E and F), the number of cells containing the phosphorylated histone is decreased by 30–50% in bnc2?/?embryos. This
m: mouth, md: mandible, n: nasal region, nc: nasal cavity, ns: nasal septum, pp: palatal processes, t: tongue.
Vanhoutteghem et al. PNAS ?
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BLASTP and TBLASTN similarity searches of the nondipteran
arthropod GenBank databases using the human bnc2 sequence
revealed that, apart from the zinc fingers, the predicted disco
proteins of Apis, Nasonia, Pediculus, and Tribolium possessed a
sequence of about 150 residues with over 65% identity to the
N-terminal region of bnc2 and bnc1 (Fig. 6A). In bnc1, bnc2, and
disco genes, this region was encoded by 3 exons, all of which were
interrupted by introns at exactly the same position, and 2 of which
were of the same size in insects and vertebrates (Fig. 6B). The
positions of the zinc finger pairs, their number, and the position of
the NLS were also similar in the nondipteran disco proteins and in
the basonuclins (Fig. 6C). Therefore the disco and the basonuclin
genes must be derived from a common ancestor. All of the
nondipteran arthropods examined possessed a single disco gene
that resembled Drosophila discorelated more than disco. We con-
Earlier studies have shown that the disco gene is expressed in the
the gut (21); disco-driven lacZ expression is also found in the joints
of the adult leg (22). Bnc2-driven lacZ expression was similarly
found predominantly in the gut of the mouse embryo (Fig. 6D), in
the mesodermal cells that surrounded the submucosa and around
the joints (Fig. S2). Therefore the Drosophila disco genes have a
pattern of expression similar to that of the bnc2 gene, and unlike
that of the bnc1 gene whose expression is confined to keratinocytes
and reproductive germ cells (5, 6). By its sequence, its tissue
distribution, and its function, bnc2 appears to be the vertebrate
homolog of the disco proteins.
From results presented here, it is clear that basonuclin 2 is
essential for the growth of a number of craniofacial bones, all of
which are of neural crest origin (23). Although expression of the
basonuclin 2 gene is also observed in mesenchymal cells not
derived from the neural crest (Fig. S2), these cells do not seem
to require basonuclin 2 for proper multiplication. Cleft of the
secondary palate can be caused by mutations in genes encoding
transcription factors, growth and signaling molecules, and their
receptors (17). Basonuclin 2 might regulate the expression of
some of these genes.
The relation between the basonuclins and the disco proteins is of
great interest. We provide here incontrovertible evidence that the
not recognized earlier because only Drosophila disco sequences
were available. The disco proteins of Drosophila have diverged
considerably more from the basonuclin sequences than those of
nondipterans, such as Tribolium. It has been suggested (24) that
Tribolium is better suited for comparisons between phyla than the
widely used dipterans and that when attempting to link human
genes to their Drosophila homologs, data from Tribolium should
approach was indeed necessary to resolve the relationship between
the Drosophila zen gene and the human Hox3 genes (25).
It has been postulated that the gene for bnc2 is the older of the
2 basonuclin genes and after its duplication to produce bnc1, the
latter was free to evolve in other directions, while the gene for bnc2
remained largely invariant and retained its original function (12).
We believe that this interpretation is correct and that bnc2 carries
out in vertebrates a function similar to that of disco in insects.
absence of the cephalopharyngeal skeletal structures that develop
from the gnathal segments (4, 26). This phenotype is strikingly
similar to that observed in mice lacking bnc2, whose most pro-
nounced defects lie in the pharyngeal region and particularly in the
gnathal bones (Fig. S1). Loss of disco in Tribolium also affects the
of the louse (PED: Pediculus humanus corporis), the flour beetle (TRI: Tribolium castaneum), the honey bee (API: Apis mellifera), and the jewel wasp (NAS: Nasonia
boundaries of bnc2, bnc1, and disco. (C) Structure of the basonuclins and disco proteins. Basonuclins contain 3 pairs of zinc fingers (vertical bars) and an NLS (red dot)
Relatedness of basonuclins and disco proteins. (A) Alignment of the N-terminal sequence and of the 3 pairs of zinc fingers of human bnc2 (GenBank
www.pnas.org?cgi?doi?10.1073?pnas.0905840106Vanhoutteghem et al.
RNA interference in larvae leads to a failure of the mandibular, Download full-text
maxillary and labial segments to extend from the head. RNA
interference in adults causes severe truncation of the maxillary and
labial appendages in both Drosophila and Tribolium (27, 28). The
truncation of the head appendages observed in the disco mutants
of these species resembles the truncation of the palatine and
pterygoid processes observed in mice lacking bnc2 (Fig. 3A). It
would be of interest to determine whether nondipteran disco genes
can rescue the phenotype of bnc2?/?mice.
We show here that bnc2 also has a function in cell multiplication,
disco proteins promote cell proliferation in the appendage primor-
dia (28). Conservation of the zinc fingers by bnc1, bnc2, and the
shared by these proteins and suggests that their molecular targets
are similar or identical.
Human monosomy 9p associates various craniofacial abnormal-
ities, which include an excessively arched palate and defects of the
nasal septum. The genomic region involved has been narrowed
down to 4.7 Mb in close proximity to the bnc2 gene (29). Dupli-
cations of the 9p22–24 region associated with breakpoints in the
region of the bnc2 gene are also associated with craniofacial
tal defects may be the consequence of bnc2 haploinsufficiency
caused by disruption of the gene.
Materials and Methods
Inverse Genomic and Multiplex PCR. DNA was prepared from mouse tails as
previously described (31). For inverse genomic PCR, DNA was digested with PstI,
(one around the insertion site and 1 in pU21) were mixed in the same PCR (30
cycles of amplification at 95° for 1 min, 56° for 1 min, and 72° for 1 min).
Southern Blot Analysis. Southern blots of mouse tail DNA were performed as
previously described (32). The probe was an 861-bp fragment of the bnc2 intron
located immediately 3? to the vector.
Northern Blot. Embryos were pulverized under liquid nitrogen and RNA was
electrophoresis on a formaldehyde/1% agarose gel, partially hydrolyzed in 50
mM NaOH to facilitate transfer of large RNA, and transferred onto a nylon
membrane by vacuum blotting. The probe was a 900-bp fragment in exon 5 of
bnc2. Hybridization was carried out in ExpressHyb (Clontech) using the manu-
facturer’s protocol except that the last washes were at 65° instead of 50°.
Expression of Genes Located in the Vicinity of the Gene-Trap Insertion Site.
following (GenBank): 1810054D0, Snapc3, Psip1, LOC100040611, 4930473A06,
LOC668010, LOC100040660, LOC100040284, D530005L17, Sh3gl2, A930009N24,
heads at E16.5 using Qiagen columns and Affymetrix mouse gene 1.0 ST arrays.
Three wt and 3 homozygous mutants were analyzed. Gene-level expression
values were derived from the CEL file probe-level hybridization intensities using
the model-based Robust Multichip Average algorithm (RMA). RMA performs
normalization, background correction, and data summarization. Differential
rate of this analysis was calculated using the Benjamini and Hochberg approach
in order to correct for multiple comparisons.
Histological Analysis. For sections stained with hematoxylin and eosin, embryos
ded in paraffin and stained.
For indirect immunofluorescence, embryos were snap frozen in OCT (Miles)
and isopentane and cut with a cryomicrotome at 7 ?m. Sections were fixed in
earlier (33). See SI Text for antibodies used.
Phosphohistone H3-positive cells were counted using the ImageJ software.
Over 40,000 cells were counted in a total of thirteen E14.5, E16.5, and E18.5
Method for staining of cartilage and bone with alizarin red/alcian blue is
available as SI Text.
ACKNOWLEDGMENTS. We thank Bruno Passet (INRA, Jouy-en-Josas) for im-
Centre National de la Recherche Scientifique, the Association pour la Recherche
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