Download Pax1/Pax9 and vertebral column development

5399
Development 126, 5399-5408 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
DEV4240
Pax1 and Pax9 synergistically regulate vertebral column development
Heiko Peters1,2, Bettina Wilm1, Norio Sakai1, Kenji Imai1, Richard Maas2 and Rudi Balling1,*
1GSF-Research Center for Environment and Health, Institute of Mammalian Genetics, 85764 Neuherberg, Germany
2Brigham and Women’s Hospital and Harvard Medical School, Genetics Division, Department of Medicine, 20 Shattuck
Street,
Boston, MA 02115, USA
*Author for correspondence (e-mail: [email protected])
Accepted 8 September; published on WWW 9 November 1999
SUMMARY
The paralogous genes Pax1 and Pax9 constitute one group
within the vertebrate Pax gene family. They encode closely
related transcription factors and are expressed in similar
patterns during mouse embryogenesis, suggesting that
Pax1 and Pax9 act in similar developmental pathways. We
have recently shown that mice homozygous for a defined
Pax1 null allele exhibit morphological abnormalities of the
axial skeleton, which is not affected in homozygous Pax9
mutants. To investigate a potential interaction of the two
genes, we analysed Pax1/Pax9 double mutant mice. These
mutants completely lack the medial derivatives of the
sclerotomes, the vertebral bodies, intervertebral discs and
the proximal parts of the ribs. This phenotype is much
more severe than that of Pax1 single homozygous mutants.
In contrast, the neural arches, which are derived from the
lateral regions of the sclerotomes, are formed. The analysis
of Pax9 expression in compound mutants indicates that
both spatial expansion and upregulation of Pax9 expression
account for its compensatory function during sclerotome
development in the absence of Pax1.
In Pax1/Pax9 double homozygous mutants, formation
and anteroposterior polarity of sclerotomes, as well as
induction of a chondrocyte-specific cell lineage, appear
normal. However, instead of a segmental arrangement of
vertebrae and intervertebral disc anlagen, a loose
mesenchyme surrounding the notochord is formed. The
gradual loss of Sox9 and Collagen II expression in this
mesenchyme indicates that the sclerotomes are prevented
from undergoing chondrogenesis. The first detectable
defect is a low rate of cell proliferation in the ventromedial
regions of the sclerotomes after sclerotome formation but
before mesenchymal condensation normally occurs. At
later stages, an increased number of cells undergoing
apoptosis further reduces the area normally forming
vertebrae and intervertebral discs. Our results reveal
functional redundancy between Pax1 and Pax9 during
vertebral column development and identify an early role of
Pax1 and Pax9 in the control of cell proliferation during
early sclerotome development. In addition, our data
indicate that the development of medial and lateral
elements of vertebrae is regulated by distinct genetic
pathways.
INTRODUCTION
tuned regulation is required to co-ordinate the prepatterning of
individual skeletal elements by region-specific mesenchymal
growth and condensations that are finally replaced by cartilage
and bone. Although in the recent years much progress has been
made in the understanding of somite patterning and sclerotome
formation (Tajbakhsh and Spörle, 1998), most aspects of the
genetic control that regulates the complex development from
the sclerotomal mesenchymes to the vertebral column remain
to be elucidated.
Pax1 and Pax9 form one group within the family of nine
vertebrate Pax genes, which are unified by the presence of the
paired box that encodes a DNA-binding domain (Walther et al.,
1991; Noll, 1993). Analyses of mouse mutants of all Pax genes
have demonstrated multiple roles in the genetic control of
mammalian organogenesis (reviewed in Dahl et al., 1997). The
128 amino acid long DNA-binding paired domains of Pax1 and
Pax9 are almost identical (98%). DNA-binding studies
have shown that the e5 site, a DNA element present in the
The vertebrate body is supported by the vertebral column, a
series of segmental skeletal elements that provide both stability
and mobility. The development of the axial skeleton is a multistep process starting with the formation of somites from the
unsegmented paraxial mesoderm on both sides of the neural
tube. Shortly after formation, the somites compartmentalise to
generate dermomyotomes and sclerotomes, the latter forming
skeletal elements of the vertebral column and ribs. Vertebral
column development requires the co-ordination of a series of
cellular events. These include de-epithelialization of somites,
proliferation and migration of sclerotomal cells, and
establishment of anteroposterior polarity of the sclerotome
(reviewed in Keynes and Stern, 1988; Christ and Wilting, 1992;
Christ and Ordahl, 1995). In mammals, a single vertebra is
composed of a variety of components that perform different
functions, depending on the level of the body axis. Thus, a fine-
Key words: Pax1, Pax9, Vertebral column, Chondrogenesis,
Proliferation, Apoptosis, Mouse
5400 H. Peters and others
Fig. 1. Phenotypes of the vertebral columns of
Pax1/Pax9 double mutants. (A-I) Ventral view of
skeletal staining of newborn mice showing
cartilaginous elements in blue and ossified elements
in red. Genotypes of the specimens are indicated on
the left side (Pax1) and on top (Pax9) of the panels. c,
t, l, and s in the wild-type skeleton (A) mark the
cervical, thoracic, lumbar and sacral region of the
vertebral column, respectively. (A-C) In the presence
of two wild-type alleles of Pax1, Pax9 deficiency
does not affect the development of the vertebral
column. (D-F) In heterozygous Pax1 mutants, mild
abnormalities in the vertebrae of the lumbar region
(D, Wilm et al., 1998) are exacerbated by introducing
heterozygosity (arrow in E) and homozygosity (F) of
the Pax9 mutant allele. (G-I) In the absence of Pax1,
loss of 50% of Pax9 gene dosage causes a further
reduction of vertebral bodies and intervertebral discs
(H). (I) In Pax1/Pax9 double homozygous mutants,
no ventral elements of the vertebral column form.
Note also the absence of caudal vertebrae and the
floating ribs in the double mutant.
Drosophila even-skipped promoter, as well as
CD19-2 (A-ins) and H2A-17C, two elements
originally identified as recognition sites for
Pax5, are bound by Pax1 and Pax9 (Chalepakis
et al., 1991; Czerny et al., 1993; Neubüser et al.,
1995; H. P. and R. B., unpublished results).
These findings suggest that Pax1 and Pax9 may
have similar functions in tissues in which they
are co-expressed.
During mouse embryogenesis, Pax1 and Pax9
are expressed in similar patterns in the
pharyngeal pouches and the developing
limbs (Neubüser et al., 1995). Pax9-specific
expression domains in craniofacial regions are
correlated with its essential role for secondary
palate and tooth formation (Peters et al., 1998). Pax1 and Pax9
are the only Pax genes expressed in the sclerotomes, the
Fig. 2. Absence of
Pax1/Pax9 affects mainly
the ventral elements of the
vertebral column. Ventral
view (A,B) and lateral view
(C,D) of the lumbar region
of the vertebral column
from wild-type (A,C) and
Pax1/Pax9-deficient (B,D)
newborn mice.
(A,C) Vertebral bodies (vb)
have formed single
ossification centres and
alternate with intervertebral
discs (id). (B,D) Vertebral
bodies and intervertebral
discs are absent in the
double mutant mice,
whereas the neural arches
(na) are formed. In addition, elements of the sacrum (sa, arrows in B)
and the proximal parts of the ribs (arrowheads in B) are missing in
the absence of Pax1/Pax9. Note also ectopic formation of cartilage
that connects adjacent neural arches (arrowheads in D).
ventromedial compartments of somites. The expression of
Pax1 precedes that of Pax9 and is initially found in all
sclerotomal cells. At later stages of sclerotome differentiation,
the expression of Pax1 acquires a maximum in the posterior,
ventromedial compartment. On the contrary, transcription of
Pax9 is predominantly detectable in the posterior, ventrolateral
region of the sclerotomes. In the region of the future vertebral
bodies, expression of Pax1 and Pax9 is downregulated when
sclerotomal cells start to chondrify, but their expression is
maintained in the perichondrium of vertebrae and in the
intervertebral disc anlagen (Deutsch et al., 1988; Neubüser et
al., 1995; Peters et al., 1995; Ebensperger et al., 1995).
Previous analyses of three natural Pax1 mutant alleles in
mice, the undulated series (un, unex, UnS), have demonstrated
an important role for Pax1 during sclerotome development
(Grüneberg, 1954; Balling et al., 1988; Wallin et al., 1994;
Dietrich and Gruss, 1995). Using a gene targeting
approach, we have recently shown that homozygous mutant
mice of a defined Pax1 null allele are viable and exhibit
morphological abnormalities of vertebrae and intervertebral
discs similar to those seen in the recessive alleles un and unex
(Wilm et al., 1998). In contrast, homozygous mutants of the
semidominant UnS allele, which has a deletion including the
Pax1 gene, die at birth and exhibit a severely affected
Pax1/Pax9 and vertebral column development 5401
Fig. 3. Upregulation and spatial expansion of the
Pax9lacZ expression in the absence of Pax1.
(A-J) Expression of the Pax9lacZ allele is visualized by
X-Gal staining of E10.5 mouse embryos. Genotypes
are indicated on top of the panels. (A,D,G) Lateral
view and (I) dorsal view of embryos showing the
expression of the Pax9lacZ allele in Pax9 single
heterozygous mutants. Pax9lacZ expression is mainly
detectable in the posterior halves of young (D) as well
as matured sclerotomes (G, I). (B,E,H) Lateral view,
and (J) dorsal view of embryos showing Pax9lacZ
expression in Pax1−/−, Pax9+/− mutants. Expression of
Pax9lacZ is ectopically induced and strongly
maintained in the anterior half of the sclerotomes
(arrows in E and H). In addition, the expression is
upregulated and dorsally expanded in the posterior
halves of the sclerotomes (arrowheads in E and H).
Upregulation of Pax9lacZ is predominantly detectable
in the lateral region of the sclerotomes (J).
(C,F) Similarly, dorsal expansion and upregulation
(arrowhead), as well as ectopic activation (arrow) of
Pax9lacZ expression is also found in Pax1/Pax9 double
homozygous mutants. Brackets in D-H indicate the
size of one somite. Asterisk in D marks a damage of
the tissue which occurred during processing of this
sample. a, anterior; p, posterior.
vertebral column (Wallin et al., 1994). Thus, the deletion in
the UnS allele interferes with vertebral column development
not only through the loss of Pax1 function. Homozygous
Pax9 mutants have skeletal defects in the limbs and in the
skull, but exhibit no obvious defects in the axial skeleton
(Peters et al., 1998).
There is accumulating evidence that closely related
transcription factors perform redundant functions during
development. This has been shown for paralogous Hox genes
(Rijli and Chambon, 1997, and references therein), the zinc
finger protein encoding genes Gli2 and Gli3 (Mo et al., 1997),
for the basic helix-loop-helix genes MyoD and MRF4 (Rawls
et al., 1998), and the homeobox-containing genes Prx1 and
Prx2 (ten Berge et al., 1998; Lu et al., 1999), and Alx1 and
Cart1 (Qu et al., 1999). The overlapping expression patterns
during mouse embryogenesis and the high sequence
conservation of the DNA-binding paired domain of members
within each group of the Pax gene family suggest redundancy
at sites where these genes are co-expressed.
To investigate a possible genetic interaction between Pax1
and Pax9 during mammalian development, we analysed double
mutant mice. These mutants reveal a gene dosage-dependent
co-operation of Pax1 and Pax9 during sclerotome
development. In the absence of Pax1 and Pax9, no vertebral
bodies and intervertebral discs are formed, a phenotype that is
not found in either single homozygous mutant, whereas mice
lacking three functional gene copies exhibit intermediate
phenotypes. Notably, the formation of neural arches is not
affected in the double mutants, demonstrating a different
genetic control for the development of these vertebral
structures. Pax1 can fully compensate for the absence of Pax9
whereas Pax9 function incompletely rescues vertebral column
formation in Pax1-deficient mice. We show that Pax1 and Pax9
are required to maintain chondrocyte-specific gene expression
in the sclerotome, but their earliest function during vertebral
column development may be involved in the control of cell
proliferation during a specific and relatively short phase of
sclerotome development.
MATERIALS AND METHODS
Fig. 4. Expression of Uncx4.1 is preserved in Pax1/Pax9 double
homozygous mutant embryos. (A) Wild-type embryo (E10.5),
hybridized with a Uncx4.1-specific RNA-probe. Uncx4.1 expression
marks the posterior halves of newly formed somites and becomes
restricted to the posterior halves of the sclerotomes as the somites
differentiate. (B) A similar expression pattern of Uncx4.1 is
detectable in Pax1/Pax9 double homozygous mutants.
Animals
Pax1null/Pax9lacZ double heterozygous mutant mice were generated by
crossing mice carrying null alleles of Pax1 (Pax1null) and Pax9
(Pax9lacZ), both of which have been described previously (Wilm et al.,
1998; Peters et al., 1998). Genotyping of mice and embryos was
performed as described previously (Wilm et al., 1998; Peters et al.,
1998).
5402 H. Peters and others
Phenotype analyses
Skeletal staining of newborn mice was performed as described by
Kessel et al. (1990). To visualize Pax9lacZ expression, mutant mouse
embryos were stained with X-gal and cleared with benzylbenzoate/
benzylalcohol as described in Gossler and Zachgo (1993). Wholemount in situ hybridizations with digoxigenin-labeled RNA probes
specific for Sox9 (Wright et al., 1995), the α1 subunit (Metsaranta et
al., 1991) of Collagen II (Col2α1), and Uncx4.1 (Mansouri et al.,
1997) were performed according to Wilkinson (1992). In situ
hybridization on paraffin sections was carried out as described in
Neubüser et al. (1995).
BrdU labeling and TUNEL staining
Cell proliferation was analysed by incorporation of BrdU into
embryos and subsequent imunohistochemical detection of BrdU on
paraffin sections according to the protocol of the ‘In Situ Cell
Proliferation Kit’ (Boehringer-Mannheim). Sections were stained
with diaminobenzidine as a colour substrate and briefly counterstained
with hematoxylin. BrdU-positive cells were counted in a defined area
around the notochord (Fig. 7). To demonstrate statistically significant
differences, standard deviations were calculated from numbers
obtained at both early and advanced phases of sclerotome
development. Numbers from at least six sections were combined in
each group for determination of the standard deviation.
Apoptotic cells were detected using the ‘In Situ Cell Death
Detection Kit’ (Boehringer-Mannheim). Fluorescein-labeled DNA
was incorporated in the terminal transferase reaction and directly
visualized after counterstaining the sections with propidium iodide.
RESULTS
Pax1 and Pax9 are required for vertebral column
development in a gene dosage-dependent manner
To reveal a potential interaction between Pax1 and Pax9 during
development, we crossed double heterozygous mutant mice
carrying the previously described Pax1null and Pax9lacZ alleles
(Wilm et al., 1998; Peters et al., 1998), which we refer to in
the following as Pax1 and Pax9 mutants, respectively. At the
newborn stage, all nine possible genotypes were obtained at
the expected Mendelian ratio, indicating that the complete loss
of Pax1 and Pax9 does not result in embryonic lethality (n=143
for newborn mice). Shortly after birth, however, all
homozygous Pax9 mutants die due to a cleft secondary palate
(Peters et al., 1998). In addition, we never observed surviving
Pax1−/−; Pax9+/− mice in our breeding colony, indicating that
this genotype causes early postnatal lethality. Except for the
vertebral column (see below), all other skeletal phenotypes
observed in the double homozygous mutants were specific to
the absence of either Pax1 or Pax9. These include
abnormalities of the shoulder skeleton in Pax1-deficient mice
(Wilm et al., 1998), and preaxial polydactyly and craniofacial
abnormalities in the absence of Pax9. Furthermore, double
homozygous mutant mice lack the derivatives of the third and
fourth pharyngeal pouches, a phenotype that is also seen in
Pax9-deficient mice (Peters et al., 1998, and data not shown).
The vertebral columns of heterozygous and homozygous
Pax9 mutants appear normal, as revealed by skeletal staining
of newborn mice (Fig. 1B,C). However, in mice that are
deficient for one functional copy of Pax1, heterozygosity and
homozygosity of the Pax9 mutation result in vertebral
malformations that are not seen in single Pax1 heterozygous
mutants. In the lumbar region, fused vertebrae, split vertebrae,
as well as ossified fusions between vertebrae and neural arches
are found (Fig. 1E,F). Thus, Pax1 and Pax9 interact during
vertebral column development. This result is more clearly
demonstrated in mutants that lack both copies of Pax1: in
Pax1−/−; Pax9−/− double homozygous mutants, no vertebral
bodies and intervertebral discs are formed (Fig. 1I), thereby
dramatically decreasing the overall length of the body axis.
Furthermore, the proximal parts of most ribs, all skeletal
elements of the tail, as well as the connection between sacrum
and pelvic girdle are missing (Figs 1I, 2B,D). A similar, but
less severe, phenotype was detectable in the vertebral column
of Pax1−/−; Pax9+/− mutants. In these mice, an irregular,
cartilaginous rod that is interrupted by ventral extensions of the
neural arches replaces the vertebral bodies and intervertebral
discs of the lumbar region (Fig. 1H). These results identify a
role for Pax9 in vertebral column formation and reveal a gene
dosage-dependent manner of Pax1/Pax9 function during
vertebral column development. The findings also indicate that
Pax1 and Pax9 may have similar or identical functions that are
redundant at many steps during sclerotome development.
Interestingly, the lateral derivatives of the sclerotomes, the
neural arches, are formed in the absence of Pax1 and Pax9,
although they were found to have an abnormal shape (Fig. 2D).
Moreover, in the lumbar region, ectopic cartilage formation is
detectable in the dorsal region between adjacent neural arches
(Fig. 2D). The disturbed development of the neural arches
might be a direct consequence of Pax1 and Pax9 deficiency.
However, secondary effects of the complete absence of ventral
vertebral elements could also contribute to this phenotype.
While our analysis does not distinguish between these two
possibilities, the presence of neural arches in Pax1/Pax9 double
homozygous mutants rules out an essential role for Pax1 and
Pax9 in their formation. Furthermore, the development of
neural arches indicates that sclerotome formation can not be
completely abolished in the absence of Pax1 and Pax9.
Upregulation and spatial expansion of sclerotomal
Pax9 expression in the absence of Pax1
To explore the basis of the functional redundancy between
Pax1 and Pax9 during vertebral column development and to
test whether Pax1 and Pax9 functions are required to maintain
Pax9 promoter activity, we analysed lacZ expression of
Pax1/Pax9 compound mutants. Previous studies have shown
that the expression pattern obtained by X-gal staining of
heterozygous Pax9 mutants recapitulates the endogenous
expression of Pax9 (Peters et al., 1998, and Fig. 3A). In the
sclerotomes of E10.5 embryos, lacZ expression is mainly
found in the posterior halves, whereas a narrow expression
domain is detectable in the anterior half, adjacent to the
preceding somite boundary (Fig. 3D,G). By gross inspection,
lacZ expression along the body axis appears upregulated in
Pax1−/−; Pax9+/− embryos (Fig. 3B). In the sclerotomes of these
mutants, the lacZ expression domain is dorsally expanded in
the posterior halves and exhibits a rectangular instead of the
triangular shape (Fig. 3E). In addition, lacZ expression is
induced in the anterior half of the sclerotomes (Fig. 3E),
where it is strongly maintained during further sclerotome
development (Fig. 3H). A similar, expanded expression pattern
is found in double homozygous mutant embryos (Fig. 3C,F).
From these observations, we conclude that, in wild-type
embryos, the function of Pax1 is involved in the spatial
Pax1/Pax9 and vertebral column development 5403
restriction of Pax9 expression to the posterior, ventral domain
of the sclerotomes. Therefore, spatial expansion and
upregulation of Pax9 in the sclerotomes is a likely mechanism
for the partial rescue of vertebral column development in Pax1
mutant mice. Furthermore, the maintenance of lacZ expression
in the double homozygous mutant shows that Pax9
transcription during early sclerotome development does not
require the function of Pax1, or of Pax9 itself (Fig. 3C,F).
Anteroposterior polarity of somites and
chondrogenesis
To identify and characterise the onset of phenotypic
abnormalities in Pax1/Pax9 double mutants, we performed in
situ hybridisation of genes that are expressed during early
sclerotome development. The expression of Uncx4.1, a pairedtype homeobox-containing gene, is induced in the posterior
half of newly formed somites and thereafter is maintained in
the posterior halves of the sclerotomes (Mansouri et al., 1997).
At E10.5 (around 30 somites), the expression patterns of
Uncx4.1 were indistinguishable among all possible allelic
combinations of Pax1 and Pax9 mutations (Fig. 4A, B, and data
not shown). This result shows that Pax1 and Pax9 are not
necessary to maintain the expression of Uncx4.1 and indicates
that the anteroposterior polarity of the sclerotomes is not
disturbed in Pax1/Pax9 double homozygous mutants.
To investigate the induction and fate of the chondrogenic cell
lineage in the sclerotomes of Pax1/Pax9 double mutants,
expressions of Sox9, encoding a HMG-box-containing
transcription factor, and Collagen II (Col2α1) were analysed.
Recent studies have shown that Sox9 is required for cartilage
formation (Bi et al., 1999). Col2α1, a direct target of Sox9 in
precartilaginous mesenchymes (Bell et al., 1997), encodes a
major component of the extracellular matrix of cartilage and
was shown to be required for normal vertebral column
development (Li et al., 1995). At E10.5, both genes are
transcribed in a similar, segmented pattern along the body axis
of normal embryos (Fig. 5A,D). This pattern was detectable in
all Pax1/Pax9 compound mutants including double
homozygous Pax1/Pax9 mutants (Fig. 5B,C,E,F, and data not
shown). In addition to the segmented expression pattern, a
continuous expression of Col2α1 is normally detectable in the
ventral domain within the somites, between forelimb and
hindlimb of E10.5 embryos (Fig. 5D,E,G). In contrast, this
domain was barely detectable in double homozygous mutants
(Fig. 5F,H). Cross sections through this region revealed that the
reduction of the Col2α1 expression domain in double
homozygous mutants is caused by a size reduction of the
ventromedial region of the sclerotome, rather than by the
inability of the sclerotome to express Col2α1 (Fig. 5J). These
results confirm that the absence of Pax1 and Pax9
predominantly affects the ventromedial region of the
sclerotomes, whereas the lateral sclerotome appears to develop
normally at this stage.
The presence of Col2α1 expression in double homozygous
mutants at E10.5 indicates that a chondrogenic cell fate is
induced in the ventromedial regions of the sclerotomes in the
absence of Pax1 and Pax9 and that chondrogenesis must be
arrested at a later stage of embryonic development.
Chondrogenesis of the vertebral column starts in the cervical
region around E12.5 of mouse development. At this stage,
the sclerotomes have formed chondrifying rudiments of the
vertebrae that express Col2α1 and alternate with yet
undifferentiated mesenchymes of the future intervertebral
discs (Fig. 6A,E). In the absence of Pax1 and Pax9, a loose
mesenchyme surrounding the notochord has developed that
lacks signs of cartilage formation and is completely devoid of
Col2α1 transcripts (Fig. 6D,H). In Pax1 single homozygous
mutants, the overall size of vertebral precursors is reduced
(Fig. 6B,F), whereas in Pax1−/−; Pax9+/− embryos, cartilage
formation was found in the ventral sclerotome only. In the
latter mutants, the cartilaginous rudiments exhibit an irregular
spacing (Fig. 6G). Expression patterns in the sclerotomes
similar to those shown for Col2α1 have been obtained using a
Sox9-specific probe (data not shown). Together, these findings
demonstrate that the control of normal size, shape and
spacing of the cartilaginous precursors of the vertebrae and
intervertebral discs require Pax1 and Pax9 in a gene-dosagedependent manner.
Programmed cell death and cell proliferation in
Pax1/Pax9-deficient sclerotomes
To investigate the mechanisms that cause the reduced size of
the ventromedial sclerotome in Pax1/Pax9 double homozygous
mutants, the number of cells undergoing apoptosis and the
number of proliferating cells were determined during early
sclerotome development. We analysed serial transverse
sections of tail somites of E12.5 embryos in these experiments
because, at this level of the body axis, the formation of skeletal
elements is entirely dependent on Pax1/Pax9 function.
The histological analysis of caudalmost somites revealed no
obvious defects of the sclerotomes. Both formation as well as
ventromedial migration of sclerotomal cells occur in the
absence of Pax1 and Pax9 (Fig. 7A,C). However, as somites
mature, the sclerotomes of double homozygous mutant
embryos become less condensed, as compared to the wild-type
control (Fig. 7I,K). Notably, Tunel-labeling revealed that the
number of cells undergoing programmed cell death in the
sclerotomes of this region was not increased in Pax1/Pax9
double homozygous mutant embryos (data not shown). On the
contrary, when the sclerotomes of wild-type embryos have
formed precartilaginous condensations, the proportion of
apoptotic cells decreases almost completely (Fig. 7O) whereas,
at the same axial levels of Pax1/Pax9 double homozygous
mutant embryo, dying cells were abundant (Fig. 7P). Thus,
prevention of apoptosis in the sclerotomes may require the
functions of Pax1/Pax9.
To determine whether cell proliferation is affected in the
developing Pax1/Pax9-deficient sclerotomes, we analysed
incorporation of BrdU in dividing cells of E12.5 embryos.
Starting shortly after sclerotome formation, we analysed
approximately 20 successive somites in 50 µm intervals. (Fig.
7M). The comparison of BrdU-labelling profiles of wild-type
and Pax1/Pax9 mutant sclerotomes reveals two phases. In the
first phase (intervals 1-7, Fig. 7N), no differences in the number
of BrdU-positive cells were found in wild-type and Pax1/Pax9
mutant embryos (84±11.5 in wild type versus 82±11.7 in
Pax1/Pax9 double homozygous mutants). In the second phase,
however, the number of dividing cells decreases in the mutant
sclerotomes (65.7±7.7) whereas significantly higher numbers
of labelled cells were found in the wild-type sclerotomes
(94.7±8.7). Conversely, a higher number of proliferating cells
were found in the developing neural tube of Pax1/Pax9 double
5404 H. Peters and others
Fig. 5. Induction of a chondrogenic cell fate in
Pax1/Pax9 mutant embryos, visualised by whole
mount in situ hybridization of Sox9 (A-C) and
Col2α1 (D-H) at E10.5. In the somites, a segmented
expression pattern of Sox9 is detectable in wild-type
(A), Pax1−/−, Pax9+/+ (B), and Pax1−/−, Pax9−/−
embryos. (D-F) In embryos of the same genotypes, a
similar pattern is found for Col2α1 expression.
(G) Higher magnification of a control embryo
showing a continuous band of Col2α1 expression
(arrows) between forelimb (fl) and hindlimb (hl).
(H) This expression domain is absent in Pax1/Pax9
double homozygous mutant. (I) Transverse section of
a wild-type embryo at E10.5 stained for Col2α1
expression in the developing sclerotomes at the
prospective trunk level. The regions of the future
vertebral bodies and neural arches are outlined by
Col2α1 expression. (J) Col2α1 expression is also
detectable in the Pax1−/−, Pax9−/− mutant embryo,
however, note the dorsoventral reduction of the
sclerotome size in the Pax1/Pax9 double homozygous
mutant as compared to the wild-type control (vertical
bars).
Fig. 6. Normal formation of cartilaginous vertebral precursors is Pax1/Pax9 gene dosage dependent. (A-D) Sagittal sections of the cervical
region of E12.5 embryos stained with Hematoxylin/Eosin (HE). Genotypes are indicated on top of the panels. (E-H) In situ hybridization of
Col2α1 to sections adjacent to those shown in A-D. (A,E) In wild-type embryos, Col2α1 is strongly expressed in the chondrifying anlagen of
the vertebral bodies (vb), in the notochord (nc), and weakly in the future intervertebral discs (id). (B,F) In the absence of Pax1, vertebral bodies
are smaller. (C,G) In Pax1−/−, Pax9+/− embryos, remnants of the vertebral bodies exhibit an irregular spacing. In addition, expression of Col2α1
in the region dorsal to the notochord is almost absent (arrowheads). (D,H) In Pax1/Pax9 double homozygous mutants, the mesenchyme
surrounding the notochord is not condensed and lacks Col2α1 expression.
Pax1/Pax9 and vertebral column development 5405
Fig. 7. Cell proliferation and programmed cell
death in the sclerotomes of Pax1/Pax9 double
homozygous mutants. Transverse sections of
the developing tail of E12.5 embryos that were
labeled with BrdU were collected on adjacent
slides. One set of sections from either wildtype (A,E,I) or Pax1/Pax9 double homozygous
mutant (C,G,K) was stained with
Hematoxylin/Eosin. Adjacent sections were
stained for the presence of incorporated BrdU
to label dividing cells (wild type: B,F,J;
Pax1/Pax9 mutant: D,H,L). Number of BrdUpositive cells was always determined in the
same area, as indicated by the red frame in B.
(A,E) Compartmentalisation of somites into
dermomyotomes (d) and sclerotome (sc) in a
wild-type embryo. (C,G) No obvious defects
in sclerotome formation and ventromedial
migration are detectable in young somites of
Pax1/Pax9 double homozygous mutants. Note
the larger size of the neural tube (arrow in G).
(I) Cell density in the sclerotomes has
increased in the wild type. (K) Condensation
around the notochord is not detectable in the
sclerotome of Pax1/Pax9 mutants. (M) The
region that was analysed for BrdU counting is
indicated in the tail region of a E12.5 embryo.
Values of BrdU-positive cells in the
sclerotomes and in the neural tube represent
50 µm distances and are given in a posterior to
anterior direction. Superscripts in the first two
columns refer to the sections shown in the
upper half of this figure. (N) Graph of
numbers of BrdU-positive cells in the
sclerotome (squares) and in the neural tube (circles) of wild-type (filled symbols)
and Pax1/Pax9-deficient (open symbols) embryos. Note the lower number of BrdU
positive cells in the sclerotomes of Pax1/Pax9 mutant embryos as development of
the sclerotomes proceeds. (O) Transverse section of the developing vertebral
column of a wild-type embryo at the level as indicated by the double-arrow in (M),
stained for the presence of apoptotic cells. Only a few cells undergo programmed
cell death in the precartilaginous mesenchyme. (P) At the same level of a
Pax1/Pax9-deficient embryo, a high number of apoptotic cells is typically present.
hg, hindgut; nt, neural tube.
homozygous mutants (Fig. 7N), a finding that is consistent with
the successive increase of the size of the neural tube in the
mutant embryo (Fig. 7G,K).
These results show that in the absence of Pax1 and Pax9 the
sclerotomal cell population is reduced by a decrease in cell
proliferation, followed by an abnormally high rate of apoptosis
that later contributes to the substantial loss of the sclerotome size.
DISCUSSION
Redundant functions of Pax1 and Pax9 during
vertebral column development
In this report, we demonstrate that the closely related
transcription factors Pax1 and Pax9 have conserved biological
functions during the development of the sclerotome. In the
absence of Pax1 and Pax9, no ventral elements of the vertebral
column develop along the entire body axis. The comparison of
this phenotype to that of either single homozygous mutants
reveals that Pax1 can completely compensate for the absence
of Pax9, whereas loss of Pax1 is rescued by Pax9 to a
considerable extent (Fig. 1). In addition, the severity of the
phenotype in the absence of either gene is significantly
exacerbated by introducing heterozygosity of the other gene.
Thus Pax1 and Pax9 functions are redundant and their
requirement for normal formation of the vertebral column is
gene dosage dependent. Functional redundancy has recently
been identified for Pax2 and Pax5, two paralogous genes of the
Pax2/Pax5/Pax8 subgroup, which have overlapping functions
during midbrain and cerebellum development (Schwarz et al.,
1997). Similarly, earlier onset of embryonic lethality than in
homozygous Pax3 mutants was found in mouse embryos
lacking both Pax3 and Pax7, which together constitute another
class within the Pax gene family (Mansouri and Gruss, 1998).
Together, these data suggest that functional redundancy
between paralogous Pax genes is a general mechanism that can
act in those tissues in which members of a given subgroup have
overlapping expression patterns.
5406 H. Peters and others
In the absence of Pax1, Pax9 expression is upregulated and
is ectopically induced in the anterior domain of the sclerotomes
(Fig. 3), thereby providing a basis for functional redundancy
between Pax1 and Pax9 in the developing vertebral column.
The molecular mechanism by which upregulation of Pax9 is
achieved is presently unclear. Our data indicate that, in wildtype embryos, Pax1 is involved in the suppression of Pax9
expression in the anterior sclerotome. Interestingly, a similar
repression between paralogous Pax genes has recently been
identified during the development of the dermomyotomes,
which generate dermis and skeletal muscles. In Pax3 mutant
embryos, expression of Pax7 is ectopically activated in the
anterior half of the dermomyotome and in vitro data suggest a
direct role of Pax3 in the suppression of Pax7 in cultured
myoblasts (Borycki et al., 1999). Together, these results
strongly suggest that Pax1 and Pax3 functions contribute to the
control of the normal expression patterns of Pax9 and Pax7,
respectively, in the sclerotomes and dermomyotomes of the
developing somite. However, in the posterior sclerotomes of
wild-type embryos, Pax1 and Pax9 are co-expressed, indicating
that high levels of Pax1 do not necessarily result in the
downregulation of Pax9 and suggesting that Pax9-activating
signals are more potent in the posterior half of the sclerotome.
Cell proliferation and prevention of apoptosis
Complete failure of organ formation in Pax mutants has
demonstrated essential roles of Pax genes during
organogenesis. Although localised cell proliferation is
commonly observed in tissues participating in organogenesis,
the role of Pax genes in cell survival during embryogenesis is
not well understood. Reduced proliferation rates have been
found in the developing diencephalon of Small-eye (Pax6)
mutant embryos (Warren and Price, 1997). In addition, it was
shown that Pax5 is required to stimulate B cell proliferation in
vitro (Wakatsuki et al., 1994) and cells overexpressing Pax
genes can form tumours in nude mice (Maulbecker and Gruss,
1993). Moreover, growth inhibition as well as induction of
apoptosis has been observed by inactivating Pax genes in in
vitro systems (Gnarra and Dressler, 1995; Rossi et al., 1995;
Bernasconi et al., 1996; Hewitt et al., 1997). Increased
apoptosis in the neural tube and somites was recently described
in homozygous Pax3 mutant embryos (Borycki et al., 1999).
Likewise, results of the present work show that sclerotomal
cells of Pax1/Pax9-deficient mice undergo increased
programmed cell death (Fig. 7). However, we noted an increase
in apoptosis after a reduced sclerotome size has already
manifested in Pax1/Pax9 double homozygous mutants. Thus,
although programmed cell death clearly contributes to the
elimination of sclerotome cells in the mutants, prevention of
apoptosis is unlikely the primary function of Pax1 and Pax9.
Previous studies have revealed that Pax1 and Pax9
expression is typically found in tissues that exhibit high levels
of cell proliferation (Müller et al., 1996). Our analysis has
shown that Pax1 and Pax9 are indeed required to maintain a
high rate of cell proliferation during a restricted phase of
sclerotome development (Fig. 7). At the end of this phase,
mesenchymal condensations are formed in wild-type, but not
in the double mutant, sclerotomes. Thus, the precursor pool in
the sclerotomes of Pax1/Pax9-deficient embryos might not
reach a critical size, a process that could be essential to
continue the sclerotome-specific developmental program.
Mesenchymal condensations and cartilage
formation
Accumulation of mesenchymal cells precedes the formation of
skeletal elements. Initially, a high mitotic activity is required
to produce enough cells for the process of condensation, which
subsequently is characterised by dramatic changes in gene
expression, reorganisation of extracellular matrix and
concentration of cells towards the centre of the future skeletal
element (for review, see Hall and Miyake, 1995). Copies of
Col2α1 mRNAs were found to increases one hundred-fold in
the developing chick limb bud as condensation occurs (Kravis
and Upholt, 1985). The sclerotomes of Pax1/Pax9 double
homozygous mutants initially express Col2α1 and its
transcriptional activator Sox9, but the enhancement of their
transcription does not occur (Fig. 5 and Fig. 6). Instead, the
expression of Sox9 and Col2α1 is gradually lost and
mesenchymal condensations do not form.
Disturbed mesenchymal condensations have been identified
in all alleles of the undulated series, which carry different
mutations in the Pax1 gene (Grüneberg, 1954; Wallin et al.,
1994; Dietrich and Gruss, 1995). Our analysis indicates that
reduced cell proliferation and increased apoptosis in the
sclerotomes of Pax1/Pax9 double mutants are causally related
to the absence of mesenchymal condensations. Thus, in the
compound mutants, partial loss of Pax1 and Pax9 could result
in a small, but significant reduction of proliferating rates, which
in turn would cause a partial size reduction of the mesenchymal
condensation. The gradual increase in the severity of the
phenotype observed in Pax1/Pax9 compound mutants would be
compatible with this idea. However, it should be noted that the
absence of Pax1 and Pax9 might also simultaneously affect the
expression of cell adhesion molecules and/or other molecules
required for the process of condensation.
Heterogeneity in the development of vertebral
elements
During embryonic development of vertebrates, the somites are
differentially influenced by neighbouring structures such as the
notochord, neural tube, the intermediate mesoderm and surface
ectoderm (reviewed in Christ et al., 1998). A central role for
vertebrae formation, ventralization of somites, as well as for
the formation and survival of sclerotomes has been identified
for the notochord, which is required for the sclerotomal
expression of Pax1 and Pax9 (Watterson et al., 1954; Koseki
et al., 1993; Brand-Saberi et al., 1993; Pourquie et al., 1993;
Dietrich et al., 1993; Goulding et al., 1994; Neubüser et al.,
1995). SHH, which is secreted by the notochord has been
shown to be the key molecule regulating these processes
(Johnson et al., 1994; Fan and Tessier-Lavigne, 1994; Teillet
et al., 1998). Indeed, Pax1 expression is rapidly lost and no
skeletal elements of the vertebral column form in Shh-deficient
mice (Chiang et al., 1996). The ventromedially restricted
vertebral defects in Pax1 mutants have raised the possibility
that Pax9, which is expressed in the lateral regions of the
sclerotomes, can partly rescue the consequences of Pax1
deficiency. Thus, the presence of neural arches, though
malformed, in Pax1/Pax9 double homozygous mutants was an
unexpected result of this study. Nonetheless, this result shows
that different genetic pathways regulate the ventromedial and
mediolateral regions of the sclerotomes. Whereas the former
requires Pax1 and Pax9 function, Pax gene expression in the
Pax1/Pax9 and vertebral column development 5407
lateral region of the sclerotome appears to be dispensable for
skeletal development. In addition to the mediolateral
differences, the development of the dorsoventral regions of the
sclerotomes have also been shown to be regulated by distinct
molecular pathways: experimental evidence in chick
demonstrated that, shortly after sclerotome formation, the
dorsalmost sclerotome cells become independent of SHH
signalling. These cells develop under the control of dorsally
expressed Bmp and Msx genes to form the spinous process, a
mechanism that involves downregulation of Pax1 expression in
the dorsal cells (Monsoro-Burq et al., 1994, 1996). Although
recent data indicated that at later stages also the development
of the ventral sclerotome requires signalling by Bmps
(Murtaugh et al., 1999), these findings support the idea that
each vertebra is assembled from multiple elements that are
regulated by different developmental pathways. Among those,
the Pax1/Pax9-dependent pathway is only required for the
development of ventromedial structures. Moreover, the ectopic
dorsal cartilage formed in the lumbar region of Pax1/Pax9
double homozygous mutants (Fig. 2) could indicate that dorsal
and ventral regulatory pathways compete for a common pool
of sclerotome cells.
We thank M. Schieweg for technical assistance and P. Gruss, K.
von der Mark and G. Scherer for DNA probes. This work was
supported by the Deutsche Forschungsgemeinschaft (DFG).
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