Pax1/Pax9 and Vertebral Column Development 5401

Development 126, 5399-5408 (1999) 5399 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 normal. However, instead of a segmental arrangement of within the vertebrate Pax gene family. They encode closely vertebrae and intervertebral disc anlagen, a loose related transcription factors and are expressed in similar mesenchyme surrounding the notochord is formed. The patterns during mouse embryogenesis, suggesting that gradual loss of Sox9 and Collagen II expression in this Pax1 and Pax9 act in similar developmental pathways. We mesenchyme indicates that the sclerotomes are prevented have recently shown that mice homozygous for a defined from undergoing chondrogenesis. The first detectable Pax1 null allele exhibit morphological abnormalities of the defect is a low rate of cell proliferation in the ventromedial axial skeleton, which is not affected in homozygous Pax9 regions of the sclerotomes after sclerotome formation but mutants. To investigate a potential interaction of the two before mesenchymal condensation normally occurs. At genes, we analysed Pax1/Pax9 double mutant mice. These later stages, an increased number of cells undergoing mutants completely lack the medial derivatives of the apoptosis further reduces the area normally forming sclerotomes, the vertebral bodies, intervertebral discs and vertebrae and intervertebral discs. Our results reveal the proximal parts of the ribs. This phenotype is much functional redundancy between Pax1 and Pax9 during more severe than that of Pax1 single homozygous mutants. vertebral column development and identify an early role of In contrast, the neural arches, which are derived from the Pax1 and Pax9 in the control of cell proliferation during lateral regions of the sclerotomes, are formed. The analysis early sclerotome development. In addition, our data of Pax9 expression in compound mutants indicates that indicate that the development of medial and lateral both spatial expansion and upregulation of Pax9 expression elements of vertebrae is regulated by distinct genetic account for its compensatory function during sclerotome pathways. development in the absence of Pax1. In Pax1/Pax9 double homozygous mutants, formation and anteroposterior polarity of sclerotomes, as well as Key words: Pax1, Pax9, Vertebral column, Chondrogenesis, induction of a chondrocyte-specific cell lineage, appear Proliferation, Apoptosis, Mouse

INTRODUCTION tuned regulation is required to co-ordinate the prepatterning of individual skeletal elements by region-specific mesenchymal The vertebrate body is supported by the vertebral column, a growth and condensations that are finally replaced by cartilage series of segmental skeletal elements that provide both stability and bone. Although in the recent years much progress has been and mobility. The development of the axial skeleton is a multi- made in the understanding of somite patterning and sclerotome step process starting with the formation of somites from the formation (Tajbakhsh and Spörle, 1998), most aspects of the unsegmented paraxial mesoderm on both sides of the neural genetic control that regulates the complex development from tube. Shortly after formation, the somites compartmentalise to the sclerotomal mesenchymes to the vertebral column remain generate dermomyotomes and sclerotomes, the latter forming to be elucidated. skeletal elements of the vertebral column and ribs. Vertebral Pax1 and Pax9 form one group within the family of nine column development requires the co-ordination of a series of vertebrate Pax genes, which are unified by the presence of the cellular events. These include de-epithelialization of somites, paired box that encodes a DNA-binding domain (Walther et al., proliferation and migration of sclerotomal cells, and 1991; Noll, 1993). Analyses of mouse mutants of all Pax genes establishment of anteroposterior polarity of the sclerotome have demonstrated multiple roles in the genetic control of (reviewed in Keynes and Stern, 1988; Christ and Wilting, 1992; mammalian organogenesis (reviewed in Dahl et al., 1997). The Christ and Ordahl, 1995). In mammals, a single vertebra is 128 amino acid long DNA-binding paired domains of Pax1 and composed of a variety of components that perform different Pax9 are almost identical (98%). DNA-binding studies functions, depending on the level of the body axis. Thus, a fine- have shown that the e5 site, a DNA element present in the 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 ventromedial compartments of somites. The expression of are the only Pax genes expressed in the sclerotomes, the Pax1 precedes that of Pax9 and is initially found in all sclerotomal cells. At later stages of sclerotome differentiation, Fig. 2. Absence of the expression of Pax1 acquires a maximum in the posterior, Pax1/Pax9 affects mainly ventromedial compartment. On the contrary, transcription of the ventral elements of the Pax9 is predominantly detectable in the posterior, ventrolateral vertebral column. Ventral region of the sclerotomes. In the region of the future vertebral view (A,B) and lateral view bodies, expression of Pax1 and Pax9 is downregulated when (C,D) of the lumbar region sclerotomal cells start to chondrify, but their expression is of the vertebral column from wild-type (A,C) and maintained in the perichondrium of vertebrae and in the Pax1/Pax9-deficient (B,D) intervertebral disc anlagen (Deutsch et al., 1988; Neubüser et newborn mice. al., 1995; Peters et al., 1995; Ebensperger et al., 1995). (A,C) Vertebral bodies (vb) Previous analyses of three natural Pax1 mutant alleles in have formed single mice, the undulated series (un, unex, UnS), have demonstrated ossification centres and an important role for Pax1 during sclerotome development alternate with intervertebral (Grüneberg, 1954; Balling et al., 1988; Wallin et al., 1994; discs (id). (B,D) Vertebral Dietrich and Gruss, 1995). Using a gene targeting bodies and intervertebral approach, we have recently shown that homozygous mutant discs are absent in the mice of a defined Pax1 null allele are viable and exhibit double mutant mice, morphological abnormalities of vertebrae and intervertebral whereas the neural arches ex (na) are formed. In addition, elements of the sacrum (sa, arrows in B) discs similar to those seen in the recessive alleles un and un and the proximal parts of the ribs (arrowheads in B) are missing in (Wilm et al., 1998). In contrast, homozygous mutants of the the absence of Pax1/Pax9. Note also ectopic formation of cartilage semidominant UnS allele, which has a deletion including the that connects adjacent neural arches (arrowheads in D). 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 Cart1 (Qu et al., 1999). The overlapping expression patterns the UnS allele interferes with vertebral column development during mouse embryogenesis and the high sequence not only through the loss of Pax1 function. Homozygous conservation of the DNA-binding paired domain of members Pax9 mutants have skeletal defects in the limbs and in the within each group of the Pax gene family suggest redundancy skull, but exhibit no obvious defects in the axial skeleton at sites where these genes are co-expressed. (Peters et al., 1998). To investigate a possible genetic interaction between Pax1 There is accumulating evidence that closely related and Pax9 during mammalian development, we analysed double transcription factors perform redundant functions during mutant mice. These mutants reveal a gene dosage-dependent development. This has been shown for paralogous Hox genes co-operation of Pax1 and Pax9 during sclerotome (Rijli and Chambon, 1997, and references therein), the zinc development. In the absence of Pax1 and Pax9, no vertebral finger protein encoding genes Gli2 and Gli3 (Mo et al., 1997), bodies and intervertebral discs are formed, a phenotype that is for the basic helix-loop-helix genes MyoD and MRF4 (Rawls not found in either single homozygous mutant, whereas mice et al., 1998), and the homeobox-containing genes Prx1 and lacking three functional gene copies exhibit intermediate Prx2 (ten Berge et al., 1998; Lu et al., 1999), and Alx1 and 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 Animals homozygous mutant embryos. (A) Wild-type embryo (E10.5), Pax1null/Pax9lacZ double heterozygous mutant mice were generated by hybridized with a Uncx4.1-speciﬁc RNA-probe. Uncx4.1 expression crossing mice carrying null alleles of Pax1 (Pax1null) and Pax9 marks the posterior halves of newly formed somites and becomes (Pax9lacZ), both of which have been described previously (Wilm et al., restricted to the posterior halves of the sclerotomes as the somites 1998; Peters et al., 1998). Genotyping of mice and embryos was differentiate. (B) A similar expression pattern of Uncx4.1 is performed as described previously (Wilm et al., 1998; Peters et al., detectable in Pax1/Pax9 double homozygous mutants. 1998). 5402 H. Peters and others

Phenotype analyses as well as ossified fusions between vertebrae and neural arches Skeletal staining of newborn mice was performed as described by are found (Fig. 1E,F). Thus, Pax1 and Pax9 interact during Kessel et al. (1990). To visualize Pax9lacZ expression, mutant mouse vertebral column development. This result is more clearly embryos were stained with X-gal and cleared with benzylbenzoate/ demonstrated in mutants that lack both copies of Pax1: in benzylalcohol as described in Gossler and Zachgo (1993). Whole- Pax1−/−; Pax9−/− double homozygous mutants, no vertebral mount in situ hybridizations with digoxigenin-labeled RNA probes bodies and intervertebral discs are formed (Fig. 1I), thereby specific for Sox9 (Wright et al., 1995), the α1 subunit (Metsaranta et α dramatically decreasing the overall length of the body axis. al., 1991) of Collagen II (Col2 1), and Uncx4.1 (Mansouri et al., Furthermore, the proximal parts of most ribs, all skeletal 1997) were performed according to Wilkinson (1992). In situ hybridization on paraffin sections was carried out as described in elements of the tail, as well as the connection between sacrum Neubüser et al. (1995). and pelvic girdle are missing (Figs 1I, 2B,D). A similar, but less severe, phenotype was detectable in the vertebral column BrdU labeling and TUNEL staining of Pax1−/−; Pax9+/− mutants. In these mice, an irregular, Cell proliferation was analysed by incorporation of BrdU into cartilaginous rod that is interrupted by ventral extensions of the embryos and subsequent imunohistochemical detection of BrdU on neural arches replaces the vertebral bodies and intervertebral paraffin sections according to the protocol of the ‘In Situ Cell discs of the lumbar region (Fig. 1H). These results identify a Proliferation Kit’ (Boehringer-Mannheim). Sections were stained role for Pax9 in vertebral column formation and reveal a gene with diaminobenzidine as a colour substrate and briefly counterstained dosage-dependent manner of Pax1/Pax9 function during with hematoxylin. BrdU-positive cells were counted in a defined area vertebral column development. The findings also indicate that around the notochord (Fig. 7). To demonstrate statistically significant differences, standard deviations were calculated from numbers Pax1 and Pax9 may have similar or identical functions that are obtained at both early and advanced phases of sclerotome redundant at many steps during sclerotome development. development. Numbers from at least six sections were combined in Interestingly, the lateral derivatives of the sclerotomes, the each group for determination of the standard deviation. neural arches, are formed in the absence of Pax1 and Pax9, Apoptotic cells were detected using the ‘In Situ Cell Death although they were found to have an abnormal shape (Fig. 2D). Detection Kit’ (Boehringer-Mannheim). Fluorescein-labeled DNA Moreover, in the lumbar region, ectopic cartilage formation is was incorporated in the terminal transferase reaction and directly detectable in the dorsal region between adjacent neural arches visualized after counterstaining the sections with propidium iodide. (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 RESULTS vertebral elements could also contribute to this phenotype. While our analysis does not distinguish between these two Pax1 and Pax9 are required for vertebral column possibilities, the presence of neural arches in Pax1/Pax9 double development in a gene dosage-dependent manner homozygous mutants rules out an essential role for Pax1 and To reveal a potential interaction between Pax1 and Pax9 during Pax9 in their formation. Furthermore, the development of development, we crossed double heterozygous mutant mice neural arches indicates that sclerotome formation can not be carrying the previously described Pax1null and Pax9lacZ alleles completely abolished in the absence of Pax1 and Pax9. (Wilm et al., 1998; Peters et al., 1998), which we refer to in the following as Pax1 and Pax9 mutants, respectively. At the Upregulation and spatial expansion of sclerotomal newborn stage, all nine possible genotypes were obtained at Pax9 expression in the absence of Pax1 the expected Mendelian ratio, indicating that the complete loss To explore the basis of the functional redundancy between of Pax1 and Pax9 does not result in embryonic lethality (n=143 Pax1 and Pax9 during vertebral column development and to for newborn mice). Shortly after birth, however, all test whether Pax1 and Pax9 functions are required to maintain homozygous Pax9 mutants die due to a cleft secondary palate Pax9 promoter activity, we analysed lacZ expression of (Peters et al., 1998). In addition, we never observed surviving Pax1/Pax9 compound mutants. Previous studies have shown Pax1−/−; Pax9+/− mice in our breeding colony, indicating that that the expression pattern obtained by X-gal staining of this genotype causes early postnatal lethality. Except for the heterozygous Pax9 mutants recapitulates the endogenous vertebral column (see below), all other skeletal phenotypes expression of Pax9 (Peters et al., 1998, and Fig. 3A). In the observed in the double homozygous mutants were specific to sclerotomes of E10.5 embryos, lacZ expression is mainly the absence of either Pax1 or Pax9. These include found in the posterior halves, whereas a narrow expression abnormalities of the shoulder skeleton in Pax1-deficient mice domain is detectable in the anterior half, adjacent to the (Wilm et al., 1998), and preaxial polydactyly and craniofacial preceding somite boundary (Fig. 3D,G). By gross inspection, abnormalities in the absence of Pax9. Furthermore, double lacZ expression along the body axis appears upregulated in homozygous mutant mice lack the derivatives of the third and Pax1−/−; Pax9+/− embryos (Fig. 3B). In the sclerotomes of these fourth pharyngeal pouches, a phenotype that is also seen in mutants, the lacZ expression domain is dorsally expanded in Pax9-deficient mice (Peters et al., 1998, and data not shown). the posterior halves and exhibits a rectangular instead of the The vertebral columns of heterozygous and homozygous triangular shape (Fig. 3E). In addition, lacZ expression is Pax9 mutants appear normal, as revealed by skeletal staining induced in the anterior half of the sclerotomes (Fig. 3E), of newborn mice (Fig. 1B,C). However, in mice that are where it is strongly maintained during further sclerotome deficient for one functional copy of Pax1, heterozygosity and development (Fig. 3H). A similar, expanded expression pattern homozygosity of the Pax9 mutation result in vertebral is found in double homozygous mutant embryos (Fig. 3C,F). malformations that are not seen in single Pax1 heterozygous From these observations, we conclude that, in wild-type mutants. In the lumbar region, fused vertebrae, split vertebrae, 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 vertebrae that express Col2α1 and alternate with yet of the sclerotomes. Therefore, spatial expansion and undifferentiated mesenchymes of the future intervertebral upregulation of Pax9 in the sclerotomes is a likely mechanism discs (Fig. 6A,E). In the absence of Pax1 and Pax9, a loose for the partial rescue of vertebral column development in Pax1 mesenchyme surrounding the notochord has developed that mutant mice. Furthermore, the maintenance of lacZ expression lacks signs of cartilage formation and is completely devoid of in the double homozygous mutant shows that Pax9 Col2α1 transcripts (Fig. 6D,H). In Pax1 single homozygous transcription during early sclerotome development does not mutants, the overall size of vertebral precursors is reduced require the function of Pax1, or of Pax9 itself (Fig. 3C,F). (Fig. 6B,F), whereas in Pax1−/−; Pax9+/− embryos, cartilage formation was found in the ventral sclerotome only. In the Anteroposterior polarity of somites and latter mutants, the cartilaginous rudiments exhibit an irregular chondrogenesis spacing (Fig. 6G). Expression patterns in the sclerotomes To identify and characterise the onset of phenotypic similar to those shown for Col2α1 have been obtained using a abnormalities in Pax1/Pax9 double mutants, we performed in Sox9-specific probe (data not shown). Together, these findings situ hybridisation of genes that are expressed during early demonstrate that the control of normal size, shape and sclerotome development. The expression of Uncx4.1, a paired- spacing of the cartilaginous precursors of the vertebrae and type homeobox-containing gene, is induced in the posterior intervertebral discs require Pax1 and Pax9 in a gene-dosage- half of newly formed somites and thereafter is maintained in dependent manner. the posterior halves of the sclerotomes (Mansouri et al., 1997). At E10.5 (around 30 somites), the expression patterns of Programmed cell death and cell proliferation in Uncx4.1 were indistinguishable among all possible allelic Pax1/Pax9-deficient sclerotomes combinations of Pax1 and Pax9 mutations (Fig. 4A, B, and data To investigate the mechanisms that cause the reduced size of not shown). This result shows that Pax1 and Pax9 are not the ventromedial sclerotome in Pax1/Pax9 double homozygous necessary to maintain the expression of Uncx4.1 and indicates mutants, the number of cells undergoing apoptosis and the that the anteroposterior polarity of the sclerotomes is not number of proliferating cells were determined during early disturbed in Pax1/Pax9 double homozygous mutants. sclerotome development. We analysed serial transverse To investigate the induction and fate of the chondrogenic cell sections of tail somites of E12.5 embryos in these experiments lineage in the sclerotomes of Pax1/Pax9 double mutants, because, at this level of the body axis, the formation of skeletal expressions of Sox9, encoding a HMG-box-containing elements is entirely dependent on Pax1/Pax9 function. transcription factor, and Collagen II (Col2α1) were analysed. The histological analysis of caudalmost somites revealed no Recent studies have shown that Sox9 is required for cartilage obvious defects of the sclerotomes. Both formation as well as formation (Bi et al., 1999). Col2α1, a direct target of Sox9 in ventromedial migration of sclerotomal cells occur in the precartilaginous mesenchymes (Bell et al., 1997), encodes a absence of Pax1 and Pax9 (Fig. 7A,C). However, as somites major component of the extracellular matrix of cartilage and mature, the sclerotomes of double homozygous mutant was shown to be required for normal vertebral column embryos become less condensed, as compared to the wild-type development (Li et al., 1995). At E10.5, both genes are control (Fig. 7I,K). Notably, Tunel-labeling revealed that the transcribed in a similar, segmented pattern along the body axis number of cells undergoing programmed cell death in the of normal embryos (Fig. 5A,D). This pattern was detectable in sclerotomes of this region was not increased in Pax1/Pax9 all Pax1/Pax9 compound mutants including double double homozygous mutant embryos (data not shown). On the homozygous Pax1/Pax9 mutants (Fig. 5B,C,E,F, and data not contrary, when the sclerotomes of wild-type embryos have shown). In addition to the segmented expression pattern, a formed precartilaginous condensations, the proportion of continuous expression of Col2α1 is normally detectable in the apoptotic cells decreases almost completely (Fig. 7O) whereas, ventral domain within the somites, between forelimb and at the same axial levels of Pax1/Pax9 double homozygous hindlimb of E10.5 embryos (Fig. 5D,E,G). In contrast, this mutant embryo, dying cells were abundant (Fig. 7P). Thus, domain was barely detectable in double homozygous mutants prevention of apoptosis in the sclerotomes may require the (Fig. 5F,H). Cross sections through this region revealed that the functions of Pax1/Pax9. reduction of the Col2α1 expression domain in double To determine whether cell proliferation is affected in the homozygous mutants is caused by a size reduction of the developing Pax1/Pax9-deficient sclerotomes, we analysed ventromedial region of the sclerotome, rather than by the incorporation of BrdU in dividing cells of E12.5 embryos. inability of the sclerotome to express Col2α1 (Fig. 5J). These Starting shortly after sclerotome formation, we analysed results confirm that the absence of Pax1 and Pax9 approximately 20 successive somites in 50 µm intervals. (Fig. predominantly affects the ventromedial region of the 7M). The comparison of BrdU-labelling profiles of wild-type sclerotomes, whereas the lateral sclerotome appears to develop and Pax1/Pax9 mutant sclerotomes reveals two phases. In the normally at this stage. first phase (intervals 1-7, Fig. 7N), no differences in the number The presence of Col2α1 expression in double homozygous of BrdU-positive cells were found in wild-type and Pax1/Pax9 mutants at E10.5 indicates that a chondrogenic cell fate is mutant embryos (84±11.5 in wild type versus 82±11.7 in induced in the ventromedial regions of the sclerotomes in the Pax1/Pax9 double homozygous mutants). In the second phase, absence of Pax1 and Pax9 and that chondrogenesis must be however, the number of dividing cells decreases in the mutant arrested at a later stage of embryonic development. sclerotomes (65.7±7.7) whereas significantly higher numbers Chondrogenesis of the vertebral column starts in the cervical of labelled cells were found in the wild-type sclerotomes region around E12.5 of mouse development. At this stage, (94.7±8.7). Conversely, a higher number of proliferating cells the sclerotomes have formed chondrifying rudiments of the 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 wild- type (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 BrdU- positive 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 reveals that Pax1 can completely compensate for the absence the successive increase of the size of the neural tube in the of Pax9, whereas loss of Pax1 is rescued by Pax9 to a mutant embryo (Fig. 7G,K). considerable extent (Fig. 1). In addition, the severity of the These results show that in the absence of Pax1 and Pax9 the phenotype in the absence of either gene is significantly sclerotomal cell population is reduced by a decrease in cell exacerbated by introducing heterozygosity of the other gene. proliferation, followed by an abnormally high rate of apoptosis Thus Pax1 and Pax9 functions are redundant and their that later contributes to the substantial loss of the sclerotome size. 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 DISCUSSION Pax2/Pax5/Pax8 subgroup, which have overlapping functions during midbrain and cerebellum development (Schwarz et al., Redundant functions of Pax1 and Pax9 during 1997). Similarly, earlier onset of embryonic lethality than in vertebral column development homozygous Pax3 mutants was found in mouse embryos In this report, we demonstrate that the closely related lacking both Pax3 and Pax7, which together constitute another transcription factors Pax1 and Pax9 have conserved biological class within the Pax gene family (Mansouri and Gruss, 1998). functions during the development of the sclerotome. In the Together, these data suggest that functional redundancy absence of Pax1 and Pax9, no ventral elements of the vertebral between paralogous Pax genes is a general mechanism that can column develop along the entire body axis. The comparison of act in those tissues in which members of a given subgroup have this phenotype to that of either single homozygous mutants overlapping expression patterns. 5406 H. Peters and others

In the absence of Pax1, Pax9 expression is upregulated and Mesenchymal condensations and cartilage is ectopically induced in the anterior domain of the sclerotomes formation (Fig. 3), thereby providing a basis for functional redundancy Accumulation of mesenchymal cells precedes the formation of between Pax1 and Pax9 in the developing vertebral column. skeletal elements. Initially, a high mitotic activity is required The molecular mechanism by which upregulation of Pax9 is to produce enough cells for the process of condensation, which achieved is presently unclear. Our data indicate that, in wild- subsequently is characterised by dramatic changes in gene type embryos, Pax1 is involved in the suppression of Pax9 expression, reorganisation of extracellular matrix and expression in the anterior sclerotome. Interestingly, a similar concentration of cells towards the centre of the future skeletal repression between paralogous Pax genes has recently been element (for review, see Hall and Miyake, 1995). Copies of identified during the development of the dermomyotomes, Col2α1 mRNAs were found to increases one hundred-fold in which generate dermis and skeletal muscles. In Pax3 mutant the developing chick limb bud as condensation occurs (Kravis embryos, expression of Pax7 is ectopically activated in the and Upholt, 1985). The sclerotomes of Pax1/Pax9 double anterior half of the dermomyotome and in vitro data suggest a homozygous mutants initially express Col2α1 and its direct role of Pax3 in the suppression of Pax7 in cultured transcriptional activator Sox9, but the enhancement of their myoblasts (Borycki et al., 1999). Together, these results transcription does not occur (Fig. 5 and Fig. 6). Instead, the strongly suggest that Pax1 and Pax3 functions contribute to the expression of Sox9 and Col2α1 is gradually lost and control of the normal expression patterns of Pax9 and Pax7, mesenchymal condensations do not form. respectively, in the sclerotomes and dermomyotomes of the Disturbed mesenchymal condensations have been identified developing somite. However, in the posterior sclerotomes of in all alleles of the undulated series, which carry different wild-type embryos, Pax1 and Pax9 are co-expressed, indicating mutations in the Pax1 gene (Grüneberg, 1954; Wallin et al., that high levels of Pax1 do not necessarily result in the 1994; Dietrich and Gruss, 1995). Our analysis indicates that downregulation of Pax9 and suggesting that Pax9-activating reduced cell proliferation and increased apoptosis in the signals are more potent in the posterior half of the sclerotome. sclerotomes of Pax1/Pax9 double mutants are causally related to the absence of mesenchymal condensations. Thus, in the Cell proliferation and prevention of apoptosis compound mutants, partial loss of Pax1 and Pax9 could result Complete failure of organ formation in Pax mutants has in a small, but significant reduction of proliferating rates, which demonstrated essential roles of Pax genes during in turn would cause a partial size reduction of the mesenchymal organogenesis. Although localised cell proliferation is condensation. The gradual increase in the severity of the commonly observed in tissues participating in organogenesis, phenotype observed in Pax1/Pax9 compound mutants would be the role of Pax genes in cell survival during embryogenesis is compatible with this idea. However, it should be noted that the not well understood. Reduced proliferation rates have been absence of Pax1 and Pax9 might also simultaneously affect the found in the developing diencephalon of Small-eye (Pax6) expression of cell adhesion molecules and/or other molecules mutant embryos (Warren and Price, 1997). In addition, it was required for the process of condensation. shown that Pax5 is required to stimulate B cell proliferation in vitro (Wakatsuki et al., 1994) and cells overexpressing Pax Heterogeneity in the development of vertebral genes can form tumours in nude mice (Maulbecker and Gruss, elements 1993). Moreover, growth inhibition as well as induction of During embryonic development of vertebrates, the somites are apoptosis has been observed by inactivating Pax genes in in differentially influenced by neighbouring structures such as the vitro systems (Gnarra and Dressler, 1995; Rossi et al., 1995; notochord, neural tube, the intermediate mesoderm and surface Bernasconi et al., 1996; Hewitt et al., 1997). Increased ectoderm (reviewed in Christ et al., 1998). A central role for apoptosis in the neural tube and somites was recently described vertebrae formation, ventralization of somites, as well as for in homozygous Pax3 mutant embryos (Borycki et al., 1999). the formation and survival of sclerotomes has been identified Likewise, results of the present work show that sclerotomal for the notochord, which is required for the sclerotomal cells of Pax1/Pax9-deficient mice undergo increased expression of Pax1 and Pax9 (Watterson et al., 1954; Koseki programmed cell death (Fig. 7). However, we noted an increase et al., 1993; Brand-Saberi et al., 1993; Pourquie et al., 1993; in apoptosis after a reduced sclerotome size has already Dietrich et al., 1993; Goulding et al., 1994; Neubüser et al., manifested in Pax1/Pax9 double homozygous mutants. Thus, 1995). SHH, which is secreted by the notochord has been although programmed cell death clearly contributes to the shown to be the key molecule regulating these processes elimination of sclerotome cells in the mutants, prevention of (Johnson et al., 1994; Fan and Tessier-Lavigne, 1994; Teillet apoptosis is unlikely the primary function of Pax1 and Pax9. et al., 1998). Indeed, Pax1 expression is rapidly lost and no Previous studies have revealed that Pax1 and Pax9 skeletal elements of the vertebral column form in Shh-deficient expression is typically found in tissues that exhibit high levels mice (Chiang et al., 1996). The ventromedially restricted of cell proliferation (Müller et al., 1996). Our analysis has vertebral defects in Pax1 mutants have raised the possibility shown that Pax1 and Pax9 are indeed required to maintain a that Pax9, which is expressed in the lateral regions of the high rate of cell proliferation during a restricted phase of sclerotomes, can partly rescue the consequences of Pax1 sclerotome development (Fig. 7). At the end of this phase, deficiency. Thus, the presence of neural arches, though mesenchymal condensations are formed in wild-type, but not malformed, in Pax1/Pax9 double homozygous mutants was an in the double mutant, sclerotomes. Thus, the precursor pool in unexpected result of this study. Nonetheless, this result shows the sclerotomes of Pax1/Pax9-deficient embryos might not that different genetic pathways regulate the ventromedial and reach a critical size, a process that could be essential to mediolateral regions of the sclerotomes. Whereas the former continue the sclerotome-specific developmental program. 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 Pax-1 for the development of vertebral and extravertebral structures. Dev. skeletal development. In addition to the mediolateral Biol. 167, 529-548. differences, the development of the dorsoventral regions of the Dietrich, S., Schubert, F. R. and Gruss P. (1993). Altered Pax gene expression in murine notochord mutants: the notochord is required to initiate sclerotomes have also been shown to be regulated by distinct and maintain ventral identity in the somite. Mech. Dev. 44, 189-207. molecular pathways: experimental evidence in chick Ebensperger, C., Wilting, J., Brand-Saberi, B., Mizutani, Y., Christ, B., demonstrated that, shortly after sclerotome formation, the Balling, R. and Koseki, H. (1995). Pax-1, a regulator of sclerotome dorsalmost sclerotome cells become independent of SHH development is induced by notochord and floor plate signals in avian embryos. Anat. Embryol. 191, 297-310. signalling. These cells develop under the control of dorsally Fan, C. M. and Tessier-Lavigne, M. (1994). Patterning of mammalian expressed Bmp and Msx genes to form the spinous process, a somites by surface ectoderm and notochord: evidence for sclerotome mechanism that involves downregulation of Pax1 expression in induction by a hedgehog homolog. Cell 79, 1175-1186. the dorsal cells (Monsoro-Burq et al., 1994, 1996). Although Gnarra, J. R. and Dressler, G. R. (1995). Expression of Pax-2 in human renal recent data indicated that at later stages also the development cell carcinoma and growth inhibition by antisense oligonucleotides. Cancer Res. 55, 4092-4098. of the ventral sclerotome requires signalling by Bmps Gossler, A. and Zachgo, J. (1993). 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