1 Research Institute of Molecular Pathology (IMP), Dr Bohr-Gasse 7, A-1030
Vienna, Austria
2 Institut Pasteur, Unite des Virus Oncogenes, URA1644 du CNRS, 25, Rue du Dr
Roux, 75724 Paris Cedex 15, France
3 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto,
Canada
* Present address: Mammalian Genetics Laboratory, Cancer Research UK, London
Research Institute, Lincoln's Inn Fields Laboratories, 44, Lincoln's Inn
Fields, London WC2A 3PX, UK
Present address: Samuel Lunenfeld Research Institute, Mount Sinai Hospital,
Toronto, Canada
Author for correspondence to (e-mail:
wagner{at}nt.imp.univie.ac.at)
Accepted 26 September 2002
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SUMMARY |
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Key words: Jun, Notochord, Skeleton, cre/loxP, Mouse
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INTRODUCTION |
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The development of the axial skeleton is a multi-step 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 (Gossler and Hrabe de Angelis,
1998; McGrew and Pourquie,
1998
; Theiler,
1988
). 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-tuned regulation is required to coordinate the
prepatterning of individual skeletal elements by region-specific mesenchymal
growth and condensations that are finally replaced by cartilage and bone. The
notochord has the most diverse functions during vertebrate development. The
early notochord is a rod-like axial structure of mesodermal origin that plays
an important role in the dorsoventral patterning of both the neural tube and
the somitic mesoderm. In the neural tube, it induces the formation of the
floor plate; in the somites, it induces the differentiation of the ventral
somitic derivatives into the sclerotome
(Fan and Tessier-Lavigne,
1994
; Pourquie et al.,
1993
). After these inductive events, some sclerotomal cells
migrate towards the notochord, where they form a continuous and initially
unsegmented perichordal tube. Later on, this axial mesenchyme acquires a
characteristic metameric pattern of condensed and noncondensed areas. While
the regularly spaced condensations represent intervertebral disc rudiments and
will give rise to the annulus fibrosus of the future intervertebral disc
(IVD), the noncondensed perichordal cells form the cartilaginous primordia of
the vertebral bodies (Theiler,
1988
). During mammalian embryonic development, the inner part of
the annulus fibrosus differentiates into hyaline-like cartilage, which,
together with the vertebral bodies, forms an uninterrupted cartilaginous
column around the notochord. Parallel with the ongoing chondrification, the
notochord vanishes in areas where vertebral bodies develop but expands between
the vertebrae to form the center of the IVD, where it forms the nucleus
pulposus (Theiler, 1988
).
Several transcription factors have been shown to be required for
skeletogenesis (McGrew and Pourquie,
1998; Summerbell and Rigby,
2000
; Wagner and Karsenty,
2001
). Targeted inactivation of Sox9 results in skeletal defects
due to impaired cartilage differentiation and absence of osteoblasts in
Cbfa1-deficient mice (Runx2 Mouse Genome
Informatics) mimics the human heritable skeletal disorder cleidocranial
dysplasia (Bi et al., 2001
;
Otto et al., 1997
). In
addition, the Pax family members Pax1 and Pax9, the proto-oncogene Myc, the
homeobox gene Bapx1 and several other transcription factors have been
implicated in the genetic control of skeletogenesis
(Gossler and Hrabe de Angelis,
1998
; Lettice et al.,
1999
; Nagy et al.,
1998
; Peters et al.,
1999
; Tribioli and Lufkin,
1999
). However, many of the pivotal signalling molecules
regulating skeletal development are likely to still await identification.
The transcription factor AP-1 consists of a variety of dimers composed by
members of the Fos and Jun families of proteins
(Jochum et al., 2001). While
the Fos proteins [Fos, Fosb, Fra1 (Fosl1 Mouse Genome Informatics),
Fra-2 (Fosl2 Mouse Genome Informatics)] can only heterodimerise with
members of the Jun family, the Jun proteins (Jun, JunB, JunD) can both
homodimerise and heterodimerise with other Jun or Fos members to form
transcriptionally active complexes (Angel
and Karin, 1991
). In addition to Fos proteins, Jun proteins can
also heterodimerise efficiently with other transcription factors such as
members of the ATF/CREB families (Hai et
al., 1999
). Jun is a major component of the AP-1 transcription
factor complex and, together with JunB and JunD, forms the family of mammalian
Jun proteins (Angel and Karin,
1991
). Targeted disruption of the Jun gene results in
embryonic lethality presumably because of defective hepatogenesis caused by
increased apoptosis of hepatoblasts and hematopoietic cells
(Hilberg et al., 1993
;
Johnson et al., 1993
). In
addition, Jun-/- embryos show malformations of the heart
outflow tract (Eferl et al.,
1999
) that resemble the human disease truncus arteriosus
persistens, a developmental defect probably caused by impaired neural crest
cell function. Although the extent of functional conservation between
individual members of the Jun family is not fully understood, knock-in
experiments have demonstrated that Junb can substitute for some of the
biological functions of Jun during embryogenesis
(Passegue et al., 2002
).
As the embryonic lethality of Jun-deficient foetuses was likely to prevent the identification of other biological functions of Jun at later stages of development, we decided to use a genetic strategy based on cre/loxP-mediated somatic recombination to screen for novel loss-of-function phenotypes.
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MATERIALS AND METHODS |
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Histological analysis, BrdU immunohistochemistry, TUNEL assay,
in-situ hybridisation and skeletal analysis
Embryos were dissected from timed matings of Junf/f
homozygous males with Junf/+ heterozygous females
harbouring the Balancre1-cre or murine or human collagen2a-cre transgenes,
respectively. Yolk-sac DNA was used for genotyping by PCR. Embryos for
histological analysis sections were fixed in 4% paraformaldehyde and 5 µm
sections were stained with Harris Haematoxylin and Eosin. BrdU
immunohistochemistry was performed as described
(Behrens et al., 2000). For
detection of apoptotic cells, histological sections prepared as above were
used for TUNEL assay (Boehringer Mannheim) according to the manufacturer's
instructions (Behrens et al.,
1999
). Skeletal analysis was carried out according to established
procedures (Peters et al.,
1999
), whole-mount in situ hybridisation and non-radioactive in
situ hybridisation on paraffin wax-embedded sections were performed as
described previously (Haigh et al.,
2000
).
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RESULTS |
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Newborn Junf/f mice carrying the Bal1 transgene
(Bal1;Jun/
) were smaller and could be
identified by open eyelids, suggesting a function of Jun in eyelid
closure and providing the proof-of-principle for the feasibility of our
genetic approach. The severity of the eyelid closure defect was variable,
being bilateral or only unilateral in individual mice, presumably reflecting
differences in Junf inactivation (data not shown). Mutant
mice were not present in numbers expected from Mendelian inheritance at birth,
which could be due to Bal1-mediated recombination of Jun in the
embryonic liver and heart, which are essential sites of Jun function
(Eferl et al., 1999
;
Hilberg et al., 1993
). In
addition, Bal1;Jun
/
mice also had reduced
viability before weaning age [4/186 survived until adulthood (2%) compared
with 25% expected], indicating an essential, yet unidentified, function of
Jun in postnatal development. All surviving
Bal1;Jun
/
mice had a shortened body axis and
showed scoliosis and kyphosis, an abnormal curvature and bending of the spinal
column (Fig. 1A). To evaluate
the nature of the skeletal abnormalities present in
Bal1;Jun
/
mice, skeletal preparations of
Bal1;Jun
/
E18.5 foetuses and littermate
Junf/f controls were made. Whereas
Junf/f controls showed a discrete metameric patterning of
the spinal column, Bal1;Jun
/
foetuses
displayed numerous defects, including fusions of the neural arches
(Fig. 1B,C). At E16.5, in
Bal1;Jun
/
embryos the chondrocytes of the
spinal column, which will subsequently ossify and form the vertebral bodies,
were not present in clearly demarcated domains, as in
Junf/f controls, but cell populations of adjacent
segmental units were connected at several different levels along the entire
longitudinal axis (Fig. 1D,E).
Therefore, the genetic analysis using mosaic
Bal1;Jun
/
mice has identified a role for
Jun in the developing spinal column.
|
As the ubiquity and variability of Bal1-mediated cre expression did not
allow the identification of the developmental stage and cell type requiring
Jun function, we next investigated Jun expression during
skeletogenesis. During somitogenesis, Jun mRNA is expressed in a
dynamic pattern in presomitic mesoderm at the level of the most recently
forming somite (Fig. 2A,B).
Jun expression was also detected in the midline (arrow in
Fig. 2B), and cross-sectioning
revealed expression in the somitic mesoderm (data not shown). To examine a
possible function of Jun during epitheliasation of the paraxial
mesoderm, the expression of molecular markers of various cell lineages and
derivatives of the somites were determined in the original Jun
knockout mice and wild-type littermate controls
(Hilberg et al., 1993). At
E12.5, the expression pattern of Sox9 and collagen 2a1
(Col2a1) was not affected by the absence of Jun, indicating
that the initial phase of chondrogenesis had proceeded normally
(Fig. 2C-F). Likewise,
Pax1, Myod1, Uncx4.1, Scx and Bapx1 expression, which
characterise various cell populations of the developing axial skeleton
(Gossler and Hrabe de Angelis,
1998
; McGrew and Pourquie,
1998
; Summerbell and Rigby,
2000
), were unaltered in Jun-/- embryos
(Fig. 2G-L and data not shown).
We concluded that, until E12.5, skeletogenesis had proceeded normally in
Jun mutants with proper anteroposterior patterning of the
somites.
|
To assess Jun function in the notochord and sclerotome,
Junf was inactivated using a transgenic line that
expresses the cre recombinase under the transcriptional control of human
collagen 2a1 promoter and enhancer sequences (Col2a1-cre). This line has been
previously demonstrated to excise lox-P flanked reporter sequences in collagen
2-expressing cells, including the notochord and sclerotome
(Haigh et al., 2000). At
E12.5, Jun protein was strongly expressed in the floor plate and in the
notochord, but weaker expression was also detected in the neural tube and in
sclerotomal cells. Junf was deleted by Col2a1-cre in the
notochord and sclerotome, but not in the floor plate (data not shown). The
axial skeletal defects of both the
Col2a1;Jun
/
mice resembled the
Bal1;Jun
/
phenotype arguing for an essential
function of Jun in the sclerotome and/or notochord. X-ray analysis
and skeletal preparations of adult
Col2a1;Jun
/
mice revealed a malformed,
scoliotic vertebral column and abnormal morphogenesis of the rib cage
(Fig. 3A-D). Interestingly,
although Col2a1-cre is expressed and mediates recombination in growth plate
chondrocytes of developing bones (Haigh et
al., 2000
), the length and overall gross morphology of the bones
of the appendages appeared to be normal in adult
Col2a1;Jun
/
mice
(Fig. 3A,B and data not shown).
We then analysed the expression of genes characterising the maturational
stages of chondrogenesis at E14.5. In
Col2a1;Jun
/
and Junf/f
control sclerotomal cells, Col2a1, a marker for proliferating
chondrocytes, was highly expressed (Fig.
3E,F). Collagen X (ColX; Col10a1 Mouse Genome
Informatics), indicative of hypertrophic chondrocytes, was not detected in the
vertebral columns of embryos of either genotype
(Fig. 3G,H). However,
ColX was expressed in other skeletal structures at E14.5. In the
developing baso-occipital bone, the ColX expression domain was
broader at the expense of Col2a1 expression in
Junf/f controls than in
COL2A1;Jun
/
mutant foetuses
(Fig. 3G,H; arrowheads). The
increased expression of ColX was accompanied by decreased
Col2a1 expression in Junf/f controls
(Fig. 3E arrow). However, this
delay in chondrocyte differentiation appeared to be transient as defects in
adult Col2a1;Jun
/
mice were restricted to
the axial skeleton and the formation of the baso-occipital bone was unaffected
(Fig. 3A,B). Therefore, the
absence of Jun did not have a severe effect on chondrogenesis and the
skeletal defect of Jun mutant mice appears to be restricted to the
developing spinal column, where Jun protein is most abundantly expressed in
the notochord.
|
To understand the function of Jun during skeletogenesis, we
performed a histological analysis of
Col2a1;Jun/
and littermate
Junf/f foetuses at various embryonic stages starting at
E11.5. As expected from the normal expression of marker genes
(Fig. 2C-L), sagittal sections
of the developing spinal column of E11.5-E13.5
Col2a1;Jun
/
foetuses revealed no apparent
alterations (Fig. 4A,B; data
not shown), but at E14.5 Col2a1;Jun
/
embryos
could be unambiguously identified histologically. Between E13.5 and E14.5, the
sclerotomal cells of the prospective vertebral bodies had progressed from a
proliferating to a hypertrophic chondrocyte morphology in
Junf/f, but not in
Col2a1;Jun
/
embryos. Secondly, a dramatic
reduction in size of the nucleus pulposus, the central part of the
intervertebral disc anlage, was apparent in
Col2a1;Jun
/
foetuses
(Fig. 4C,D).
|
To investigate the reason for the inefficient formation of the nucleus
pulposus, whose cells are derived from the notochord
(Theiler, 1988), the extent of
cellular proliferation and apoptosis was analysed at E13.5, before
histological differences became apparent. Bromodeoxyuridine (BrdU) was
injected into pregnant mothers and cells that had incorporated BrdU indicative
of cellular proliferation were quantified by manual counting. 26±4
BrdU-positive cells/metameric segment were detected in the sclerotome of
Col2a1;Jun
/
foetuses compared with
28±7 present in littermate Junf/f controls. In the
notochord 1.5±0.4 and 0.7±0.5 BrdU-positive cells were detected
in Junf/f and
Col2a1;Jun
/
foetuses, respectively
(Fig. 4E,F). Thus, the absence
of Jun results in a reduced, albeit statistically insignificant,
number of proliferating cells in both the sclerotome and the notochord, which
appears to be unable to explain fully the failure of IVD development. By
contrast, the absence of Jun had a profound effect on the extent of
cell death. Whereas only a minimal number of TUNEL-positive cells were
detected in E13.5 Junf/f vertebral columns, a significant
number of cells in the notochord, but not in the sclerotome, of
Col2a1;Jun
/
foetuses were found to be
undergoing apoptosis (Fig.
4G,H).
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DISCUSSION |
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In different biological processes, Jun can have both pro- and
anti-apoptotic functions. During embryogenesis, Jun appears to elicit a
crucial anti-apoptotic signal that is required for the survival of notochord
cells. In addition, Jun-deficient embryos die at midgestation,
displaying increased apoptosis of hepatoblasts and hematopoietic cells, and
adult mice with a liver-specific Jun deletion show hepatocyte death
during liver regeneration (Behrens et al.,
2002; Eferl et al.,
1999
; Hilberg et al.,
1993
; Johnson et al.,
1993
). By contrast, Jun and Jun phosphorylation appears to be
required for the execution of apoptosis in both neuronal cells and thymocytes
(Behrens et al., 2001
;
Behrens et al., 1999
). The
molecular mechanism that underlies the dichotomy of Jun functions in
apoptosis is not understood. The DNA-binding properties and sequence
specificity of Jun can be altered by dimerisation with different interacting
proteins. Jun/Fos heterodimers bind the DNA sequence 5'-TGA G/C
TCA-3', whereas the DNA motif 5'-TGACATCA-3' is
preferentially recognised by Jun/Atf2 complexes
(Karin and Hunter, 1995
).
Possibly, different Jun-containing heterodimeric transcription factor
complexes could be required for the transcriptional regulation of two classes
of target genes, which are independently required for cell survival and
apoptosis.
Reciprocal interactions between the notochord and the sclerotome are
essential for axial skeleton development
(Gossler and Hrabe de Angelis,
1998; McGrew and Pourquie,
1998
). The notochord induces sclerotomal formation of the paraxial
mesoderm and subsequently regulates the cartilagenous differentiation of
sclerotomal cells mainly through production and secretion of the signalling
molecule sonic hedgehog (Shh)
(Fan and Tessier-Lavigne,
1994
; Pourquie et al.,
1993
). However, Pax1 expression in the developing
sclerotome, which is dependent on and a mediator of the Shh signal
(Fan and Tessier-Lavigne,
1994
; Koseki et al.,
1993
), is unaltered in Jun-/- embryos
(Fig. 2K,L). Moreover, the
absence of Jun did not affect the induction of chondrogenic
differentiation, as judged by marker gene expression
(Fig. 3C-L).
Pax1-mutant mice have provided evidence for a sclerotomal signal back
to the notochord, as the absence of Pax-1 expression in the
sclerotome resulted in a hyperproliferation of notochordal cells
(Wallin et al., 1994
). Owing
to the bidirectionality of the sclerotome-notochord interaction, the cell type
that requires Jun function during skeletogenesis cannot be
unambiguously identified. Jun is expressed and
Junf is inactivated in both the notochord and sclerotome.
The high expression of Jun in the notochord argues in favour of a
cell-autonomous function, but we cannot exclude the possibility that Jun
expression in the sclerotome is required for the generation of a sclerotomal
survival signal for notochordal cells. The removal of Jun function by
conditional gene targeting only in the notochord or only in the sclerotome,
will be necessary to distinguish between these alternatives.
Jun becomes essential at the developmental stage when the
notochord transforms into the centre of the vertebral disc anlagen. Initially,
an uninterrupted longitudinal structure, the notochord, vanishes in areas
where the vertebral bodies develop, but expands between the vertebrae to form
the nucleus pulposus (Theiler,
1988). It is thought that the developing vertebral bodies exert a
mechanical stimulus on the notochord that induces the migration of notochordal
cells towards the presumptive intervertebral disc area
(Aszodi et al., 1998
;
Rufai et al., 1995
;
Theiler, 1988
). In
Jun-mutant mice, the regionalisation of the notochord proceeds
normally as notochordal cells are removed from the area of developing
vertebral bodies, but notochordal cell number is reduced. Previously genes
have been identified that are required for notochord survival at early stages
of somitogenesis (Maatman et al.,
1997
), but Jun is the first factor shown to be essential
for the survival of notochordal cells at a later stages of skeletogenesis. The
Jun-mutant phenotype suggests that two distinct mechanisms control
notochordal viability, a Jun-independent pathway during early
development and a Jun-dependent pathway during later phases of axial
skeletogenesis. The identification of Jun target genes required for
notochord survival is expected to result in a better understanding of this new
aspect of notochord biology.
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ACKNOWLEDGMENTS |
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REFERENCES |
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---|
Angel, P. and Karin, M. (1991). The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochem. Biophys. Acta 1072,129 -157.[CrossRef][Medline]
Aszodi, A., Chan, D., Hunziker, E., Bateman, J. F. and Fassler,
R. (1998). Collagen II is essential for the removal of the
notochord and the formation of intervertebral discs. J. Cell
Biol. 143,1399
-1412.
Behrens, A., Sibilia, M. and Wagner, E. F. (1999). Amino-terminal phosphorylation of c-Jun regulates stress-induced apoptosis and cellular proliferation. Nat. Genet. 21,326 -329.[CrossRef][Medline]
Behrens, A., Jochum, W., Sibilia, M. and Wagner, E. F. (2000). Oncogenic transformation by ras and fos is mediated by c-Jun N-terminal phosphorylation. Oncogene 19,2657 -2663.[CrossRef][Medline]
Behrens, A., Sabapathy, K., Graef, I., Cleary, M., Crabtree, G.
R. and Wagner, E. F. (2001). Jun N-terminal kinase 2
modulates thymocyte apoptosis and T cell activation through c-Jun and nuclear
factor of activated T cell (NF-AT). Proc. Natl. Acad. Sci.
USA 98,1769
-1774.
Behrens, A., Sibilia, M., David, J. P., Mohle-Steinlein, U.,
Tronche, F., Schutz, G. and Wagner, E. F. (2002). Impaired
postnatal hepatocyte proliferation and liver regeneration in mice lacking
c-jun in the liver. EMBO J.
21,1782
-1790.
Betz, U. A., Vosshenrich, C. A., Rajewsky, K. and Müller, W. (1996). Bypass of lethality with mosaic mice generated by Cre-loxP-mediated recombination. Curr. Biol. 6,1307 -1316.[Medline]
Bi, W., Huang, W., Whitworth, D. J., Deng, J. M., Zhang, Z.,
Behringer, R. R. and de Crombrugghe, B. (2001).
Haploinsufficiency of Sox9 results in defective cartilage primordia and
premature skeletal mineralization. Proc. Natl. Acad. Sci.
USA 98,6698
-6703.
Eferl, R., Sibilia, M., Hilberg, F., Fuchsbichler, A.,
Kufferath, I., Guertl, B., Zenz, R., Wagner, E. F. and Zatloukal, K.
(1999). Functions of c-Jun in liver and heart development.
J. Cell Biol. 145,1049
-1061.
Fan, C. M. and Tessier-Lavigne, M. (1994). Patterning of mammalian somites by surface ectoderm and notochord: evidence for sclerotome induction by a hedgehog homolog. Cell 79,1175 -1186.[Medline]
Gossler, A. and Hrabe de Angelis, M. (1998). Somitogenesis. Curr. Top. Dev. Biol. 38,225 -287.[Medline]
Hai, T., Wolfgang, C. D., Marsee, D. K., Allen, A. E. and Sivaprasad, U. (1999). ATF3 and stress responses. Gene Exp. 7,321 -335.
Haigh, J. J., Gerber, H., Ferrara, N. and Wagner, E. F.
(2000). Conditional inactivation of VEGF-A in areas of
collagen2a1 expression results in embryonic lethality in the heterozygous
state. Development 127,1445
-1453.
Hilberg, F., Aguzzi, A., Howells, N. and Wagner, E. F. (1993). c-jun is essential for normal mouse development and hepatogenesis. Nature 365,179 -181.[CrossRef][Medline]
Jochum, W., Passegue, E. and Wagner, E. F. (2001). AP-1 in mouse development and tumorigenesis. Oncogene 20,2401 -2412.[CrossRef][Medline]
Johnson, R. S., van Lingen, B., Papaioannou, V. E. and Spiegelmann, B. M. (1993). A null mutation at the c-jun locus causes embryonic lethality and retarded cell growth in culture. Genes Dev. 7,1309 -1317.[Abstract]
Karin, M. and Hunter, T. (1995). Transcriptional control by protein phosphorylation: signal transmission from the cell surface to the nucleus. Curr. Biol. 5, 747-757.[Medline]
Koseki, H., Wallin, J., Wilting, J., Mizutani, Y., Kispert, A.,
Ebensperger, C., Herrmann, B. G., Christ, B. and Balling, R.
(1993). A role for Pax-1 as a mediator of notochordal signals
during the dorsoventral specification of vertebrae.
Development 119,649
-660.
Lettice, L. A., Purdie, L. A., Carlson, G. J., Kilanowski, F.,
Dorin, J. and Hill, R. E. (1999). The mouse bagpipe gene
controls development of axial skeleton, skull, and spleen. Proc.
Natl. Acad. Sci. USA 96,9695
-700.
Maatman, R., Zachgo, J. and Gossler, A. (1997).
The Danforth's short tail mutation acts cell autonomously in notochord cells
and ventral hindgut endoderm. Development
124,4019
-4028.
McGrew, M. J. and Pourquie, O. (1998). Somitogenesis: segmenting a vertebrate. Curr. Opin. Genet. Dev. 8,487 -493.[CrossRef][Medline]
Nagy, A., Moens, C., Ivanyi, E., Pawling, J., Gertsenstein, M., Hadjantonakis, A. K., Pirity, M. and Rossant, J. (1998). Dissecting the role of N-myc in development using a single targeting vector to generate a series of alleles. Curr. Biol. 8, 661-664.[Medline]
Otto, F., Thornell, A. P., Crompton, T., Denzel, A., Gilmour, K. C., Rosewell, I. R., Stamp, G. W., Beddington, R. S., Mundlos, S., Olsen, B. R. et al. (1997). Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 89,765 -771.[Medline]
Passegue, E., Jochum, W., Behrens, A., Ricci, R. and Wagner, E. F. (2002). JunB can substitute for Jun in mouse development and cell proliferation. Nat. Genet. 30,158 -166.[CrossRef][Medline]
Peters, H., Wilm, B., Sakai, N., Imai, K., Maas, R. and Balling,
R. (1999). Pax1 and Pax9 synergistically regulate vertebral
column development. Development
126,5399
-5408.
Pourquie, O., Coltey, M., Teillet, M. A., Ordahl, C. and le Douarin, N. M. (1993). Control of dorsoventral patterning of somitic derivatives by notochord and floor plate. Proc. Natl. Acad. Sci. USA 90,5242 -5246.[Abstract]
Rufai, A., Benjamin, M. and Ralphs, J. R. (1995). The development of fibrocartilage in the rat intervertebral disc. Anat. Embryol. 192, 53-62.[Medline]
Summerbell, D. and Rigby, P. W. (2000). Transcriptional regulation during somitogenesis. Curr. Top. Dev. Biol. 48,301 -318.[Medline]
Theiler, K. (1988). Vertebral malformations. Adv. Anat. Embryol. Cell Biol. 112, 1-99.[Medline]
Tribioli, C. and Lufkin, T. (1999). The murine
Bapx1 homeobox gene plays a critical role in embryonic development of the
axial skeleton and spleen. Development
126,5699
-5711.
Wagner, E. F. and Karsenty, G. (2001). Genetic control of skeletal development. Curr. Opin. Genet. Dev. 11,527 -532.[CrossRef][Medline]
Wallin, J., Wilting, J., Koseki, H., Fritsch, R., Christ, B. and
Balling, R. (1994). The role of Pax-1 in axial skeleton
development. Development
120,1109
-1121.