Download The role of Pax-1 in axial skeleton development

1109
Development 120, 1109-1121 (1994)
Printed in Great Britain © The Company of Biologists Limited 1994
The role of Pax-1 in axial skeleton development
Johan Wallin1, Jörg Wilting2, Haruhiko Koseki1, Rüdiger Fritsch3, Bodo Christ2 and Rudi Balling1,4,*
1Department of Developmental Biology, Max-Planck Institute of Immunobiology,
2Department of Anatomy, University of Freiburg
3Max-Planck Institute for Biophysical Chemistry, Göttingen
4Institut für Säugetiergenetik, GSF-Forschungszentrum Neuherberg, FRG
Stübeweg 51, D-79108 Freiburg, FRG
*Author for correspondence at address4
SUMMARY
Previous studies have identified a single amino-acid substitution in the transcriptional regulator Pax-1 as the cause
of the mouse skeletal mutant undulated (un). To evaluate
the role of Pax-1 in the formation of the axial skeleton we
have studied Pax-1 protein expression in early sclerotome
cells and during subsequent embryonic development, and
we have characterized the phenotype of three different
Pax-1 mouse mutants, un, undulated-extensive (unex) and
Undulated short-tail (Uns). In the Uns mutation the whole
Pax-1 locus is deleted, resulting in the complete absence of
Pax-1 protein in these mice. The other two genotypes are
interpreted as hypomorphs. We conclude that Pax-1 is
necessary for normal vertebral column formation along the
entire axis, although the severity of the phenotype is
strongest in the lumbar region and the tail. Pax-1-deficient
mice lack vertebral bodies and intervertebral discs. The
proximal part of the ribs and the rib homologues are also
missing or severely malformed, whereas neural arches are
nearly normal. Pax-1 is thus required for the development
of the ventral parts of vertebrae. Embryonic analyses
reveal that although sclerotomes are formed in mutant
embryos, abnormalities can be detected from day 10.5 p.c
onwards. The phenotypic analyses also suggest that the
notochord still influences vertebral body formation some
days after the sclerotomes are formed. Furthermore, the
notochord diameter is larger in mutant embryos from day
12 p.c., due to increased cell proliferation. In the strongly
affected genotypes the notochord persists as a rod-like
structure and the nucleus pulposus is never properly
formed. Since the notochord is Pax-1-negative these
findings suggest a bidirectional interaction between
notochord and paraxial mesoderm. The availability of
these Pax-1 mutant alleles permitted us to define an early
role for Pax-1 in sclerotome patterning as well as a late role
in intervertebral disc development. Our observations
suggest that Pax-1 function is required for essential steps
in ventral sclerotome differentiation, i.e. for the transition
from the mesenchymal stage to the onset of chondrogenesis.
INTRODUCTION
gives rise to vertebral bodies and intervertebral discs whereas
the caudal halves of the paired lateral sclerotome areas are the
origins of neural arches, pedicles and ribs (Verbout, 1985;
Christ and Wilting, 1992). The axial regions of highest cell
density, the intervertebral disc anlagen, are positioned approximately midsegmentally with respect to the former somite
boundaries. By cell labelling experiments it has been shown
that one vertebral body is derived from cells originating from
two adjacent somites (Bagnall, 1992; Ewan and Everett, 1992).
The mechanisms that control these morphological events in
axial and lateral regions have not been studied, however.
The existence of a large number of mouse mutants with
specific malformations in vertebral column development
(Grüneberg, 1963; Theiler, 1988) is an important resource for
investigating the molecular and morphogenetic events that lead
to the development of this structure. One of these mutants,
undulated (un; Wright, 1947), carries a point mutation in the
Pax-1 gene (Balling et al., 1988), which is transcriptionally
activated in sclerotome cells at the time of differentiation of
the epithelial somites (Deutsch et al., 1988). A detailed mor-
The vertebral column is derived entirely from cells in the
ventral halves of the somites. In morphological terms the
events leading to axial skeleton formation have been extensively studied in several species, and are well described
(Verbout, 1985; Christ and Wilting, 1992). The vertebral
column is the most conspicuously segmented structure of the
vertebrate body. The metameric pattern is initially laid down
during the formation of somites, yet there is not a clear relationship between the vertebral units and the initial somites.
Upon somite differentiation the ventral parts de-epithelialize
and form the mesenchymal sclerotomes, which give rise to precartilaginous structures as well as connective tissue. This
process is dependent on signals from the notochord (Watterson
et al., 1954; Pourquie et al., 1993). Some sclerotome cells
migrate medially and form the perichordal tube, which is
initially unsegmented and uniform in density. Thereafter a
segmented pattern of condensations starts to appear, the lateral
regions slightly preceding the axial ones. The perichordal tube
Key words: Pax-1, vertebral column, sclerotome, notochord, mouse
mutants
1110 J. Wallin and others
Fig. 1. Analysis of Pax-1 expression in the developing vertebral column. (A) Whole-mount in situ hybridization of a day 10.5 wild-type
embryo, showing strong Pax-1 expression in the sclerotome. In each segment, the caudal half displays stronger expression than the cranial half.
In addition, a thin domain of strong expression can be seen in the top of the cranial half. Arrows indicate segment boundaries. (B) Pax-1
immunostaining of a frontal section from a day 10.5 wild-type embryo. Cranial is towards the top and lateral to the left of the picture. The
notochord (n) can be seen on the right-hand side and the spinal nerve (sn) in the cranial part of each segment. Most sclerotome (s) cells, seen
between the dermomyotome (dm) and the notochord, are Pax-1-positive, whereas dermomyotome, nerve and notochord cells are negative. The
localization of the spinal nerve as well as cell density, might explain the banded staining pattern in the whole-mount staining in A. (C) A
sagittal section through the thoracic region of a wild-type day-14.5 embryo; ventral is to the left. Expression is now confined to the anlagen of
the intervertebral discs (ivd) and to a layer of cells in the perichondrium surrounding the vertebral body (vb) anlage. Scale bars: (B,C) 100 µm.
phological analysis of the skeletal phenotype and the
embryonic development of this mutant has been made by
Grüneberg (1950, 1954). He described malformations of
vertebral bodies and intervertebral discs as well as vertebral
processes, and came to the conclusion that these result from
decreased sizes of the mesenchymal condensations that
precede chondrification. Two alleles of un, undulatedextensive (unex; Wallace, 1985) and Undulated short-tail (Uns;
Blandova and Egorov, 1975), both being phenotypically more
affected than un, have subsequently been isolated. Both these
mutants have Pax-1 gene deletions, the latter one removing the
entire Pax-1 locus (Balling et al., 1992; Dietrich S., Gruss P.,
and R.B., unpublished observations).
The Pax genes form a family of developmental control genes
that have recently been the focus of great interest (Chalepakis
et al., 1992; Gruss and Walther, 1992; Noll, 1993). They encode
sequence-specific transcription factors that contain the DNAbinding paired-domain (Bopp et al., 1986; Chalepakis et al.,
1991; Adams et al., 1992; Zannini et al., 1992), and in addition
some of the Pax proteins also contain a paired-type homeodomain (Walther et al., 1991). In the mouse, nine Pax genes
have been identified to date (Walther et al., 1991; Wallin et al.,
1993), all being expressed in a spatially restricted manner
beginning during early organogenesis. In addition to Pax-1, two
other members of the Pax gene family have been shown to play
important roles in mouse embryogenesis. Mutations in the Pax3 and Pax-6 genes cause the Splotch and Small-eye phenotypes,
respectively (Epstein et al., 1991; Hill et al., 1991).
To gain further insight into early mechanisms of vertebral
column formation we have carried out a comparative phenotypic analysis of the three Pax-1 mouse mutants, un, unex and
Uns, of which the latter two have not been investigated before.
By analysis of mutant embryonic and new-born mice we show
that Pax-1 is required for normal development of ventral
vertebral structures, but is dispensable for sclerotome
formation per se.
MATERIALS AND METHODS
Mice
undulated (un) mice were purchased from the Jackson laboratory.
Undulated-short tail (Uns) and undulated-extensive (unex) mutant
mice were kindly provided by Dr A. M. Malashenko, Krosnogorsk,
Russia and Dr J. L. Cruickshank, Leeds, England, respectively. These
mutants were backcrossed onto the C57BL/6 strain. On this genetic
background homozygous un and unex mice show reduced fertility, particularly unex. Homozygous unex mice are often born small, in which
case they also remain smaller and die earlier than their littermates. N1
to N5 generations were used for analysis. Embryos were recovered
on days 9.5-18.5 p.c. where day 0.5 was 12 a.m. on the day of
detection of the vaginal plug. On average, litters were born on day 19
p.c. As controls, +/+ or un/+ littermates were normally used. In exceptional cases C57BL/6 embryos of the corresponding stages were used.
Genotyping
Genotypings were made either by PCR analysis on embryonic DNA
prepared from yolk-sacs or by Southern blotting of placental DNA.
Genomic Southern blots were performed according to standard procedures (Sambrook et al., 1989). For PCRs two different primer pairs
were used: (1) with primers flanking the mutated HaeIII restriction
site in un and (2) specific for the fifth exon deleted in unex. These Pax1-specific oligonucleotides were:
(1) 5′-CAGAGCAGACGTACGGCGAAG-3′ and 5′-AGGCAAAGATGCCAGGATCCC-3′ and for
(2) 5′-AGAGCCATCAGCATGGTTTCG-3′ and 5′-TGGAGGGAGTCCAGATTAAGC-3′. PCR reaction conditions were: 30 cycles
of denaturation for 1 minute at 94°C, reannealing for 1 minute at 60°C
Pax-1 and axial skeleton development 1111
Fig. 2. A schematic representation of the molecular basis of the Pax-1 deficiency of the un and Uns alleles (A), and the external appearance of
adult homozygous un (B), homozygous unex (C) and heterozygous Uns mice (D). In the upper part of A, the exon-intron organization of the
Pax-1 gene is depicted (Deutsch, 1990) The coding region is indicated as filled boxes and the paired box is hatched. The position of the point
mutation in un is marked by a cross. The exact locations of the deletion breakpoints in Uns have not been determined.
and extension for 1 minute at 72°C for 1 minute; 2.5 units Taq polymerase (Amersham) using assay conditions specified by the manufacturer in a 50 µl reaction volume. PCR products were separated in
agarose gels and visualized by ethidium bromide staining. For typing
of un, PCR products were digested with HaeIII before gel separation,
producing allele-specific band sizes that could easily be scored. For
diagnosis of Uns and unex homozygotes, a primer pair specific for an
unlinked gene (Pax-9) was included as an internal control. A large
number of un and unex embryos were also obtained from crosses of
homozygous mice.
Histology
For histological analysis, day 9.5-18.5 embryos were fixed in Bouin’s
solution and embedded in paraffin. 8 µm sections were stained either
in haematoxylin-eosin or Azan. In addition, for day 10.5-12.5 p.c.
embryos of the Uns/Uns, Uns/un and +/+ genotypes, semi-thin
sections were made, focusing on the strongly affected lumbar region.
Embryos were fixed in Karnovsky’s solution, embedded in plastic and
0.75 µm sections were stained with toluidine blue.
Skeletal preparations
Skeletal preparations were made by a slight modification of the Alcian
blue/alizarin red staining procedure described by Kessel et al. (1990).
Specimens were fixed in 99% ethanol for 24 hours (fetuses older than
day 15 p.c. were first deskinned and eviscerated), and then kept in
acetone for another 24 hours. Incubation in staining solution (1 vol.
of 0.3% Alcian blue in 70% ethanol, 1 vol. of 0.1% alizarin red S in
96% ethanol, 1 volume of absolute acetic acid, and 17 volumes of
70% ethanol) was performed for 4-6 hours at 37°C and then overnight at room temperature. Samples were rinsed in water and kept in
1% potassium hydroxide/20% glycerol at 37°C over-night, with additional incubation at room temperature until complete clearing. For
long term storage, specimens were transferred into 50%, 80% and
finally 100% glycerol.
In situ hybridization
Pax-1 riboprobes were generated from a HincII-SacI paired box
fragment using either 35S-UTP or digoxigenin-11-UTP labelling for
in situ hybridization on sections or in whole embryos, respectively.
Fixation, hybridization and subsequent detection procedures were
essentially as described by Kessel and Gruss (1991) and Rosen and
Beddington (1993).
BrdU labelling
Cell proliferation was monitored by incorporation of BrdU into
embryos in utero. Pregnant mice were injected intraperitoneally with
a 5 mg/ml BrdU (Sigma) solution in PBS. The amount injected was
approximately 50 µg/g body weight. After 2 hours the mice were
killed and embryos isolated, fixed and embedded in paraffin. 8 µm
sections were stained with an anti-BrdU antibody as described below
and counterstained with basic fuchsin or hematoxylin. The number of
labelled cells as well as total cell numbers were scored.
Immunohistochemistry
For immunostainings, embryos were fixed in 3% acetic acid in
absolute ethanol at 4°C over-night, incubated first in absolute ethanol
and then in xylene, both twice for 30 minutes, followed by a 1:1
mixture of xylene:paraffin for 30 minutes at 55°C, infiltrated with
paraffin by three incubations for 1 hour at 55°C and embedded.
The generation of the Pax-1-specific antiserum has been previously
described (Chalepakis et al., 1991). The mouse monoclonal anti-BrdU
antibody was purchased from Dako.
For BrdU immunocytochemistry, sections were deparaffinized and
air-dried over-night. They were then incubated in 2 N HCl for 30
minutes, washed in PBS, pH 6.0, for 1 minute and in PBS, pH 7.4
(hereafter called PBS) for 5 minutes. Primary antibody was diluted
1:50 in PBS, 1% BSA, 0.25% Tween-20 and incubated for 1 hour.
Following washes, detection was made with the biotin-streptavidinperoxidase amplification procedure, UniTect ABC-kit (Dianova).
Pax-1 immunostaining was made on deparaffinized sections that
were first bleached in 0.3% H2O2 in methanol for 30 minutes, washed
in PBS, blocked with 10% normal goat serum, in PBS for 60 minutes
and then incubated with the antiserum diluted 1:200 in the blocking
solution. Detection was made with peroxidase-conjugated goat-antirabbit IgG (Sigma), and diaminobenzidine was used as chromogen.
1112 J. Wallin and others
RESULTS
root ganglia) remain strongly Pax-1-positive at day 14.5 p.c.
(Figs 1C, 7G). In the vertebral column of new-born mice only
a small number of Pax-1-positive cells can be observed in the
ventral part of the annulus fibrosus (data not shown).
Pax-1 expression during sclerotome development
To determine the role of Pax-1 in sclerotome differentiation,
we have analysed Pax-1 RNA and protein expression in early
Comparative phenotypic analysis of three Pax-1
somites and in subsequent axial skeleton development. Pax-1
mutant alleles
transcription can first be detected around day 8.5 p.c. in the
ventromedial part of newly formed somites (data not shown).
We have chosen to study the phenotype of three spontaneously
This corresponds to the time of deepithelialization of the ventral
somite half, i.e. the emergence of
the sclerotome. At first, expression
is uniformly distributed in sclerotome cells. In whole-mount
expression analysis of day-10.5
embryos with a Pax-1 RNA probe,
it was possible to distinguish
between cranial and caudal parts of
the sclerotomes, the caudal part of
each segment being more strongly
positive and extending more
dorsally than the cranial one (Fig.
1A). At day 12.5, the Pax-1 transcripts are mainly confined to the
anlagen of the intervertebral disc
and weaker domains can be seen in
the perichondria lining the cartilage
blastemas of the vertebral bodies,
the pedicles and the proximal ribs
(data not shown). These findings
are in good accordance with those
of Deutsch et al. (1988).
Pax-1 protein expression was
detected with a rabbit antiserum
raised against a peptide in the
carboxy-terminal end of the protein
(Chalepakis et al., 1991). The
protein is expressed with a delay of
about 1 day compared to the onset
of transcription. First, at day 9.5,
Pax-1 protein can be detected in
sclerotome cells, albeit weakly
(data not shown). Immunostaining
of day-10.5 embryos revealed
expression in almost all sclerotome
cells (Fig. 1B). These cells
continue to express Pax-1 up to the
time when mesenchymal cells start
to chondrify in order to form the
vertebral bodies, which takes place
around day 12.0 p.c. At day 12.5
p.c., the cells of the anlagen of the Fig. 3. Histological analysis at day 18.5 p.c. Sagittal sections of +/+ (A) and Uns/Uns (B). Note the
vertebral bodies are not stained, complete absence of vertebral column structures adjacent to the aorta in the mutant (arrowhead).
of the internal organs and a reduced
while the anlagen of the interverte- The shortening of the vertebral column leads tos a compression
s
bral discs remain strongly positive thoracic cavity. The airways of the lungs in Un /Un are not dilated and the lung appears more
litter-mate. Arrows indicate lungs. Higher magnification of thoracic
(Fig. 7E). Cells surrounding the compact than in the wild-type
vertebrae of +/+ (C) and Uns/Uns (D) are shown. The skeletal elements in the mutant section are
vertebral body anlagen, those in the much thinner dorsoventrally and resemble ventral arches rather than proper vertebral bodies (vb).
disc anlagen as well as those in the Note also the absence of intervertebral discs (ivd). Although consecutive boundaries can be
cranial half of each lateral sclero- identified, the nucleus pulposus and annulus fibrosus are missing. (E) Example of fused dorsal root
tome (forming connective tissue ganglia (drg) over three segments in a parasagittal section of Uns/Uns at day 18.5 p.c. Such fusions
around the spinal nerves and dorsal are found along the entire axis in this genotype. Scale bars: (A,B) 2 mm, (C-E) 200 µm.
Pax-1 and axial skeleton development 1113
arisen mouse mutants, un, unex and
Uns, since the molecular characterization of these mice has shown that
there is a point mutation in the
paired domain of un (Balling et al.,
1988), whereas unex and Uns
display Pax-1 gene deletions. In
Uns the entire locus is deleted
(schematically depicted in Fig. 2A).
The absence of the Pax-1 gene in
this mutant has previously been
demonstrated for the paired box
region (Wallin et al., 1993), and has
been extended to the 5′-flanking
sequences and 3′-untranslated
region (data not shown). The size of
the Uns deletion has not been
exactly determined, but a major
chromosomal deletion has been
excluded by karyotyping and by
demonstration of the presence, in
Uns homozygous DNA, of several
closely linked DNA markers
(GBASE, March, 1993, A. Y.
Hillyard, D. P. Doolittle, M. T.
Davisson, and T. H. Roderick, The
Jackson Laboratory, Bar Harbor,
ME) (data not shown). In unex the
fifth exon, which includes part
of the carboxy-terminal coding
sequence, is deleted (Dietrich S.,
Gruss P., and R. B., unpublished
observations). The allelic nature of
all three mutations has also been
confirmed by inter-crosses. In no
combination of the different
mutants was a complementation
effect observed.
The phenotypic appearance of
adult mutant mice is characterized
by their shortened and kinky tails
(Fig. 2B-D). un is known as a
recessive mutation (Wright, 1947),
but in our colony we have observed
a small percentage of heterozygous
animals that have slight but significant distal tail kinks, which is also
true for unex (data not shown). Thus
although un/+ embryos occasionally
do display very mild abnormalities,
they serve as good controls for the
stronger genotypes for all practical
purposes. Uns is invariably semidominant and produces a clear
phenotype in the heterozygous
situation. Uns/Uns mice die shortly
after birth. Their tails are only rudimentary and the trunks considerably
shorter than those of wild-type littermates (Fig. 3A,B), nevertheless
pups show normal movements at
Fig. 4. Comparison of the vertebral column phenotypes at the new-born stage. Whole-mount skeletal
preparations of the following genotypes are shown: +/+ (A), un/un (B), unex/unex (C), Uns/+ (D),
Uns/un (E) and Uns/Uns (F). Note the phenotypic similarity between the un/un and unex/unex skeletons.
The strongest abnormalities can be seen in the lumbar region where split vertebrae and dual
ossification centers as well as affected transverse processes are found. In Uns/+ (D), the ossification
centers are slightly smaller and the lateral sclerotome derivatives, transverse processes and ribs, are
clearly affected. Note for example the floating 13th rib pair. In Uns/un (E), ventral vertebral structures
are severely affected in the cervical and lower thoracic-lumbar region. The last four rib pairs are
floating. The most extreme phenotype, that of Uns/Uns (F), is an accentuation of what is seen in
Uns/un. Affected structures are now further decreased in size, displaying a complete lack of ventral
skeletal structures in lower thoracic-lumbar region. Arrows point out abnormal proximal ribs.
1114 J. Wallin and others
Pax-1 and axial skeleton development 1115
Fig. 6. Alcian blue stainings of fetal
skeletons. Dorsal view of day 13.5 p.c.
+/+ (A) and Uns/Uns (B) specimens, and
detail of a day 14.5 p.c. unex/unex
skeleton (C). In the cervical and lower
thoracic-lumbar region the cartilage
precursors of the vertebral bodies are
absent (B). The change in phenotypic
pattern between the sacral and caudal
regions can also be seen. Cartilage
structures surrounding the notochord
can be seen in the midline of the tail,
while there is an absence of pedicle
anlagen bilaterally. (C) The vertebral
body formation is significantly more
advanced at the side of the notochord,
which has been laterally displaced
(arrow).
birth. The reason for the postnatal lethality, which is also
observed for the Uns/un and Uns/unex compound heterozygotes,
could not be exactly determined. The most likely explanation
is respiratory failure as a result of lung space constraints. Histological sections of Uns/Uns at day 18.5 p.c. show lungs with
drastically reduced airway lumina (Fig. 3A,B).
Fig. 5. Analysis of separate skeletal elements at the new-born stage.
Frontal views of the lower thoracic-upper lumbar region of the +/+,
un/un, Uns/+, Uns/un and Uns/Uns genotypes (A-E). In the new-born
wild-type skeleton (A) the ribs form joints at the lateral aspects of
the intervertebral discs, which at this stage are well-defined
structures. In un/un, however, the 13th rib pair fuses to the vertebral
body (arrowhead) and the discs are virtually absent (arrow). In Uns/+
(C), the annulus fibrosus is thinner than in the wild-type (arrow),
whereas Uns/un (D) and Uns/Uns (E) have no bodies and discs. Note
also the notochord remnant in Uns/un (D) which is covered by a
cartilage layer. The ninth thoracic vertebrae of the same genotypes as
A-E are compared in F-J, respectively. The principal similarity of the
phenotypes can clearly be seen; vertebral bodies are reduced or
absent and the proximal part of the rib is abnormal or missing. A
comparison of isolated vertebrae from different craniocaudal levels
of +/+ (K-O) and Uns/Uns (P-T) new-born mice are also shown.
Atlas (K,P), sixth cervical vertebrae (L,Q), fourth lumbar vertebrae
(M,R), second sacral (N,S) and tails (O,T). The +/+ tail specimen in
O shows the last sacral and the first three caudal vertebrae, while the
Uns/Uns (T) tail is composed of the last two sacral vertebrae as well
as remnants of several caudal vertebrae. Arrows indicate the position
of the first caudal vertebrae. With the exception of the atlas and the
tail, the vertebral abnormalities at different levels are principally
similar, i.e. lack of ventral and ventrolateral elements. A slight effect
on of the neural arches can also be noted as a reduction of the
spineous processes. The dens axis is fused to the atlas in Uns/Uns
(arrowhead; P). The tails are viewed at a slight angle and the
abnormal tail of Uns/Uns has a trifurcated appearance (T). One
ventral and two lateral structures are observed. These vertebrae
remnants are fused along the craniocaudal axis. ivd (intervertebral
disc), vb (vertebral body), n (notochord), L1 (first lumbar vertebra),
T (thoracic), C (cervical), S (sacral), vt (ventral tubercle), tp
(transverse process).
Whereas the kinks in un/un are soft and can easily be
corrected upon pressure, the tail abnormalities in unex/unex and
Uns/+ are more pronounced and rigid, partially due to vertebral
fusions. The increase in severity of the phenotypes, can be
interpreted as a gradual loss of Pax-1 function in the different
mutants. In general, the axial skeleton abnormalities in these
mutants are qualitatively similar but quantitatively different.
This is more apparent when a detailed analysis of the vertebral
components is made. As the strongest phenotypes result in
postnatal death, we have concentrated our analyses and comparisons on embryos up to the new-born stage.
Abnormalities in the vertebral column of new-born
mice
All undulated mutants display phenotypic abnormalities of the
vertebral column along the entire axis. A list of typical
vertebral column aberrations is given in Table 1. The abnormalities affecting vertebral bodies or other axial skeletal
elements are similar in all the mutants, but can be found to
different degrees; the Uns/Uns genotype being the most
strongly affected. The severity of defects is not evenly distributed along the axis and is most pronounced in the lumbar
Table 1. Types of skeletal abnormalities that are observed
in the vertebral column of un, unex and Uns mice
Perichordal tube derivatives
Absence of vertebral body
Split vertebral body
“Ventral arches”
Fusions between atlas and dens axis
Reduced and malformed vertebral
body (dual ossification centers)
Absent or reduced intervertebral
discs
“Haemal arches” (in proximal part
of tail)
Fusions between vertebral bodies
(mainly seen in tail)
Lateral sclerotome derivatives
Absent or reduced transverse
processes
Absent or reduced ribs
Fusions between ribs and vertebral
bodies
Fusions between adjacent neural
arches
Reduced spinous processes
Absent pedicles (only in caudal
vertebrae)
1116 J. Wallin and others
region and the tail. A comparison of skeletal preparations at
the new-born stage is shown in Fig. 4. In the wild type, single
ossification centres are formed in all vertebral bodies (Fig. 4A).
The un and unex homozygous new-borns show split vertebrae,
vertebrae with dual ossification centers and missing transverse
processes primarily in the lumbar region (Fig. 4B,C). These
phenotypes are very similar, although unex has slightly more
severe malformations and a few more segments are affected.
In the case of Uns a mild phenotype can already be seen in heterozygous mice (Fig. 4D). Vertebral body ossification centers
are smaller than normal and transverse processes as well as the
proximal parts of ribs are missing or reduced. Uns/Uns mice,
with a total Pax-1 deficiency, completely lack vertebral bodies
in the lower thoracic and lumbar regions (Fig. 4F). The
compound heterozygote Uns/un is intermediate in phenotype
between Uns/Uns and un/un mice (Fig. 4E).
On closer inspection of the lower thoracic and upper lumbar
regions, it is evident, that with the exception of Uns/+, the
different mutants lack intervertebral discs (Figs 3C,D and 5AE). In Uns/+ the discs are thinner in the periphery, indicating
a disturbance in the formation of the annulus fibrosus. The
intervertebral disc is, at the new-born stage, composed partly
of hyaline cartilage, and can therefore be stained with Alcian
blue. In adult mice the hyaline cartilage has been replaced by
fibrous cartilage (Theiler, 1988) and appears clear in wholemount skeletal preparations. In adult skeletal preparations the
lack of discs in undulated could be confirmed (data not shown).
In extreme cases the lack of discs leads to vertebral body
fusions. On rare occasions mice of the Uns/unex genotype have
survived after birth and at most lived up to five months of age.
The vertebral column of a few of these mice displayed multiple
fusions (data not shown).
Thus, in all mutants we detect disturbed formation of both
vertebral bodies and intervertebral discs. Furthermore, in all
cases the proximal parts of the ribs as well as the rib homologues, the transverse processes, are affected. In un and unex
the last pairs of ribs are shorter and fuse abnormally to the
vertebral bodies. Uns/+, Uns/un and Uns/Uns display more
severe abnormalities, with several floating rib pairs and the
13th pair is completely missing in Uns/Uns (Fig. 5A-E).
A systematic comparison of the +/+, un/un, Uns/+, Uns/un
and Uns/Uns genotypes at the level of the ninth thoracic
vertebra clearly shows that the basic phenotype is essentially
the same, and varies only in degree of severity between the
alleles (Fig. 5F-J). Although we observe small variations in
phenotypic severity within one allele (none of the strains are
completely inbred), these are insignificant compared to the
variation between alleles.
Comparison of single vertebrae was made by dissection of
whole-mount skeletal preparations. In Fig. 5K-T representative
vertebrae from cervical, lumbar, sacral and caudal regions from
wild-type and Uns/Uns mice are shown. From this analysis it
is evident that vertebral body and rib/transverse process
formation are abnormal at all levels. Where a ventral ossified
structure can be seen, it resembles a pair of ventrally fused
neural arches rather than a vertebral body (Fig. 5J,S).
Therefore, Uns/Uns mice are deficient in structures derived
both from the perichordal tube and the ventralmost parts of
lateral sclerotomes. The neural arches are fairly normally
shaped. Reductions of the spinous and articular processes as
well as rare fusions between adjacent neural arches can be
detected. These deformities are discrete in comparison to the
ventral ones and may to a large extent be due to the compression of the vertebral column. The tail vertebrae display a
phenotype which is different from the rest of axis. The tailremnant in Uns/Uns gives a trifurcated appearance, representing vestigial vertebral bodies and paired neural arches (Fig.
5T). The pedicles, the structures connecting bodies and neural
arches, are lost. These malformed structures display only scant
signs of segmentation and are fused to the tip of the tail.
First phenotypic sclerotome abnormality at day 10.5
p.c. of development
To study when the Pax-1 protein starts to exert its effect we
have conducted a detailed morphological analysis of mutant
embryos. We have concentrated our study primarily on the
lumbar region in the different mutants with particular emphasis
on the strongest genotypes Uns/un and Uns/Uns. The compound
heterozygote Uns/un has the advantage that the Pax-1 RNA and
protein expressed from the un allele can be detected in the
mutant embryos, although functionally this mutant is almost as
affected as the null-allele Uns/Uns.
Analyses of cartilage formation in skeletal clearance preparations, revealed that axial structures in the vicinity of the
notochord never chondrify in mutant embryos (Fig. 6B). The
mutant phenotype is visible from day 11.5 p.c., the first timepoint when Alcian blue can be used to stain the developing
skeleton. Therefore, in agreement with the earlier results of
Grüneberg (Grüneberg, 1954), it can be concluded that the
malformations are formed at an early embryonic stage and that
the primary function of Pax-1 should be looked for before
chondrogenesis commences. This lack of axial chondrogenesis most often leads to a lateral displacement of the notochord.
Vertebral body formation is always more normal on the side
of the notochord. This phenomenon, best observed in unex/unex
skeletal preparations (Fig. 6C), suggests that vertebral body
formation is also dependent on a late notochord function.
Histological analysis of early vertebral column formation,
reveals that sclerotome formation occurs largely normally in
the Pax-1-deficient mice. Morphological abnormalities can
first be detected at day 10.5 p.c., when the lumbar sclerotome
region already is abnormal compared with controls. In the wild
type, densely and loosely arranged areas are present and the
axial cells are oriented towards the notochord, whereas in the
most affected mutants the sclerotome cells are evenly distributed (Fig. 7A,B).
Abnormalities in the perichordal tube and lateral
sclerotome regions
At day 12.5 p.c. more profound changes can be detected. In
mid-sagittal sections of Uns/Uns and Uns/un embryos, in the
region of the perichordal tube, cell numbers are strongly
reduced, corresponding to the loss of the anlagen for the
vertebral bodies and intervertebral discs (Fig. 7E,F). The few
cells present in the perichordal tube of Uns/un embryos at this
stage do not display the normal metameric arrangement of
condensed intervertebral disc anlagen and cell-sparse vertebral
body anlagen. We performed immunostainings on mutant and
wild-type embryos to compare the distribution of Pax-1expressing cells. As can be seen in Fig. 7, the Pax-1-positive
domains are much wider in the craniocaudal dimension in the
Uns/un mutant. The observation that most cells are Pax-1-
Pax-1 and axial skeleton development 1117
Fig. 7. Histological (A-D) and immunohistochemical (E-H) analysis of mutant and wild-type embryos. Sclerotome (s) is formed but displays
abnormal organization in transverse semi-thin sections of day 10.5 +/+ (A) and Uns/un (B) embryos. In the wild-type embryo a clear
mediolateral organization can be observed, with a cell-dense region next to the dermomyotome (dm) and fewer cells surrounding the
notochord. In the mutant, however, the mesenchymal cells are disorganized and very few cells can be observed in the perichordal space.
Arrowheads indicate the position of the notochord. Frontal sections of day 12.5 +/+ (C) and Uns/un (D) embryos through the lumbar-sacral
region. Note the reduced size of the cell condensations that surround the notochord (n). Also the lateral sclerotome regions are affected. Loss of
caudal (ca) halves as well as over-abundance of cells surrounding the spinal nerves in the cranial (cr) halves are observed. Arrowheads indicate
the position of segment boundaries. In Pax-1 immunostainings of sagittally sectioned day 12.5 un/+ (E) and Uns/un (F) embryos, the reduction
of perichordal tube size can be clearly observed. Note also the absence of well-defined segmentation. Parasagittal sections of sacral regions in
day 14.5 un/+ (G) and Uns/un (H) embryos show the absence of ventrolateral skeletal structures, i.e. transverse processes (tp), are observed in
the mutant. Instead, spinal nerves are surrounded by Pax-1-positive cells only. nt (neural tube), na (neural arch). Scale bars: (A-D,G,H) 100 µm,
(E,F) 250 µm.
positive reflects the lack of chondrogenesis, which normally
coincides with loss of Pax-1 expression as described previously. In Uns/Uns embryos, a more severe loss of cells in the
perichordal tube could be observed and the total absence of
Pax-1 protein could be confirmed (data not shown).
To analyse axial and lateral relationships, frontal sections of
mutant embryos were made at day 10.5-12.5 p.c. In sections
of the ventral part of the developing vertebral column in day12.5 p.c. mutant embryos, a profoundly altered organization of
the lateral lumbosacral sclerotome domains, in addition to the
poorly developed perichordal tube, was revealed. Instead of the
normal division in cranial and caudal halves, only one ‘compartment’ with equal, fairly high, cell density is detected. The
spinal nerve is found in the middle, surrounded by concentric
layers of fairly dense cells (Fig. 7C,D). These cells are all
staining positive for Pax-1 (Fig. 7G,H). The effect is an
expansion of mesenchymal regions at the expense of cartilage
formation. Lack of the caudal condensations in these ventrolateral regions later results in the lack of transverse processes
and proximal rib structures in the vertebral column of newborn mice. In more dorsal sections, through the anlagen of the
neural arches, the craniocaudal division of each segment
appears more normal. Thus, Pax-1 mutants do not have difficulty in establishing craniocaudal polarity per se, but rather
display a loss of such polarity in the ventral domain of the sclerotome due to the loss of skeletal precursor structures.
The notochord and dorsal root ganglia do not
develop normally in undulated mice
When analysing sclerotome morphology in mutant embryos,
we noticed that the notochord appeared larger than in wild-type
controls at day 12.5-13.5 p.c. Transverse sections reveal more
cells per section and a changed notochordal sheath in the
mutants (Fig. 8). Consistent with this observation we found a
dramatic increase in the rate of cell proliferation at the corresponding stage. In mutant embryonic notochord, a seven to tenfold increase in the number of BrdU-labelled nuclei was
detected, at day 12.5 p.c. (Fig. 8E,F and Table 2). The finding
1118 J. Wallin and others
Table 2. Determination of notochordal cell proliferation
rates at day 12.5 p.c.
+/+
un/+
Percentage of labelled
cells
1.8
2.5
Average number of
labelled cells/section
0.45
0.45
Uns/un
17
3.9
Uns/Uns
22
6.4
Replicating embryonic cells were labelled with BrdU in utero. For each
genotype 18 transverse sections from the lumbar region of two separate
embryos have been analysed.
that the development of the notochord is impaired is evident
also at later stages. Whereas the normal notochord vanishes
from the vertebral bodies and is supposed to contribute
to the formation of the nucleus pulposus, the
notochord in the strong Pax-1 mutants persists as a
rod-like structure up to the new-born stage. This can
be seen in skeletal preparations of the Uns/un
genotype most clearly (Fig. 5D).
Uns homozygotes also display fused dorsal root
ganglia (DRG) at day 18.5 p.c. (Fig. 3E). To some
degree DRG fusions can be seen in all un alleles (data
not shown). In early stages, up to around day 12.5 p.c.,
no fusions are detected. These arise later during fetal
development, possibly as a consequence of the severe
shortening of the trunk (Fig. 3B).
all or most cells that participate in the formation of ventral
vertebral structures have expressed Pax-1 in their early developmental stages.
In Grüneberg’s original description of un, he stated that the
intervertebral discs are larger than in normal mice (Grüneberg,
1954). This observation could not be confirmed in the present
study. On the contrary, the annulus fibrosus is much reduced
and cannot be observed in whole-mount skeletal preparations.
The reason for this discrepancy is likely due to the fact that the
anlagen of the intervertebral discs are less well defined and
occupy a larger space in craniocaudal dimension in mutant
embryos. These anlagen will not differentiate into proper discs,
however. Our morphological observations and conclusions
made from these, that are greatly facilitated by the allelic series
DISCUSSION
Pax-1 is required for normal development of
ventral vertebral structures
The proper formation of ventral vertebral structures
requires Pax-1 function. Vertebral bodies and intervertebral discs are virtually absent in Pax-1-negative
mice while the neural arches are fairly normal. This
conclusion is obvious for the lumbar region of
Uns/Uns mutants where ventral structures are completely missing, while other regions as the upper
thoracic region present a more complex phenotype.
Here the vertebrae have a ventral structure that
connects the neural arches. Due to its form and small
size we suggest that these are ventral neural arches
and the perichordal tube contribution to this tissue, if
any, is very small. Thus Pax-1-negative mice lack
vertebral bodies at all levels of the vertebral column
down to the beginning of the tail. The tail phenotype
of Uns/Uns mice is different, however, due to the fact
that the ventral cartilage remnants are not continuous
with the paired lateral ones. The different phenotype
in the tail may represent a different mechanism for
vertebra formation by cells originating from the tail
bud. When the perichordal tube has acquired a welldefined segmentation at about day 12.5 p.c., the intervertebral disc anlagen are Pax-1-positive whereas the
vertebral body anlagen are negative. It may not be
surprising, however, that both vertebral bodies and
intervertebral discs are heavily affected in the
mutants since the immunostaining data indicate that
Fig. 8. Notochord morphology and proliferation rate are changed in Pax-1deficient embryos. In lumbar transverse sections of day 13.5 +/+ (A) and
Uns/Uns (B) embryos, the typical phenotype with absent ventral vertebral
structures can be observed in the mutant. Moreover, the size of the notochord
(arrowhead) is increased in the mutant. Higher magnification of +/+ (C) and
Uns/Uns (D) reveals a significant increase in notochord diameter and cell
number. Note also the difference in the notochordal sheaths (arrows), the
mutant sheath being thinner. The larger size of the notochord can be attributed
to a higher notochordal proliferation rate at day 12.5 p.c. When comparing
transverse sections of un/+ (E) and Uns/un (F) embryos, a higher number of
cells in the mutant notochord (n) have been labelled with BrdU. The figure
shows two representative sections through the lumbar region. Scale bars:
(A,B) 100 µm, (C,D) 10 µm, (E,F) 20 µm.
Pax-1 and axial skeleton development 1119
now available, are otherwise in good agreement with
Grüneberg’s work on un (1950, 1954).
The role of Pax-1 in intervertebral disc development might
represent a late function compared to the patterning of early
sclerotome cells. In later stages, Pax-1 expression is found in
disc anlagen and lining the skeletal structures. Pax-1 may here
have a role in the differentiation of fibrous tissue, and
therefore, in the maintenance of the boundaries between
skeletal elements. Indeed, fusions between axial skeletal
elements are frequently seen in the mutants.
The strong phenotypic similarities in the three Pax-1
mutants leave little doubt that undulated is due to a reduction
of Pax-1 function. While in un the phenotype can be linked to
a single point mutation in the Pax-1 gene, the deletion sizes of
the other two alleles are not known. It thus remains an open
question whether other deleted genes also significantly contribute to the strong phenotype seen in Uns/Uns mice. We
believe this not to be the case, because of the apparent lack of
any phenotypic changes that cannot be observed in a milder
form in un mice. This will not be strictly proven, however, until
a clean Pax-1 null mutation has been accomplished via homologous recombination in ES cells and introduced into the
germline or until Uns/Uns mice have been rescued via transgenesis. We suggest that Uns/Uns mice display a Pax-1 null
phenotype and that the weaker mutations are hypomorphs. An
observation in support of that idea is that Pax-1un protein still
retains DNA-binding activity, albeit much weaker than the
wild-type protein (Chalepakis et al., 1991). Further support for
this notion is provided by the demonstration that the causative
genetic change in the Splotch-delayed mutant, is a point
mutation in the paired domain, similar to the situation in un.;
the phenotype of this mutant was interpreted to be due to partial
loss of Pax-3 function (Vogan et al., 1993).
Recently, a detailed study of the interaction of paired
domains with DNA target sequences revealed that this DNA
binding domain is composed of two subdomains (Czerny et al.,
1993). These authors discuss that a mutation in one of the subdomains may affect the binding to only a subset of target genes.
This speculation is of great interest for the present study, as un
is one of the potential models. Indeed, a differential effect on
separate targets is consistent with our phenotypic analyses.
Although all abnormalities to some degree can be seen in all
mutants, the relative strength of phenotypic alterations in
different regions are not always proportional. Thus, when the
phenotype of un is compared to that of Uns, it can be seen that
the point-mutation in un affects development in axial sclerotome derivatives relatively more than lateral ones, i.e. the
rib/transverse process phenotype is fairly weak in un.
We have recently identified a Pax gene that is highly homologous to Pax-1 (Wallin et al., 1993). This gene, Pax-9, is
expressed in a similar but not identical pattern during sclerotome development. To analyse whether the expression of this
gene is dependent on Pax-1 we have compared Pax-9
expression domains in wild-type and Uns/un embryos (A.
Neubüser, J. W. and R. B., unpublished observations). We
have not been able to detect any changes in expression patterns
that may not primarily be due to morphological changes.
Sclerotome and notochord are interdependent
structures
A common denominator of the affected vertebral structures is
their proximity to the notochord. The development of the
vertebral column is dependent on the presence of the notochord
(Watterson et al., 1954), and a supernumerary notochord
induces the formation of additional vertebral body-like structures, but represses myotome formation, in surgically manipulated chicken embryos (Brand-Saberi et al., 1993; Pourquie et
al., 1993). Interestingly, it was also shown that the notochord
is able to induce Pax-1 expression in dorsal somite derivatives,
that are normally Pax-1 negative (Brand-Saberi et al., 1993).
The induced Pax-1 expression is accompanied by a loss of dermomyotome cells and the appearance of sclerotome-like mesenchymal cells. These observations make Pax-1 a very good
candidate for a mediator of notochordal signals to the sclerotome, and as such might function as an embryonic competence
factor. Based on the analysis of Pax-1 expression in the
notochord mouse mutant Danforth’s short-tail (Sd) and comparison of the Sd and un phenotypes, we have suggested that
Pax-1 might be one of the major mediators of inductive signals
from the notochord to the sclerotome (Koseki et al., 1993). Our
observation that vertebral body formation is often differentially
affected on either side of the midline is correlated with a displacement of the notochord to the more normal side. We
suggest that the development of the bodies is dependent on
notochord signals that come significantly later than the
factor(s) that initially induce Pax-1 expression. The simplest
interpretation would be that sustained notochord signalling is
required for normal maintenance of Pax-1 expression, which
in turn controls the downstream cascade involved in the
formation of vertebral bodies and intervertebral discs. Alternatively, Pax-1 acts as a competence factor in sclerotome cells
enabling them to respond to further notochord signals.
A strong rib phenotype was observed in Uns/Uns. The
proximal part is absent or reduced while lateral rib structures
are unaffected. This division corresponds fairly well to those
parts of the ribs that are apparently derived from medial and
lateral somite-halves, respectively; the medial half gives rise
to proximal ribs, whereas the lateral half gives rise to the more
distal part of the rib (Charles Ordahl, personal communication). It may be that only the medial somite-half is dependent
on notochord signals; in Sd, the rib phenotype is similar to the
one in Uns/Uns (Koseki et al., 1993). Mutant mice with a
phenotype that is virtually the opposite of that for Uns with
respect to the ribs have been recently described (Braun et al.,
1992). These mice carry targeted mutations in the myf-5 gene.
To our surprise the morphological changes in the mutant
also involve the notochord. Enlarged notochords were shown
to have increased proliferation rates, and abnormal development results in the persistence of the notochord in vertebrae.
These observations indicate that proper notochord development is dependent on surrounding sclerotome cells, which
means that signalling may be bidirectional. Normally the
notochord expands significantly at the level of the intervertebral discs, whereas it vanishes from the centres of the vertebral
bodies. We suggest that notochordal cell proliferation is
normally suppressed by surrounding perichordal tube cells and
that subsequent differentiation of intervertebral discs leads to
an expansion of the notochord only at these levels. A schematic
model for the induction, expression and functional role of Pax1 is presented in Fig. 9.
Another unexpected finding was the fusion of dorsal root
ganglia. It is conceivable that this abnormality in a neural tissue
1120 J. Wallin and others
Fig. 9. A schematic model for the induction of Pax-1 expression and the role of Pax-1 in vertebral column development. (A) Notochord signals
induce Pax-1 expression in the ventromedial part of the somite (Brand-Saberi et al., 1993; Koseki et al., 1993). (B) Pax-1-expressing
sclerotome cells give rise to ventral parts of the vertebral column. In this developmental process, there is a bidirectional dependence of cells of
the notochord and the anlagen of the vertebral bodies and intervertebral discs for their proper differentiation (arrows). (C) The major
consequences of a Pax-1 deficiency are absence of vertebral bodies and intervertebral discs (black) and the loss of proximal rib structures
(grey) or rib homologues, i.e. the transverse processes. The neural arches (white) are relatively unaffected.
is secondary to sclerotome abnormalities. It is well established
that only the cranial half of each lateral sclerotome segment is
permissive for neural crest cell migration and dorsal root
ganglion growth (Keynes and Stern, 1984). In transplantation
experiments in the avian system, it has been shown that neural
crest-derived cell development in the context of cranial half
somites only resulted in nonsegmented coalesced ganglia
(Kalcheim and Teillet, 1989). In the Pax-1 mutants, the
abnormal sclerotome cells in the cranial compartment may
affect ganglion growth in a way akin to the influence on the
notochord. Alternatively, the shortening of the vertebral
column may cause a mechanical compression that forces the
ganglia together, resulting in fusions.
Pax genes and organ development
From our observations in un mutant mice, we speculate that
Pax-1 may act as an embryonic differentiation factor, making
ventral mesenchymal cells competent to differentiate into
hyaline and fibrous cartilage. Several mutant phenotypes
resulting from genetic alterations in Pax genes have been
described in diverse species (Chalepakis et al., 1992; Gruss and
Walther, 1992; Noll, 1993), making the Pax gene family one
of the currently better understood groups of developmental
genes. Studies on the different Pax mouse mutants have documented a central role for Pax genes in morphogenesis.
Splotch, a Pax-3 mutant, displays overgrowth of the neural
tube with exencephaly and spina bifida, affecting also the
migration of pigment cells, derivatives of the neural crest
(Auerbach, 1954). Small-eye embryos, which carry a mutation
in the Pax-6 gene, also display neural crest cell migration
problems (Matsuo et al., 1993; Schmahl et al., 1993). Our data
on the undulated phenotype might be interpreted as a problem
of sclerotome cell proliferation, differentiation and/or
migration. The significant reduction of ventral sclerotome cells
has not been correlated to a readily detectable decrease in sclerotome cell proliferation rate (J. W. and R. B., unpublished
observations). These preliminary data are, however, compatible with a slight but significant change in proliferation rate.
Compared to the notochord, which displayed a large increase
in proliferation rate, the developing sclerotome is a complex
structure with regional differences in all three dimensions. An
extended analysis is therefore required to document changes in
the different sclerotome regions. One study that argues in
favour of at least some proliferative role of Pax genes is the
demonstration of an oncogenic activity of these genes
(Maulbecker et al., 1993). Recently, a role for Pax-2 in the conversion of mesenchyme to epithelium during kidney development was demonstrated in an organ culture system (Rothenpieler and Dressler, 1993). This situation, where early
mesenchymal cells fail to aggregate and differentiate, bears a
strong resemblance to the phenotype in undulated mice where
mesenchymal sclerotome cells fail to condense and initiate
chondrogenesis. We propose that Pax-1 plays a crucial role in
the early differentiation of ventral sclerotome cells, leading to
profound changes in morphology and extracellular matrix
composition. Very little is known about downstream target
genes under control of Pax transcription factors. So far only
three candidate target genes have been suggested; CD19 for
Pax-5 and thyroperoxidase and thyroglobulin for Pax-8
(Kozmik et al., 1992; Zannini et al., 1992). The identification
of Pax-1 as an essential gene for early sclerotome development
provides a starting point, however, for a characterization of the
cascade of events that underlie the morphogenesis of the axial
skeleton.
We thank Peter Gruss for the gift of Pax-1 cDNA, Mary Dickinson
and Andy McMahon for the BrdU-labelling protocol, Bärbel Strack,
Monika Schüttoff, Günther Frank and Sybille Antoni for technical
assistance, Uli Birsner for oligonucleotide synthesis and Lore Lay for
photographic work. J. W. was supported by an EMBO fellowship and
the Swedish Medical Research Council. H. K. was supported by the
Human Frontier Science Program. The work was supported by the
Max-Planck-Society and the Deutsche Forschungsgemeinschaft (Ba
869/3-1).
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(Accepted 8 February 1994)