/ . Embryol exp. Morph. Vol. 55, pp. 227-245, 1980
Printed in Great Britain © Company of Biologists Limited 1980
227
Histochemistry of the developing notochord,
perichordal sheath and vertebrae in Danforth's
short-tail (Sd) and normal C57BL/6 mice
By LAURIE G. PAAVOLA, 1 DORIS B. WILSON
AND ELIZABETH M. CENTER
From the Department of Anatomy, Temple University School of Medicine,
Philadelphia; Division of Anatomy, Department of Surgery, University of
California, San Diego; and Department of Biology, College of Notre Dame,
Belmont
SUMMARY
The development of the notochord, perichordal sheath and vertebrae was studied in
C57BL/6 and Danforth's short-tail (Sd) mutant mice on days 9-14 of gestation, using
histochemical stains to detect possible extracellular matrix (ECM) components or precursors. Stains used were periodic-acid Schiff (PAS) after diastase treatment (glycoproteins,
neutral polysaccharides) and alcian blue (glycosaminoglycans). Embryos from C57BL/6Sfd
mice were analyzed to establish a normal baseline. In 9-day normal (C57BL/6Sfd; + / + )
embryos the notochord is an uninterrupted structure and contains PAS-positive, diastaseresistant granules, whereas in abnormals (Sd/ + ; Sd/Sd) the notochord is discontinuous and
exhibits few, if any, granules. A notochordal sheath is present in normal and abnormal
embryos on day 10 and stains with PAS, alcian blue and aniline blue; subsequently, it
increases in thickness in normal, but not defective, embryos. In normal embryos, the notochord shows dilatations, and notochordal cells become vacuolated from 13 to 14 days. In
contrast, the notochordal fragments of abnormals never develop dilatations, nor do the cells
vacuolate. Organization of mesenchymal cells into specific patterns is observed initially in
11-day normal embryos; further mesenchymal organization into vertebral and intervertebral
disc analgen occurs during days 12-14. In abnormal embryos, disturbance of mesenchymal
cell organization is evident as early as day 11, and by day 12 aberrant patterns of organization
have emerged. Mesenchymal cells of abnormal embryos also lack the typical distribution of
PAS-positive, diastase-resistant granules that occurs in normal specimens. The possible
relationship of these granules in notochordal and mesenchymal cells to ECM materials is
discussed.
INTRODUCTION
Many studies have focused on the tissue interactions involved in chondrogenesis in an attempt to clarify the mechanisms underlying this process. The
role of the notochord and neural tube in vertebral formation has been of particular interest, and the interactions between these structures and somitic
1
Author's address: Department of Anatomy, Temple University School of Medicine,
3400 N. Broad Street, Philadelphia, PA 19140 (215) 221-3194. U.S.A.
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L. G. PAAVOLA, D. B. WILSON AND E. M. CENTER
mesenchyme during chondrogenesis have been examined extensively both in
vivo and in vitro (see reviews by Holtzer, 1968; Lash, 1968a, b).
Danforth's short-tail (Sd) mouse, which shows defective vertebral development in heterozygotes (Sd/ + ) and in homozygotes (Sd/Sd), provides a system
in which these interactions can bs examined. This mutant, first described by
Dunn, Gluecksohn-Schoenheimer & Bryson (1940), exhibits a syndrome of
defects, including anomalies of the axial skeleton, and of the urogenital and
digestive systems (Gluecksohn-Schoenheimer, 1943). The disturbances of the
axial skeleton include an abnormal articulation between the atlas and axis,
reduction or absence of nuclei pulposi, smaller (split or indented) vertebrae,
and a paucity of tail vertebrae (Theiler, 1951 a, b, 1954; Griineburg, 1953;
Diirr, 1958). The last defect leads to the most conspicuous feature, a shortening
and kinking of the tail.
The Sd mutant mouse is particularly interesting because of the controversy
concerning the relationship of the notochord to the constellation of defects
observed in this animal. Griineburg (1958) suggested that the notochord is
directly responsible for this syndrome, whereas Gluecksohn-Schoenheimer
(1945) indicated that notochordal involvement occurs later and is secondary to
degenerative changes in the mesenchyme of the tail. These uncertainties point
to the need for further examination of the Sd mutant mouse during embryonic
development.
Considerable evidence has accumulated indicating that extracellular matrix
(ECM) materials (e.g. glycosaminoglycans such as chondroitin sulfates, and
glycoproteins such as collagen) mediate vertebral chondrogenesis (Minor, 1973;
Kosher & Lash, 1975). The presence of these substances or their precursors can
be detected by various histochemical stains, including the periodic-acid Schiff
(PAS) reagent and alcian blue. A positive PAS reaction that persists subsequent
to diastase digestion suggests the presence of glycoproteins (LeBlond, Glegg &
Eidinger, 1957), neutral polysaccharides (Kvist & Finnegan, 1970), or other
substances containing neutral sugar residues. Neutral polysaccharides have
been reported to be components of adult (Dische, Danilczenko & Zelmenis,
1958) and embryonic (Kvist & Finnegan, 1970) cartilage. In addition, the linkage
regions connecting the precursors of chondroitin sulfates to core proteins contain neutral sugars (Roden, 1970) that are potentially amenable to periodate
oxidation. A positive alcian blue reaction is generally considered to detect
glycosaminoglycans (acid mucopolysaccharides) (Yamada, 1963; Pearse, 1968).
Thus, these stains are capable of detecting various components of the ECM or
their precursors and were used as the main probes in this investigation.
The purpose of this study therefore has been to: (1) analyze and compare the
histochemical staining properties of the normal and abnormal notochord, and
(2) to ascertain the stage at which notochordal development in the abnormal
embryo deviates from that in the normal embryo. Finally, particular attention
will be focused on those aspects of vertebral development in the Sd mutant
Histochemistry of notochordal development in mice
229
mouse which yield information on the role of the notocbord in normal vertebral
development.
MATERIALS AND METHODS
The mutant embryos used in this study were obtained from matings of
heterozygous (Sd/+ x Sd/+ ) adult mice. Litters from C57BL/6Sfd adult mice
were used as controls to establish a morphological baseline. The embryos were
timed by the vaginal plug method (vaginal plug = day zero) and staged according to Griineburg's criteria (1943).
Embryos ranging in age from 9 to 14 days' gestation were used in this study.
After removal from the uterus, the embryos were placed in physiological saline
and dissected free from the fetal membranes. The embryos were routinely fixed
in either Bouin's or Carnoy's solution. The fixed specimens were dehydrated
in graded ethanols, cleared in xylene or cedarwood oil and embedded in paraplast. During the final phases of embedding, the embryos were carefully oriented
so that transverse or frontal sections could be obtained. Serial sections of 9- and
10-day embryos were cut at 7-8 /im and at 10 fim for 11-14 days' gestation.
The serial sections were then stained with alcian blue (Pearse, 1968), MalloryHeidenhain stain for connective tissue (aniline blue), or periodic-acid Schiff
reagent (Pearse, 1968) with or without a 20 min predigestion with saliva or
0-5% diastase to remove glycogen (Culling, 1963).
A total of 34 litters was obtained and 250 embryos were dissected out; only
embryos of 12 days' gestation and older could be identified as Sd/ + or Sd/Sd
by external inspection alone. Microscopic examination was necessary to confirm the genotypes of younger embryos.
The distribution in 112 embryos from Sd/+ xSd/ + matings was 24 + / + ,
68 Sd/ + , and 20 Sd/Sd. The expected ratio would be 28 + / + , 56 Sd/ + , and
28 Sd/Sd. A similar deviation toward an excessive number of heterozygotes
was observed by Dunn et al. (1940).
RESULTS
In the present study, most of the PAS-positive granules described in notochordal cells and many of those in mesenchymal cells, chondroblasts and
chondrocytes (as well as the notochordal sheath) remain PAS-positive after
diastase digestion in normal and abnormal embryos at all stages examined.
No differences in notochordal or vertebral development were observed between
C57BL/6Sfd embryos and the + / + embryos from the Sd line. Therefore,
these two groups will be referred to collectively as 'normal embryos'. The
differences between Sd/ + and Sd/Sd embryos are a matter of degree; thus,
these two categories will be grouped together as 'abnormal embryos'. Most of
the normal as well as the abnormal changes described below are first observed
in the cervical or upper thoracic regions of the embryo, and later in more caudal
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L. G. PAAVOLA, D. B. WILSON AND E. M. CENTER
Allfiguresare of sections stained with PAS reagent. In black and white photographs
a positive PAS reaction is indicated by either discrete black granules within cells or
by diffusely darkened areas. All figures, except Fig. 2, are oriented with the neural
tube at the top of thefield.Unless otherwise indicated, the material was sectioned in
a transverse plane.
Fig. 1. Notochord and neural tube at 9 days of gestation, (a) Normal embryo.
Notochord is in contact with ventral neural tube. Discrete PAS-positive granules
(arrows) are apparent within notochordal cells. Randomly placed mesenchymal
cells occur in vicinity of notochord. (6) Abnormal embryo. Notochord (arrow) is
reduced in calibre and lacks PAS-positive granules. Sparse mesenchyme surrounds
notochord. (c) Abnormal embryo showing cluster (between arrowheads) of notochordal cells closely applied to neural tube. Notochordal cells are devoid of PASpositive granules, (d) Abnormal embryo. Notochord is absent from this section, and
mesenchymal cells fill the site it normally occupies, (nt = neural tube.) x 1125.
Histochemistry of notochordal development in mice
231
and cranial regions. The development of the tail notochord parallels that of
the trunk notochord, but lags behind it in time.
It should be noted that at 9 days' gestation abnormal embryos could be
distinguished from normal embryos on the basis of notochordal morphology
and histochemistry. The remaining structures appeared normal, at least under
the conditions of this study.
Notochord
Normal embryos. At 9 days' gestation the notochord is a continuous rod of
constant calibre extending from the hypophysis to almost the tip of the tail.
By 12 days' gestation the notochord in cranial regions has alternating narrower
and wider segments while in caudal areas it still displays a constant calibre
(Fig. 2a). This segmentation becomes more obvious in the 13-day embryo
(Fig. 2c) and is exaggerated by 14 days (Fig. 2e).
The cytoplasm of notochordal cells displays a faint, diffuse PAS-positive
reaction, which is more obvious in cranial regions of the embryos, and contains
discrete PAS-positive granules (Fig. 1 a). These PAS-positive granules gradually
accumulate in numbers over days 10-11 (Fig. 3a, c) and by day 12 they are
numerous (Fig. 4b, d). In 13-day embryos, the cells in inter vertebral segments of
the notochord are moderately vacuolated while those in vertebral regions remain
non-vacuolated. At this time most notochordal cells are filled with PAS-positive
non-granules (Fig. la). By 14 days, few notochordal cells remain in vertebral
anlagen (Fig. 8 a); however, vacuolation of notochordal cells is pronounced in
intervertebral areas, and they display abundant PAS-positive granules. The
PAS staining of these granules persists subsequent to treatment with diastase.
Abnormal embryos. The notochord of abnormal embryos is a discontinuous
structure even at 9 days of gestation (Fig. 1 d), at which time it may be absent
over distances up to 70/tm. In some instances, presumptive notochordal cells
do not yet form a rodlike structure but are closely applied to the neural tube
(Fig. Ic). As development continues, the notochord becomes increasingly
fragmented. The notochordal remnants vary in size and shape, are often diffuse
(Figs. Id, 5b) and are always smaller in calibre and more irregular in outline
than the normal notochord of a comparable age (Figs. lb,3d, 7 b). In abnormals,
notochordal fragments detach from the neural tube, but at any given stage less
distance separates these two structures than in normal embryos. Even at later
stages they remain abnormally close to the neural tube. On days 12-14, none of
the notochordal fragments in abnormal embryos show signs of impending
dilatation (Fig. 2b, d). In these older embryos, the remaining notochordal pieces
often occupy an eccentric site in or lie entirely outside the anlagen of the
vertebrae or disc (Figs. 6b, 1b). By 14 days, most sections of abnormal embryos
lack notochordal fragments, and the few that remain generally occur in the
sacral region.
In 9-day abnormal embryos notochordal cells display a faint PAS-positive
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L. G. PAAVOLA, D. B. WILSON AND E. M. CENTER
Fig. 2. Frontal sections of notochord. (a) Normal 12-day embryo, caudal region.
PAS-positive granules fill notochordal cells (arrowhead). A perichordal sheath is
apparent, (b) Abnormal 12-day embryo. Notochord (arrowhead) is discontinuous
and cytoplasm of notochordal cells appears clear and lacks PAS-positive granules.
An attenuated perichordal sheath is present, (c) Normal 13-day embryo. The notochord shows regularly spaced dilatations (arrowheads). Notochordal cells contain
PAS-positive granules and now display some vacuoles. (d) Abnormal 13-day embryo.
The notochord is fragmented, does not show regularly spaced dilatations, and its
calibre is reduced and variable. Note lack of granules and vacuoles in fragments
(between arrowheads). A thin perichordal sheath is present, (e) Normal 14-day
embryo. The notochord in intervertebral disc anlagen (arrowheads) has increased
in diameter. PAS-positive granules and vacuoles are characteristically present in
notochordal cells, (a, b), x 500; (c-e), x275.
Histochemistry of no to chordal development in mice
233
Fig. 3. (a) Neural tube and notochord in 10-day normal embryo. A delicate PA.Spositive sheath (arrow) is apparent, and the notochord has become separated from
the ventral neural tube. Notochordal cells contain scattered PAS-positive granules.
More mesenchymal cells have accumulated in vicinity of notochord but show little
orientation to it. (b) Neural tube and notochord in 10-day abnormal embryo. A PASpositive perichordal sheath is present (arrow) but the notochord is smaller and lies
abnormally close to the neural tube as compared with the normal. Few PAS-positive
granules occur in notochordal cells, (c) Notochord and mesenchyme of 11-day
normal embryo. The notochord shows an increased number of PAS-positive granules,
is somewhat larger in calibre, and is further separated from the neural tube than at
previous stage. The sheath (arrow) is of increased thickness. The mesenchymal cells
(arrowheads) nearest the notochord show alignment with respect to the notochord
and now contain PAS-positive granules (black dots), (d) Notochord and mesenchyme of abnormal 11-day embryo. The notochordal fragment is small, irregular
in shape (arrow) and lacks PAS-positive granules. Few mesenchymal cells (arrowheads) are aligned with respect to the notochord and few display PAS-positive
granules. x450.
reaction. However, most notochordal cells exhibit only a few PAS-positive
granules and some lack granules altogether (Fig. 1 b). By 10 days, many notochordal cells are devoid of PAS-reactive granules, and such granules are absent
from virtually all notochordal cells of 11-14-day abnormal embryos (Figs. 2b, d,
3d, 6b, 7b). In addition, vacuolation does not occur in the notochordal cells
remaining in inter vertebral regions of day-13 (Figs. 2d,lb) or day-14 abnormal
embryos.
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L. G. PAAVOLA, D. B. WILSON AND E. M. CENTER
Fig. 4. Twelve-day normal embryos, (a) Vertebral anlage showing centrum (between
arrowheads) and vertebral arches (arrows). Notochord appears black due to
numerous PAS-positive granules, (b) Higher magnification showing deposition
of hyaline ground substance in developing centrum, chondroblasts more distant
from one another, and further organization of mesenchymal cells. PAS-positive
granules are seen in some chondroblasts, especially dorsal and ventral to the
notochord. Note thick perichordal sheath, (c) Intervertebral disc anlage (between
arrowheads) with notochord and several rows of mesenchymal cells containing
PAS-positive granules, (d) Higher magnification showing further organization of
mesenchymal cells and increased content of PAS-positive granules (compare with
Fig. 3 c). Note cells not involved in disc formation (asterisks) are PAS-negative.
Perichordal sheath is thick and notochordal cells are filled with PAS-positive
granules, (a, c), x 150; (b, d), x 425.
Histochemistry of notochordal development in mice
235
Notochordal sheath
Normal embryos. A clear-cut notochordal sheath is obvious in 10-day
embryos (Fig. 3a), and is strongly PAS-positive by day 12 (Fig. 4 c, d). In 13and 14-day embryos, the sheath enclosing vertebral notochord is thick, while
that around the notochord in inter vertebral regions is attenuated. A sheath
remains in all vertebral centra of 14-day embryos, attached to the surrounding
cartilage, whether or not notochordal cells are present (Fig. 8a). In addition to
being PAS-reactive, the sheath stains with alcian blue and aniline blue at all
stages examined.
Abnormal embryos. In 10-day abnormal embryos, the notochordal fragments
are encircled by a thin, PAS-positive sheath (Fig. 3 b), which appears more or
less normal. In sections where the notochord is absent, no sheath is seen, The
notochordal sheath of abnormals does not undergo an increase in thickness as
development continues (Figs. 3d, 5b). It is consistently much thinner than that
observed in normal embryos of comparable ages. By 11 days, some notochordal
fragments lack a sheath, and in 12- to 14-day abnormal embryos the remaining
notochordal fragments commonly lack a sheath (Fig. 7b). When present, the
sheath stains positively with the PAS-reagent, aniline blue and alcian blue.
Mesenchymal cells
Normal embryos. In 9-day embryos the notochord is surrounded by sparse,
unorganized mesenchyme, whose constituent cells are PAS-negative. At 10 days
the mesenchyme around the notochord varies from loose to moderately dense,
and lacks any specific pattern of organization (Fig. 3d). By 11 days a definite
alignment of mesenchymal cells with respect to the notochord is obvious, with
two or more rows of cells forming concentric rings around it (Fig. 3 c), and
PAS-positive granules occur in the cytoplasm of the mesenchymal cells closest to
the notochord (Fig. 3 c).
Further organization of mesenchymal cells is observed in embryos at 12 days'
gestation, at which time presumptive vertebral and intervertebral disc areas
are readily recognized (Fig. 4). In regions where vertebrae will develop, the
cells comprising the future centrum are separated from each other by considerable ground substance and contain PAS-positive granules (Fig. 4a, b), whereas
the cells of the developing arches are densely packed, with little ground substance between them, and lack PAS-positive granules (Fig. 4a). By 13 days the
vertebral models are enlarged and chondrified; their ground substance is
moderately PAS-reactive, and more chondrocytes in the centrum contain PASpositive granules (Fig. 6a). By 14 days, the ground substance is more intensely
PAS-reactive, and nearly all chondrocytes are hypertrophied and contain
abundant PAS-positive granules (Fig. 8a).
Intervertebral disc anlagen of 12-day embryos consist of the notochord surrounded by closely spaced rows of mesenchymal cells. PAS-positive granules
236
L. G. PAAYOLA, D. B. WILSON AND E. M. CENTER
i. •> r-
Histochemistry of notochordal development in mice
237
Fig. 6. Vertebral anlagen at 13 days' gestation, (a) Normal anlage shows chondrocytes (arrows) with PAS-positive granules. Ground substance is moderately PASpositive. Notochordal cells persist in anlage and contain PAS-positive granules.
(b) Abnormal anlage showing T-shaped notochord with bar of T lying outside and
dorsal to vertebral anlage and tail of T (arrow) trailing into vertebra. Notochordal
cells lack PAS-positive granules. Vertebral anlage is chondrified but reduced in size
(compare with a). x275.
FIGURE 5
Fig. 5. Twelve-day abnormal embryos, (a) The vertebral centrum (between arrowheads) shows randomly dispersed mesenchymal cells, (b) Higher magnification of
centrum with indistinct notochordal remnant (between arrows). Perichordal sheath
is thinner than normal and notochordal cells lack PAS-positive granules. Mesenchymal cells fail to be aligned relative to notochord (compare with Fig. 4a, b).
Ground substance deposition appears to be reduced, and chondroblasts tend to lack
PAS-positive granules, (c) Aberrant centrum composed of two side-by-side whorls
(arrowheads), (d) Higher magnification of side-by-side whorls. Note lack of notochord, sparse ground substance with dense packing of presumptive chondroblasts,
and fewer cells with PAS-positive granules than in normals, (e) Anlage of intervertebral disc (between arrowheads) is poorly organized. (/) Higher magnification
of disc anlage shows lack of a notochord and less precise alignment of mesenchymal
cells than in normals (compare with Fig. 4c, d). (a, c, e), x 150; (b, d,f), x 425.
16
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L. G. PAAVOLA, D. B. WILSON AND E. M. CENTER
Fig. 7. Intervertebral disc at 13 days' gestation, (a) Normal embryo. The notochord
(arrow) is greatly enlarged due to hypertrophy and vacuolation of its cells, which are
still filled with PAS-positive granules. Mesenchymal cells form many rows around
notochord and are diffusely PAS-positive. (b) Abnormal embryo. The notochord.
(arrow) lies outside the disc anlage, lacks PAS-positive granules and a sheath, and is
not vacuolated. Mesenchymal cells lack organization and are randomly placed.
x275.
are abundant in the cells of the concentric rows, but absent from the more
peripheral cells (Fig. 4b, c). By 13 days the disc anlagen has enlarged and in
contrast to the previous stage, most of its cells are devoid of PAS-stainable
granules (Fig. la). Their cytoplasm, however, remains moderately PAS-positive.
The cellular organization and staining of the disc anlagen at 14 days is similar
to that at 13 days' gestation.
Abnormal embryos. The mesenchyme surrounding the notochords of 9- and
10-day abnormal embryos (Figs. \b, 3b) resembles that described for normal
specimens of these ages. When the notochord is absent, mesenchymal cells fill
Histochemistry of notochordal development in mice
239
Fig. 8. Vertebral anlagen at 14 days' gestation, (a) Normal embryo. Nearly all of the
chondrocytes have hypertrophied and are filled with PAS-positive granules
(arrows). Notochordal cells are lacking but perichordal sheath remains, (b) Abnormal
embryo. Note indented centrum, reduced dorsoventral dimensions in midline, and
chondrification occurring in side-by-side whorls. Notochord and perichordal sheath
are lacking. Hypertrophied chondrocytes tend to lie in dorsal portions (arrow) of
anlage. x250.
the site that this structure would normally occupy (Fig. 1 d). On day 11, these
cells fail to be aligned in rows with respect to the notochord; instead, they
remain randomly arranged (Fig. 3d). In marked contrast to the normal situation,
far fewer mesenchymal cells near the notochord contain PAS-positive granules.
As development proceeds the lack of organization in these mesenchymal
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L. G. PAAVOLA, D. B. WILSON AND E. M. CENTER
derivatives persists. In the developing centra of 12-day embryos: (1) the cells
nearest the notochord fail to tightly encircle it (Fig. 5 b), (2) the more peripheral
cells of the precartilage model lack an ordered arrangement (Fig. 5 a, b), (3) far
less ground substance separates the chondroblasts, and fewer chondrocytes
contain PAS-stained granules than in normal specimens (Fig. 5 b), and (4)
fewer cells appear to comprise the anlagen. Often, when the notochord is
absent the mesenchymal cells of the centrum assume a characteristic configuration, two side-by-side whorls (Fig. 5 c, d).
By 13 days, the vertebral model is chondrified, but is always much reduced
in the dorsoventral dimension (Fig. 6b), and its ground substance appears to
be less PAS-positive than that in normal specimens. Of the chondrocytes in the
centrum fewer have begun to hypertrophy and fewer contain PAS-positive
granules than in normal embryos. In 14-day embryos, the centra are generally
partially or completely bifurcated (Fig. 8fr). The ground substance of these
models is PAS-positive but the reaction is variable even within a given specimen,
and few chondrocytes have hypertrophied.
Intervertebral disc anlagen also show disturbances of mesenchymal cell
organization. The cells of the disc may form concentric rows but the rows are
not precisely arranged (Fig. 5e,f), or they may be randomly placed, lacking any
indication of order (Fig. 7b), or they may be arranged in side-by-side whorls.
All arrangements are seen in any given specimen and no one type predominates
over the others. In any of these situations, a notochordal fragment may or may
not be present. As in vertebral regions fewer cells appear to form the disc
primordia and fewer contain PAS-positive granules than in normal embryos.
DISCUSSION
The results presented here demonstrate that the histochemical properties of
notochordal development in Sd mutant mice are clearly anomalous as early as
the ninth day of gestation. At this stage, most notochordal cells of abnormal
embryos are deficient in PAS-positive granules, whereas such granules are
typically present in the notochords of normal specimens. Moreover, as embryogenesis continues, the notochordal sheath of Sd mutants fails to develop
normally.
One of the striking findings of the present study, revealed by the use of the
periodic-acid Schiff reagent, was the presence of discrete PAS-positive, diastaseresistant granules in the cytoplasm of notochordal cells. They are apparent in
notochordal cells of normal mouse embryos at 9 days and subsequently increase
in number until they become abundant. In earlier studies that employed the
PAS reagent, similar PAS-stained granules were not described in notochordal
cells (Leeson & Leeson, 1958; Leeson & Threadgold, 1960; Kvist & Finnegan,
1970).
The persistence of PAS-stainable material after diastase digestion is generally
Histochemistry of notochordal development in mice
241
regarded as implying the presence of glycoproteins (LeBlond et ah 1957) or
neutral polysaccharides (Kvist & Finnegan, 1970). Glycosaminoglycans such
as chondroitin sulfates theoretically can react with the PAS reagent (Pearse,
1968) but such a reaction is unlikely when a routine PAS procedure is used
(LeBlond et ah 1957; Zugibe, 1963), since more rigorous oxidation conditions
are required to demonstrate these compounds (Scott & Dorling, 1969). The
PAS-positive, diastase-resistant granules described in the current report accumulated in substantial numbers only in notochordal cells and those cells destined
to become chondrocytes, making it tempting to speculate that they are involved
in chondrogenesis.
Recently, considerable biochemical (Minor, 1973; Hay & Meier, 1974;
Kosher & Lash, 1975) and morphological (Lauscher & Carlson, 1975; Kenney &
Carlson, 1978) evidence has accumulated, strongly indicating that notochordal
cells produce extracellular matrix (ECM) materials, glycosaminoglycans
(chondroitin-4, and • 6, sulfate, and heparan sulfate) and collagen. Moreover,
these substances are produced at the time of somite chondrogenesis, and have
been implicated as mediators of the interactions between the notochord and
somitic mesoderm that bring about cartilage formation (Kosher & Lash, 1975;
Hay & Meier, 1974). ECM components initially accumulate in the perichordal
sheath, then appear to diffuse away from the notochord to become distributed
among nearby mesenchymal cells (Ruggeri, 1972). If these proteoglycans are
removed from the notochord by various digestion procedures, the ability of the
notochord to support chondrogenesis is considerably impaired (Kosher &
Lash, 1975).
Thus, the PAS-positive, diastase-resistant material seen as 'granules' in
notochordal cells of the present study may reflect one or more of the substances
produced by the notochord such as (1) collagen, a glycoprotein containing
variable amounts of carbohydrate (Spiro, 1970; Dische, 1970), (2) the population of non-collagenous glycoproteins that typically occur in association with
glycosaminoglycans (Roden, 1970), or (3) precursors of glycosaminoglycans.
Although it is not possible at this time to rule out the possibility that unrelated
compounds are responsible for the observed staining, such an alternative seems
less likely since the notochord appears to produce a limited number of substances (Kosher & Lash, 1975).
The decreased number or lack of notochordal granules in &/mutant embryos
appears to suggest that the secretory capacity of notochordal cells is in some way
impaired. Our findings, however, do not rule out the unlikely possibility that
the synthesis and turnover of these granules in abnormal embryos is accelerated,
which would also lead to decreased numbers of granules in notochordal cells.
The diminished ability of the young notochord in the present study to accumulate
these granules also implies that notochordal cells of abnormal embryos are
inherently defective, a proposal that gains support from our observation that
these cells fail to vacuolate at the appropriate point in development. In chick
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L. G. PAAVOLA, D. B. WILSON AND E. M. CENTER
and mouse embryos, the inability to vacuolate is characteristic of notochordat
cells that fail to effectively promote chondrogenesis (Cooper, 1965). The decrease in PAS-positive granules in mesenchymal cells of disc regions and centra
of abnormal embryos also suggests that the ability of the notochord to mediate
chondrogenesis in Sd mutants may be faulty.
Our results tend to corroborate Griineburg's (1958) proposal of early involvement of the notochord in the Sd syndrome, as opposed to GluecksohnSchoenheimer's (1945) contention that notochordal involvement is secondary
to defects in the tail.
Of particular interest is the failure of somitic mesenchyme in the 11-day
Sd mutant embryos to attain the typical organization observed in vertebral and
intervertebral disc anlagen of normal specimens. Defects in the notochord or
sheath, or both, may be responsible for the lack of cellular organization in
mutants. Flower & Grobstein (1967) have indicated that although surface
contact is not critical in the initiation of chondrogenesis, pre-existing surfaces
may well have a role in later events in vertebral morphogenesis. Moreover, the
properties of substrata or interfaces are reported to influence the morphogenetic
movements of mesenchymal cells in embryonic and other systems (Weiss, 1961;
Cohen & Hay, 1971; Toole et al. 1977). In view of these findings, it seems possible
that in normal development the notochord-sheath complex contributes to
vertebral formation by providing the appropriate surface about which mesenchymal cells initially align, especially since we have shown that when the
notochord-sheath complex is demonstrably defective (in histochemical, and
possibly chemical terms), the mesenchymal cells rarely show a definite orientation to the notochord (Figs. 3d, 4b). Although anomalies of mesenchymal cell
organization in Sd mutants can be correlated with a dysfunctional notochord/
sheath, the possibility that mesenchymal cells themselves have limited capabilities cannot be excluded. This question hopefully will be answered by means of
experimental manipulation involving reciprocal interchanges of notochord and
mesenchymal cells between Sd and normal embryos.
Disturbances of axial organization as a consequence of notochordal anomalies
occur in other mouse mutants, including brachyury, truncate, pintail and anury
(see review by Griineburg, 1963).
The axial defect common to the above mutants is reduction in number of tail
vertebrae leading to shortening or absence of the tail; however, the involvement
in these mice may be extensive, as in Sd, in which the entire vertebral column
is involved, or localized, as in pintail, in which only axial structures of the tail
are affected. Although the vertebral defects are often morphologically similar,
the initial demonstrable abnormality of the notochord may be quite different.
For example, the notochord of pintail is reduced in calibre, while that of
truncate ends abruptly in the posterior region of the embryo, and that of anury
is retained or incorporated into the hindgut or neural tube. On a cellular level,
recent work suggests that brachyury (T) probably results from defects in cell
Histochemistry of notochordal development in mice
243
surface components, which are expressed early in development and which may
compromise the structural integrity of the notochord (Bennett, 1975). Differences in cell surfaces between normals and abnormals have also been implied
by dissociation-reaggregation experiments using brachyury embryos (Yanagisawa & Fujimoto, 1978).
In conclusion, our histochemical results suggest that the development of
normal vertebrae seems to be dependent, to a considerable extent, on the presence of a functional notochord. However, the possibility raised by Gruneburg
(1958) as to whether the abnormal Sd notochord, in turn, reflects improper
development and regression of the primitive streak, remains to be explored.
The authors would like to thank Mr Charles O. Boyd for preparing thefigures.This work
was supported in part by Research Grant HD-09993 from the NICHD.
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{Received 30 March 1979, revised 20 August 1979)