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Surface ultrastructure of the isolated avian notochord in vitroThe effect of the perinotochordal sheath.

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THE ANATOMICAL RECORD 197:257-276 11980)
Surface Ultrastructure of the Isolated Avian
Notochord In Vitro: The Effect of
the Perinotochordal Sheath
Department of Human Anatomy, University of California,
Davis, California 9561 6
The perinotochordal sheath (PNS) is a "tube" of extracellular
matrix (ECM) that surrounds the avian notochord beginning in the second day
of development. Somites, like the notochord, derive from chordamesoblast but
are encased by a less substantial perisomitic matrix (PSM). Initially both tissue
types exhibit epithelioid characteristics. Somitic cells subsequently disperse,
however, while notochordal histoarchitecture is maintained until much later. To
test the possible shape-preserving role of the PNS, notochords were isolated
from chick embryos by homogenization (which retains the sheath) or by trypsinization (which removes the sheath). Somites were similarly isolated. Tissues
were cultured 12-72 hours and studied by LM, SEM and TEM. Mechanically
isolated notochords are initially rigid with smooth surfaces. During the culture
period a few cells grow outward from cut ends of the notochord, but its overall
rod shape and intact PNS are maintained. In contrast, uncultured trypsinized
notochords a r e flaccid, denuded cylinders with numerous cytoplasmic blebs.
They adhere to the substratum within 12 hours of culture when a few cells break
away from the central tissue rod, migrate laterally, and appear mesenchymal.
This cellular dispersion is directional (perpendicular to the long notochordal
axis) and continuous (up to 72 hours). At this time a flattened ovoid growth area
is formed. Cultured somites form flat circular growth areas within 12 hours of
culture irrespective of the isolation method. These data suggest that the maintenance of a n epithelial configuration by notochords in vivo may be due in part to
physical restraints of the PNS. It seems possible that notochordal secretions
(manifested by the formation of a PNS) could result in its compartmentation
and axial confinement while its unrestrained somitic relatives are free to disperse.
The early chick embryo is particularly well- Histochemical and enzyme digestion studies
suited for studies of notochordal morphogene- of t h e sheath demonstrate t h e presence of
sis (Jurand, '62; Bancroft and Bellairs, '76). acid mucopolysaccharides (O'Connel a n d
This structure is of special interest because it Low, '70; Frederickson and Low, '71; Ruggeri,
induces neural plate formation from overly- '70, '72) and collagen (Frederickson and Low,
ing ectoderm (Holtfreter, '68) and chondro- '71; Kenney and Carlson, '78). This latter obgenesis in somitic mesoderm (Lash, '63; 68a, servation is consistent with biochemical inb, c). Therefore it has been the subject of nu- vestigations which show that a cartilage-like
merous morphological and biochemical inves- collagen (type 11) is associated with the nototigations.
chord in vivo (Miller and Mathews, '74).
At the level of transmission electron microThe P N S was first described by Duncan
scopy (TEM), extracellular matrix (ECM) - ('57), but its origin was at first controversial
including basal lamina, microfibrils, a n d because of its association with a n epithelioid
other amorphous materials - forms a perino- structure prior to the differentiation of sectochordal sheath (PNS) surrounding the noto- ondary mesenchyme from t h e sclerotome.
chord beginning in the second day of development (Low, '68; Bancroft and Bellairs, '76).
Received July 11, 1979; Accepted November 21, 1979.
0003-276X/80/1972-0257$03.50 0 1980 ALAN R. LISS, INC.
More recently Carlson and co-workers ('74)
showed that cultured mesenchyme-free, isolated notochords produced connective tissue
materials that were ultrastructurally indistinguishable from those which comprised the
PNS in vivo. This "reappearance" of ECM in
vitro h a s been confirmed by other studies
(Carlson a n d Upson, '74; L a u s c h e r a n d
Carlson, '75; Kenney and Carlson, '78) and it
is now generally believed that the notochord
is the progenitor of its own sheath.
Other in vitro studies (Hay and Meier, '74;
Kosher and Lash, '75) have shown t h a t t h e
notochord s y n t h e s i z e s s u l f a t e d glycosaminoglycans (GAGS) and collagen(s), t h e
majority of which consists of three identical
a1 chains (Linsenmayer et al, '73). These results are consistent with our recent studies
which demonstrate the production of ruthenium red-positive, hyaluronidase-sensitive
ECM, and striated collagen fibrils by notochordal organ cultures within three days of
incubation (Kenney and Carlson, '78).
It is interesting t h a t during the development of the PNS, the notochord becomes more
rod-like and remains confined to its axial
position until its eventual demise. This is particularly noteworthy in light of the concomit a n t disorganization and dispersion of adjacent somitic cells. Like the notochord, somites
are derived from chordamesoblast and appear
as epithelial structures early in development.
They are surrounded by components of ECM
similar to those which comprise the PNS. In
contrast, however, somites do not remain
compartmentalized i n segmental tissue
blocks. Rather, they organize further to form
sclerotome, myotome and dermatome subdivisions, each of which eventually disperses to
give rise to various mesodermal derivatives.
Somitic mesoderm is a t least a s active a s
the notochord in the production of ECM both
in vivo (Kvist and Finnegan, '70; O'Connel
and Low, '70; Olson and Low, '71; O'Hare,
'72a, b; Minor, '73; Lipton and Jacobson, '74;
von der Mark et al, '76) and in vitro (Lash,
'68b; OHare, '72a, b; Orkin e t al, '73; Gordon
and Lash, '74; Lash and Vasan, '78). Furthermore, the ECM is composed of GAGS (Minor,
'73; O'Hare, '73; Gordon and Lash. '74; Lash
and Vasan,'78) as well as collagen (von der
Mark et al, '76), which are similar to those
produced by the developing notochord. Unlike
the PNS, however, perisomitic matrix (PSM)
does not continue to a c c u m u l a t e a t t h e
somitic external surface, nor does it restrict
subsequent dispersion of somitic cells. Indeed
most current embryology textbooks consider
somitic break-up and dispersion a necessary
prerequisite to mesodermal differentiation.
The present study examines the capability
of the intact PNS to prevent or permit notochordal cell migration in a n in vitro system
and compares the results with similar data
derived from cultured somites. Light (LM),
scanning electron (SEMI, and transmission
electron (TEM) microscopic data are correlated to demonstrate t h e surface morphology
and histoarchitecture of t h e PNS. The primary aim is to provide evidence for the maintenance of t h e notochordal epithelioid rod
shape in terms of physical restriction and
compartmentation by the PNS during early
Tissue preparation and organ culture
Fertile White Leghorn hens' eggs were incubated 24-60 hours a t 37°C (60% relative
humidity). Sterile filter paper rings aided removal of embryos from the egg as previously
described (Lauscher and Carlson, '75). Embryos were than transferred to sterile Hank's
Balanced Salt Solution (HBSS) and staged according to Hamburger and Hamilton ('51).
Cross-sectional scissor cuts were made
caudal to the heart and rostra1 to terminal
somites on embryos of the same or similar
stages. Intermediate t r u n k segments were
placed in a) cold (4°C) Karnovsky's ('65) fixative, b) cold Earle's Balanced Salt Solution
(EBSS) buffered with 0.028 M HEPES, or
c) cold tryspin (1% in HBSS).
Notochords and somites were mechanically
isolated from trunk segments in "b" (above)
by mild homogenization in a hand-held glass
tissue homogenizer with specially machined,
conical (small end directed upward) teflon
pestle. The homogenate was passed over a
153-pm mesh nylon sieve and washed generously with buffered EBSS. The residue was
rinsed into a 10-cm petri dish, and somites
and fragments of isolated notochords were removed and collected with a Pasteur pipette.
Trunk segments in "c" (above) were trypsinized for 25 minutes and then flushed gently
through Pasteur pipettes to isolate somites
and notochords.These were collected separately and washed three times in 15% fetal
calf serum to inactivate the trypsin. All isolated tissues were incubated in Falcon plastic
35-mm tissue culture dishes in M-199 culture
medium supplemented with 2% fetal bovine
serum, 8% bovine serum, ascorbic acid (100
pg/ml), penicillin-streptomycin (100 units/
ml), fungizone (2.5pgiml), 10% tryptose phosphate broth and NaHCO, (2.2 mg/ml).
Light and transmission electron microscopy
All t r u n k segments, notochords, and somites were fixed one hour in cold Karnovsky's
('65) fixative (0.2 M sodium cacodylate buffer,
pH 7.4). Cultured tissues were rinsed three
times in buffered EBSS prior to treatment
with the same fixative. Phase-contrast micrography was carried out on cultured tissues
during the aldehyde fixation using an Olympus IMT inverted microscope.
All tissues were post-fixed one hour at room
temperature with 2% OsO, (0.144 M cacodylate buffer, pH 7.4) prior to dehydration in a n
ascending series of graded ethanols. Uncultured isolated notochords or somites were
surrounded by several drops of warm 2%
Nobel agar in order to facilitate their manipulation. These agar-embedded tissues were
exposed t o propylene oxide for additional dehydration. Tissues were embedded in a n EponAraldite mixture (Anderson and Ellis, '65)
and cured overnight a t 37°C and for an additional 48 hours a t 60°C.
Sections 1 p thick were stained with toluidine blue (1% in 1% sodium borate) and observed and photographed with an Olympus
F H photomicroscope. Thin sections (500-800
A) were placed on uncoated 200- or 300-mesh
copper grids prior to staining with uranyl acetate (5% in absolute ethanol) and lead citrate
(Venable and Coggeshall, '65). These were observed and recorded a t original magnifications of 4,600 to 35,000 diameters in a Philips
EM 400 transmission electron microscope.
Scanning electron microscopy
Uncultured notochords were fixed one hour
a t room temperature i n aldehyde fixative
(Karnovsky, '651, post-fixed in OsO, (2% in
0.144 M sodium cacodylate buffer), and dehydrated in a series of graded ethanols. Notochords were critical point-dried in CO, and
mounted on aluminum specimen stubs. They
were coated with evaporated carbon and gold,
and observed with a Philips 501 scanning
electron microscope.
In vivo trunk segments
Cross sections through t r u n k segments
(seventh somite level) from chick embryos a t
stage 1 0 show prominent centrally located
neural tubes (fig. 1).The recently closed dorsal surface is closely associated with surface
ectodermal cells a n d a transitory surface
groove is present in t h e dorsal embryonic
midline. The neural tube is flanked laterally
by block-shaped somites which are separated
from surrounding tissues by a cell-free connective tissue space. Somites are composed of
irregular columnar cells surrounding a n illdefined lumen in a rosette configuration.
They have not yet organized into sclerotome,
dermatome and myotome subdivisions. The
notochord is unvacuolated and appears as a
rod of cells, flattened dorsoventrally and located in the midline approximately equidistant between the overlying neural tube and
ventral developing entoderm. A primitive but
continuous endothelium forms the walls of
paired aortae in these specimens. At t h i s
stage and segmental level the embryo is composed entirely of epithelial structures separated from a single uncompartmented tissue
space. Electron micrographs show t h a t the
perinotochordal area is occupied by electrondense extracellular connective tissue materials including a discontinuous basal lamina
(fig. 2). These matrix components, including
microfibrils, i n t e r s t i t i a l bodies and basal
lamina, constitute the PNS.
By stage 15, the dorsal ectoderm is completely separated from the underlying neural
tube, while neural crest cells stream ventrad
along its lateral margins (fig. 3). Somitic cells
reorganize to form compact dorsolateral dematomyotomes and more dispersed ventromedial sclerotomes. Identical magnification
micrographs show that at this stage sclerotoma1 cells have not begun to disperse. The entodermal lining moves ventrad away from the
developing notochord, resulting in a n appare n t enlargement in t h e connective tissue
space. Cross-sectioned notochords show small
vacuoles, and demonstrate a more circular
profile t h a n a t earlier stages. At this stage
notochordal basal lamina is relatively continuous, and numerous small (5-10 nm) microfibrils are present within the PNS (fig. 4).
The total area occupied by the sclerotome is
enlarged dramatically a t stage 17 (primarily
by increased intercellular spaces), and sclerotomal cells are closer to the notochord than at
earlier stages (fig. 5 ) . Such proximity is probably a function of increased notochordal diameter except in the area ventral to the notochord, where mesenchymal cells (possibly resulting from sclerotomal proliferation and
migration) a r e associated with aortic endothelium. The notochord increases in diameter and vacuoles are enlarged, but its crisp
circular profile and close proximity to t h e
overlying n e u r a l t u b e a r e relatively u n changed. The PNS, including the subjacent
b a s a l l a m i n a , i s s i m i l a r t o t h a t seen i n
stage-15 embryos (fig.4) but appears denser
than those seen at stage 10 + (fig. 2).
A remarkable increase in notochordal girth
between stages 17 and 20 (compare figs. 5 and
7) probably results in its lateral association
with sclerotomal cells. However, similar cells
are also present in the embryonic midline immediately dorsal and ventral to the developing notochord (fig. 7). These late-arriving
midline cells from the sclerotome occupy a
space which has been previously cell-free (fig.
1 , 3 , 5).
At stage 20 more t h a n half of t h e total
cross-sectional a r e a of t h e notochord is
occupied by spherical vacuoles (fig. 7). Its rodlike configuration is maintained and its remarkably circular shape in cross section sug-
gests confinement to a limited space by an external retainer. The PNS is more electrondense at this stage than a t shorter incubation
times (fig. 8). Its basal lamina is composed of
a lamina densa (3G40 nm thick) separated
from subjacent notochordal cells by a clear
20-30 nm area. Microfibrils parallel the notochordal surface in the adjacent connective tissue space.
Somite isolation and culture
In an effort to demonstrate the possible effects of perisomitic matrix (PSM) on growth
patterns of isolated somites in vitro, these tissues were isolated from stage-12-14 chick
embryos either by mechanical methods (to retain the PSM) or by trypsinization (to remove
the PSM) and cultured as described in Materials and Methods.
Following isolation by mild homogeniza-
Fig. 1.Light micrograph of cross section through seventh somite of chick embryo a t stage l o + . At this stage
embryos are composed almost entirely of epithelial tissues including ectoderm (ect), entoderm (ent), neural
tube (NT), notochord (N), somites (S), and primitive endothelium of the developing aortae (a).Toluidine
blue. x 220.
Fig. 2. Electron micrograph of notochord surface i n area shown by arrows in figure 1. Intermittent basal
lamina (bl) material surrounds the notochord (N) and small ( 5 C 1 0 0 A) microfibrils embedded in electron
lucent ground substance forms the early perinotochordal sheath (PNS). x 21,000.
Fig. 3. Light micrograph of cross section through seventh somite of chick embryo a t stage 15. The notochord
( N ) appears more cylindrical than at earlier stages and the connective tissue space interposed between notochord, somites, and underlying entoderm is increased in volume (compare with figure 1).At this time the
ventromedial portion of the somite or sclerotome (SC) has hegun to disperse. Somite remnant h a s moved
dorsolaterally. Aortae, a; toluidine blue. x 220.
Fig. 4. Electron micrograph of notochord (N) surface in area shown by arrows in figure 3. Perinotochordal
sheath (PNS) consists of continuous basal lamina (bl) and numerous microfibrils (mfb). These completely
surround the notochord. x 21,000,
Fig. 5. Light micrograph of cross section through chick embryo at stage 17 dorsal and j u s t rostral to the
heart. Notochord (N) is increased in diameter compared to earlier stages. Intercellular spaces in the sclerotome (SC) have enlarged, and some sclerotomal cells approach the midline between notochord and underlying
aortae ( a ) . Large vacuoles i n notochordal cells a r e normal f e a t u r e s a t t h i s incubation age. Toluidine
blue. x 220.
Fig. 6. Electron micrograph of notochord (N) surface i n area shown by arrows in figure 5. By stage 17 the
perinotochordal sheath (PNS) consists of numerous microfibrils arranged parallel to and associated with the
perinotochordal basal lamina (bl). x 21,000.
Fig. 7 . Light micrograph of cross section through embryo a t stage 20 dorsal and just rostral to the heart.
Notochord (N) is highly vacuolated and isenlarged in diameter by nearly double compared with stage 17 (see
figure 5). The notochord maintains its axial position and cylindrical configuration. The once-epithelial somite
is now highly disorganized, and sclerotomal cells (SC) merge i n the midline (dorsal and ventral to the notochord) with cells from the corresponding contralateral segment. Aorta, a. Toluidine blue. x 220.
Fig.8. Electron micrograph of notochord (N)surface in area shown by arrows in figure 7 . Perinotochordal
basal lamina (bl) is continuous and exhibits clearly demarcated lamina densa and lamina lucida. Perinotochordal sheath (PNS) contains dense concentrations of 15&200 A microfibrils (arrows) and amorphous materials. x 21,000.
tion and sieving, somites from stage-12-14
embryos appear as condensed tissue blocks
with smoothly contoured external surfaces
and average 230 x 330 pm (fig. 9). By TEM,
basal surfaces of somitic cells are covered by a
continuous basal lamina surrounded by an
electron-lucent matrix t h a t contains faintly
cross-banded, 15-nm microfibrils (fig. 10).
Somitic cells are undifferentiated and show
little smooth- or rough-surfaced endoplasmic
reticulum. Their peripheral cytoplasm is composed primarily of polyribosomes. When these
mechanically isolated somites are submerged
in embryo extract-free culture medium and
grown 12 hours on Falcon plastic, they do not
remain compartmentalized by PSM. Rather,
somitic cells become stellate, disaggregate
rapidly, and migrate radially on the substrat u m forming a circle-shaped growth a r e a
(fig. 11).
When somites are isolated by a brief treatm e n t w i t h t r y p s i n followed by f l u s h i n g
through Pasteur pipettes, they appear approximately equal in overall size and shape
but more translucent than their mechanically
isolated counterparts. Moreover, trypsinized
somites show convex surfaces virtually covered with small blebs. TEM demonstrates
t h a t cells with these cytoplasmic evaginations show no evidence of cytotoxicity and
t h a t t h e enzyme treatment completely re-
moves adjacent PSM (fig. 13). After 12 hours
of culture these denuded somites exhibit
growth patterns t h a t a r e indistinguishable
from those of somites isolated by mechanical
m e a n s (fig. 1 4 ) . I n both c u l t u r e t y p e s ,
fibroblast-like somitic cells migrate peripherally from the central cell cluster and exhibit
approximately equal mobility in all directions (compare figs. 11 and 14).
Isolation and culture of mechanically
isolated notochords
In this group of experiments we endeavored
to determine whether notochords isolated and
cultured with the PNS intact might retain
this sheath and if the presence of the PNS
could play a role in maintaining notochordal
shape under in vitro conditions.
Notochords isolated by a hand-held tissue
homogenizer and passed over nylon sieves average 20&1,500 p m in length. Typically they
express resiliency and resist mechanical distortion. I t is not possible to prepare completely clean cylindrical notochord segments
by this isolation procedure, and those grossly
contaminated with mesenchymal cells were
Following preparation for SEM, isolated
notochords appear as rigid rods with slight
regional constrictions (fig. 15). In these preparations small, irregular surface projections
Fig. 9. Bright field micrograph of unfixed somite (S) isolated from stage-12 chick embryo by mild homogenization and sieving. Somites maintain the rosette configuration and exhibit smooth external surfaces (arrows).
x 160.
Fig. 10. Electron micrograph of surface of mechanically isolated somite (S)similar to that shown in figure 9.
Basal lamina material (bl) surrounds t h e somite . A few microfibrils which exhibit faint irregular striations
(arrows) are found in the associated perisomitic matrix (PSM). x 22,000.
Fig. 11.Phase-contrast micrograph of mechanically isolated somite (S)cultured 12 hours on Falcon plastic.
Cells migrate radially (arrows) and are not restrained by somitic basal lamina and other components of the
perisomitic matrix. Most cells exhibit stellate fibroblast-like shapes. x 70.
Fig.12. Bright field micrograph of unfixed somite (S) enzymatically isolated from stage-I2 chick embryo.
Trypsinized somites exhibit blebbed surfaces (arrows) and are more transparent than those isolated by mechanical methods (compare with figure 9). x 160.
Fig. 13. Electron micrograph of surface of enzymatically isolated somite (S)similar to t h a t shown in figure
12. Cellular organelles are unaffected by this treatment but cytoplasmic blebbing (B) is common a t external
surfaces. Perisomitic matrix including basal lamina and microfibrils is completely removed (arrows) by the
enzyme. x 22,000.
Fig. 14. Phase-contrast micrograph of enzymatically isolated somite (Sj cultured 12 hours on Falcon plastic.
Somitic cell growth patterns are virtually identical to those seen in cultures of mechanically isolated somites
(compare with figure 11). Cells migrate radially (arrows) from the original cell cluster and form circular
growth areas on the culture dish. x 70.
a r e relatively common. They do not appear
cell-like, however, and serial cross-sections
show that cellular materials are rarely assoc i a t e d w i t h t h e notochordal surface. As
pointed out above, however, mesenchymal
“contamination” i s possible a n d , indeed,
phase-contrast monitofing indicates t h a t a
few sclerotomal cells are occasionally carried
into culture along with the intact PNS. The
number of such cells is not significant and
they probably do not materially affect the
outcome of the experiment. LM shows t h a t
other features of uncultured notocfiords are
within the range of normal variation (fig. 16)
and, except for occasional increased vacuolation, they are virtually indistinguishable
from their in vivo counterparts (fig. 3).
Although ECM surrounding the notochord
is poorly resolved by LM (fig. 161, the presence of an intact PNS up to 2.0 p m thick is
clearly demonstrable by TEM (fig. 17). No
electron-dense counterparts of t h e surface
projections imaged by SEM are observed, but
basal lamina, microfibrils, and other components of the PNS are ultrastructurally indistinguishable from those observed in vivo
(fig. 4).
After 12 hours of incubation on Falcon plastic, mechanically isolated notochords maintain their rod shape (fig. 18). Cells from the
cut ends of the notochord attach and appear to
“fan out” on the substratum. Cross sections
through t h e middle portion of these notochords (fig. 19) show that their circular profile
is preserved and that their central parts remain unattached to the surface of the culture
At 36 hours in vitro, notochords show dumb
bell-shaped growth areas by phase-contrast
microscopy (fig.20). Extensive cell migration
from both the cephalic and caudal ends (indistinguishable in our preparations) of the notochord continues but the mid-section remains
rod-shaped and the outline of the original tiss u e is easily discernable. Cross sections
through the central region show remarkably
circular and smoothly contoured tissues with
no evidence of lateral migration (fig. 21). The
continued presence of a well-established PNS
is confirmed by TEM (fig.22). Basal lamina
material, microfibrils, and associated ground
substance combine to compartmentalize notochordal cells and preserve its rod shape.
Surface ECM is not diminished up to 72
hours in culture and often appears more concentrated after several days in vitro. A simil a r a c c u m u l a t i o n of ECM m a t e r i a l s is
observed in notochordal interstitial spaces (in
the rod-shapped mid-section) and is associated
with cell surfaces (in the fan-shaped growth
areas). Unquestionably, however, the largest
population of microfibrils, basal lamina materials, and interstitial bodies is located in the
“tube” of matrix-the PNS surrounding the
intact notochord.
Isolation and culture of enzymatically
isolated notochords
Notochords isolated by trypsinization and
microdissection average 1-3 mm in length.
Following treatment with the enzyme, they
lose their characteristic rigidity and are easily distorted by physical manipulation. They
exhibit little elasticity and do not usually regain their straight rod shape following mechanical bending or coiling. In the following
experiments mesenchymal contaminants and
other non-notochordal debris (usually visible
by stereo microscopy) were removed by gentle
flushing with pipettes and only the cleanest
notochords were selected for in vitro morphological studies.
When isolated uncultured notochords are
prepared for SEM, they appear as cylindrical
structures with highly irregular surfaces (fig.
23). They show relatively uniform diameters
(about 25 p m ) and occasionally coil upon
themselves. Their most striking topographical feature is the bulb-shaped blebs which virtually cover the entire notochordal surface.
Most blebs are approximately 3-4 p m in diameter and do not appear stressed or torn.
Mesenchymal cells or extracellular surfaceassociated substances (PNS) are not evident
in these preparations.
Following embedding in epoxy, orientation
for cross-sectional microtomy and preparation for LM, cross sections of uncultured isolated notochords appear as solid circular tissues
with numerous cytoplasmic projections (fig.
24). These projections correspond directly
with blebs observed by SEM and are not vacuolated. Trypsinized notochords usually demonstrate few intracellular vacuoles, and dilated interstitial spaces which are prominent in
vivo at later stages of development (Carlson,
’73a) are not present. Sections of notochords
show 10-15 contiguous cells with prominent
nuclei and dark-staining nuceoli.
At the level of TEM, basal surfaces of notochordal cells are clearly resolved (fig. 25). Cytoplasmic blebs a r e lined by a continuous
plasmalemma and their internal structure
shows no evidence of cytotoxicity. They
usually demonstrate a paucity of membranebound organelles and contain numerous polyribosomes and cytoplasmic microfilaments.
When trypsinized notochords are cultured
under the same conditions as those described
for notochords isolated mechanically, their respective patterns of growth on the substratum are widely disparate. Phase-contrast microscopy shows t h a t by 12 hours of culture,
cells from enzyme-treated notochords migrate
randomly from the original tissue mass and
attach to the substratum (fig. 26). At 36, 48,
a n d 72 h o u r s of i n vitro incubation (figs.
27-29), notochordal cells expand across the
substratum so that the final growth area exhibits a relatively circular shape (fig. 29).
This is in contrast to ensheathed notochords
(mechanically isolated) where peripheral migration emanates primarily (if not totally)
from the ends of notochordal segments (figs.
18, 20).
Notochordal and somitic morphology in uiuo
The early chick notochord develops from
specialized mesoderm (chordamesoblast) near
Henson’s node (at about stage 5 ) and appears
early a s a bilaminar-flattened strip of cells
(Bancroft and Bellairs, ’76). It gradually becomes more rod-shaped as cell-cell contacts
between presumptive notochordal cells increase (Jurand, ’62). This occurs during the
first two and one-half days of development
when the notochord loses contact with surr o u n d i n g t i s s u e s a n d a s s u m e s a “frees t a n d i n g ” a x i a l position v e n t r a l to t h e
developing neural tube. The connective tissue
space surrounding t h e notochord appears
“ e m p t y ” by o r d i n a r y l i g h t microscopic
methods, possibly because of fixation shrinkage. Nevertheless several investigators (Low,
’68; Ruggeri, ’72; Lauscher and Carlson, ’75;
Revel and Brown, ’76; Bancroft and Bellairs,
’76; Ebendal, ’77) have clearly demonstrated
by SEM and TEM that this area contains numerous fibrils and other ECM components.
The developing notochord is larger in diame t e r , s h o w s a more c i r c u l a r profile, a n d
a p p e a r s more highly differentiated a t i t s
cephalic end t h a n caudally. The more advanced cephalic end is also surrounded by a
thicker, denser PNS than its caudal counterpart (Carlson, unpublished observations). In
order t o compare cross-sectional morphological characteristics of the notochord and associated PNS at successively later stages of de-
velopment, it is a requirement that sections
be taken from the same position relative to
some reference point (such as the heart). In
addition, magnifications of sections to be compared must be kept identical.
With these guidelines in mind, the present
study shows that successively older embryos
demonstrate a progressively denser, more fibrillar PNS, including a better-developed
basal lamina. The sheath encases sequent i a l l y larger, more vacuolated notochords
with crisp circular profiles, which give the
impression of highly compartmentalized tissues surrounded by an extracellular barrier
that disallows cellular dispersion. This overa l l picture of notochordal development is
consistent with the suggestion that the notochord becomes progressively more turgid and
thus provides early longitudinal support for
the embryo (Romanoff, ’60). It seems possible,
therefore, that the developing notochord may
be responsible for the production of a n extracellular “tube” of matrix that surrounds and
physically compartmentalizes itself to the ext e n t t h a t its rod shape remains unaltered
prior to involution. Furthermore, t h i s a r rangement may be a requirement for normal
embryonic axial rigidity prior to the establishment of segmental chondrification centers.
Like t h e notochord, somites a r e derived
from chordamesoblast. Pre-somitic mesoderm
(primary mesenchyme) (Hay, ’68) from t h e
primitive streak apparently disperses beneath the epiblast and in the paraxial region
is segmented by intersomitic clefts leaving bilaterally symmetrical blocks of tissue flanking the notochord (Hamilton, ’52).
The present study shows that at stage 10
(10 somites) the seventh somite exhibits a n
epithelioid, rosette-like configuration with a
poorly defined lumen. Perisomitic m a t r i x
(PSM) is sparse compared to t h e P N S although a relatively continuous basal lamina
surrounds the basal plasmalemmae. Following formation of the dermatomyotome and
disorganization of the sclerotome these latter
ventro-medial cells temporarily m a i n t a i n
their position with respect to the midsagittal
plane. By stage 17, however, sclerotomal intercellular spaces further increase and t h e
cells appear closer to the notochordal surface.
This phenomenon apparently is exaggerated
by a concomitant increase in notochordal diameter (Gasser, ’79). Nevertheless, by stage
20 the block-like somite is highly dispersed,
and sclerotomal cells interposed between no-
Fig. 15. Low power scanning electron micrograph of mechanically isolated notochord.
These tissues are quite rigid and regain their straight rod shape following mechanical
bending. Surface projections are non-cellular (see arrows in fig. 16) and are not stained
by usual TEM techniques (see fig. 17). Double-headed arrow indicates position of section in fig. 16. x 650.
Fig. 16. Light micrograph of cross section through mechanically isolated notochord
(N) (see double-headed arrow in fig. 1 5 for section location). The smooth circular profile
of in vivo notochords (see fig. 3) is maintained by this procedure and no blebbing is
observed. Arrows indicate non-cellular amorphous materials (see projections in fig. 15).
Square indicates approximate area shown in fig. 17. Toluidine blue. x 285.
Fig. 17. Low power transmission electron micrograph of notochord iN) surface (see
square in fig. 16 for approximate area shown). A clearly identifiable perinotochordal
sheath (PNS) is retained and completely encases the isolated notochords except a t its
cut ends. x 20,100.
Fig. 18. Phase-contrast micrograph of mechanically isolated notochord (N) cultured
12 hours. The cent,ral portion of the notochord has not attached to the substratum. It
maintains its crisp edges (small arrows) and rod shape. Cellular migration is evident a t
the cut ends of not,ochord. These cells firmly adhere to culture dishes. Double-headed
arrow indicates approximate position of section in fig. 19. x 150.
Fig.19. Light micrograph of cross section through notochord (N) similar to that shown
i n figure 18 (see double-headed arrow in figure 18 for approximate location of section).
The cylindrical shape of the notochord is maintained. Cells adjacent to notochord probably migrated from cut end. Toluidine Blue. x 190.
Fig. 20. Phase-contrast micrograph of mechanically isolated notochord (N) cultured
36 hours. Crisp surface contrast in mid-section (small arrows) indicates preservation of
tissue cylinder in this region. Cell migration and tissue attachment occur almost exclusively a t cut ends of notochord segment producing a dumb bell-shaped growth pattern.
Double-headed arrow indicates approximate position of section in figure 21. x 150.
Fig. 21. Light micrograph of cross section through mechanically isolated notochord (N)
cultured 36 hours (see double-headed arrow in fig. 20 for approximate location of section). A few small vacuoles are present. The maintenance of the notochord rod shape is
clearly shown by its circular profile. Square indicates approximate area shown in figure
22. Tnluidine blue. x 190.
Fig. 22. Transmission electron micrograph of surface of notochord (N) in area similar
to that shown by square in figure 21. Perinotochordal sheath is intact and is composed
of basal lamina ibl) microfibrils and other components of the extracellular matrix.
x 23,400.
Fig. 23. Scanning electron micrograph of uncultured notochord isolated by trypsinization from stage15 chick embryo. Following enzymatic isolation rigidity is lost and the
normally smooth cylindrical surface is profusely blebbed (B(.Isolated notochords average
1-3 mm in length, approximately 25 W
m in diameter and are flaccid rods which may be
bent or coiled by mechanical manipulation. Double-headed arrow, position of cross
section shown by light microscopy in figure 24. x 960.
Fig. 24. Light micrograph of cross section through notochord (N) in area similar to that
indicated by double-headed arrow in figure 23. Numerous cytoplasmic projections are
seen a t the notochordal surface. Cross sections usually show 10-15 cells. Nuclei and
nucleoli are readily distinguished. Rectangle indicates approximate area shown in figure
25. Toluidine blue. x 1.700.
Fig. 25. Transmission electron micrograph of surfce of contiguous notochordal cells in
area similar to that seen in rectangle in figure 24. Trypsinization does not alter the
morphological integrity of cellular organells. Note complete absence of extracellular
matrix on basal surfaces of cells. B. cytoplasmic blebs; nuc, nucleus. x 17,300.
Fig. 26. Enzymatically isolated notochord cultured 12 hours on Falcon plastic. Rod
shape profile of notochord segment is maintained. A few randomly scattered cells (arrows) are associated with the notochord. x 68.
Fig. 27. Enzymatically isolated notochord cultured 36 hours on Falcon plastic. The
original notochordal attachment site is recognizable but the cylindrical shape is lost.
Cells migrate a t approximately equal rates i n all directions (arrows). x 68.
Fig. 28. Enzymatically isolated notochord cultured 48 hours on Falcon plastic. Only a
poorly defined “ghost” of the initial notochordal tissue adherence is evident. The growth
area is oval, suggesting t h a t cells migrating laterally (horizontal arrows) may have
greater mobility than those a t terminal ends (vertical arrows). x 68.
Fig. 29. Enzymatically isolated notochord cultured 72 hours on Falcon plastic. By this
culture time notochordal cells form a circular growth pattern and the original notochordal tissue is nearly indistinguishable. X 68.
27 1
tochord and neural tube dorsally, and between notochord and aorta ventrally, are located approximately 8@120 p m dorsomedial
or ventromedial from their position a t stage
10+ (compare figs. 1 and 7).
Recently Gasser ('79) provided good evidence that, in the rat, sclerotomal cells do not
migrate medially and their final position can
be explained on t h e basis of dorsolateral
migration of the somite remnant and sclerotoma1 cell proliferation. Gasser ('79) also suggested t h a t gaps between tissues at early
stages of development may be due to fixation
shrinkage. Applying these arguments to the
present study it may be inferred that the apparent distance traversed by sclerotomal cells
(< 120 pm relative to the notochord) may result from random cellular redistribution due
to a high mitotic rate and also from some
preparation shrinkage. Although our d a t a
tend to support Gasser's ('79) observations,
the mechanisms by which sclerotomal cells
attain their ultimate midline location remain
In the present study our general account of
somite formation and dispersion is consistent
with the summaries of Hay ('68) and Lipton
and Jacobson ('74) as well a s with the more
general overviews offered by most current
textbooks of embryology. However, our study
emphasizes the striking contrast between
morphogenetic changes of t h e somite and
those of the notochord. It seems possible that
the lack of a dense fibrillar PSM prior to dispersion may be assistive in somitic disorganization while the relatively more advanced
PNS may be more confining.
Isolation and culture of somites
In the past decade, numerous investigations of somite explants have been carried out
primarily to study their production of cartilage in vitro (Lash, '67; O'Hare, '72b; Kosher
et al, '73; Lash et al, '73; Gordon and Lash,
'74; Minor, '73; Lash and Vasan, '78). Clusters
of somites were cultured on a variety of substrata including various types of collagens,
nucleopore filter, millipore filter, n u t r i e n t
agar, and chorioallantoic membrane. With
the exception of Minor's ('73) TEM study,
somite explant investigations h a v e been
aimed largely a t elucidating biosynthetic
products. Lash and Vasan ('78) recorded the
degree of somitic spreading on various substrata but made no attempts to relate this parameter to the presence or absence of PSM a t
the beginning of culture. Moreover, the resolution of their photomicrographs did not per-
mit determinations of growth patterns and
cell migrations. No previous ultrastructural
studies have ben carried out to describe surface characteristics of somite explants prior t o
incubation, and investigations of the effects of
PSM on growth patterns and tissue expansion
in vitro have not been reported.
Our studies show that somites isolated by
mechanical means from t r u n k segments of
stage-1P16 chick embryos exhibit clearly defined shapes with smooth contours. They appear as flattened blocks with approximately
two-thirds of their periphery smooth and undisturbed. The remaining one-third appears
rough and irregular, suggesting mechanical
dissociation of adjacent tissues. This is consistent with Hamilton's ('52) description of six
somitic surfaces, all of which are free except
one (the lateral), which is continuous with intermediate mesoderm.
Somites isolated by trypsinization compare
favorably in size (about 260 x 360 pm) and
overall shape to those isolated by homogenization. By TEM, however, enzyme-isolated
somites a r e completely denuded, while mechanically isolated somites show a PSM
which is indistinguishable from their in vivo
counterparts. In addition, trypsinized somites
are decorated with cytoplasmic blebs, an apparently innocuous feature which is also seen
i n enzyme-isolated notochords and o t h e r
embryonic epithelia from which basal lamina
has been remove (see Hay and Dodson, '73, for
When somites isolated either mechanically
or by trypsinization (ie, with or without PSM)
a r e cultured on a non-collagenous (Falcon
plastic) substratum, they spread rapidly in
radial growth patterns. Within 12 hours cells
migrate in a monolayer in all directions and
consistently form ovoid to circular growth
patterns 0.8-1.3 mm in diameter. These diameters vary within groups and are unrelated to
the method of isolation.
The data indicate that PSM associated with
somites from stage-14-16 chick embryo trunk
s e g m e n t s i s not competent to m a i n t a i n
somitic block shape nor to prevent cell migration in culture. Although it is possible that
the substratum may stimulate somite spreading, the stimulus is equally effective regardless of the presence or absence of PSM.
Isolation and culture of notochords
Mechanically isolated notochords. Most in
vitro investigations of notochordal tissues
have begun with notochords isolated with the
aid of enzymes. A notable exception is the
work of Kosher and Lash ('75) who dissected
notochords from stage-17 chicks with or without enzymatic treatment prior to co-culture
studies with somites. Their freely dissected
(no enzyme treatment) notochords were surrounded by alcian blue-positive materials as
d e m o n s t r a t e d by conventional LM histochemical techniques. No SEM or TEM observations of these notochords were carried out
but, based on the presence of a perinotochordal halo of polyanionic materials, it was concluded that the PNS was intact.
The present study shows t h a t notochords
isolated from stage-1616 trunk segments by
mild homogenization behave a s relatively
rigid cylinders that regain their straight rod
shape following mechanical bending or coiling. By conventional LM, they show sharply
demarcated circular profiles similar to those
described by Kosher and Lash ('75). By SEM
they exhibit smooth surfaces marked by irregular protrusions. Since non-notochordal
cells are infrequently observed by serial LM
sections, it seems likely that the irregularities imaged by SEM may represent attached
particles of proteoglycans or other extracellular macromolecules that are not clearly visualized by routine LM and TEM procedures.
This interpretation is consistent with other
investigations (Jacob et al, '75; Bancroft and
B e l l a i r s , '76; Revel a n d Brown, '76;
Schoenwolf, '79) t h a t show perinotochordal
ECM manifested as surface irregularities and
wispy fibrillar materials. Most importantly,
however, our TEM studies confirm the presence of a n i n t a c t P N S t h a t is u l t r a s t r u c t u r a l l y i d e n t i c a l to t h a t s e e n i n vivo
(Frederickson and Low, '71; Ruggeri, '72;
Bancroft and Bellairs, '76).
Following 12 hours in vitro a few cells begin to migrate from the cut ends of mechanically isolated notochords. These cells are not
derived from non-notochordal (mesenchymal)
cells, since our cross-sectional LM and TEM
observations show that the PNS is cell-free.
The body of t h e notochord remains solidly
cylindrical with sharp, straight borders, indicating the continued presence of a PNS. No
lateral cell migration is noted. At 24 hours,
the PNS is densely fibrillar and the notochord
mid-section remains rod-shaped while "end"
cells continue to migrate outward. The fanlike growth pattern and cellular adhesion
resulting from this peripheral expansion is
further exaggerated a t 36 hours. Still the central portion of the tissue remains cylindrical,
unattached to the substratum and invested
with a dense "tube" of matrix (PNS). Com-
27 3
partmentalization of notochordal tissues is
even more striking a t 72 hours of culture.
Cells from the cut ends continue their expansion of the substratum while the PNS-encased
mid-section retains its pre-incubation rod
Growth patterns of mechanically isolated
notochords differ m a r k e d l y from t h o s e
exhibited by cultured, mechanically isolated
(PSM intact) somites. The PSM is ineffective
i n preventing somitic cell spread (at least on
Falcon plastic substrata), while notochordal
cells are completely immobilized by the PNS.
It is noteworthy that a similar phenomenon
occurs in vivo where the dissociation of the
sclerotome (and concomitant deposition of
ECM in intercellular spaces) is contrasted by
continued restriction of notochordal cells (and
concomitant accumulation of matrix in the
In an effort to compare the in vitro growth
patterns of notochords stripped of their PNS
with those cultured with PNS intact, the PNS
was completely removed from the notochordal
surface prior to culture. The results of these
experiments are disscussed below.
Enzymatically isolated notochords
The present study shows that following isolation by trypsinization and microdissection,
notochords from s t a g e - 1 6 1 6 chick embryos
lose their smoothly contoured shapes and
show highly irregular surfaces. Their diameters are comparable to in vivo equivalents but
they do not exhibit the rigidity expressed by
mechanically isolated notochords. This suggests that the enzyme-sensitive PNS may provide some longitudinal resilience which is
transmitted to the entire embryonic axis. It
seems likely that the sheath also offers some
circumferential support since its removal results in cytoplasmic blebbing similar to that
observed in somites following enzymatic removal of PSM. Our SEM studies indicate that
the blebs virtually cover isolated notochordal
surfaces. No patterns such as segmentations
or other non-uniform distributions of blebs
a r e recognizable along t h e i r lengths. It is
noteworthy that although other investigators
(Kosher and Lash, '75) have demonstrated
reduced histochemical staining of acid mucopolysaccharides following isolation of notochords by trypsinization, cytoplasmic blebbing was not evident. This is inconsistent
with our results and other studies that show
that blebbing seems to be a common phenomenon following enzymatic removal of basal
lamina (Cohen and Hay, '71; Hay and Dodson,
’73; Carlson et al, ’74; Lauscher and Carlson,
’75; Kenney and Carlson, ’78).
Cross sections through trypsinized notochords show 7-10 contiguous cells, radially
arranged and morphologically similar to the
same structure in vivo. Intracellular vacuoles
are rarely observed in freshly isolated notochords, suggesting that these tissues may be
dehydrated to some extent during the isolation procedure. Surface-associated cytoplasmic blebbing also could be a result of such
TEM observations confirm the absence of
non-notochordal cells or contaminating ECM
i n enzymatically isolated notochords a n d
shows that trypsinization does not interrupt
or otherwise damage cell membranes.
Following 12 hours of in vitro incubation, a
few notochordal cells adjacent to the plastic
substratum break away from the central cell
cluster and begin to migrate laterally. These
early in vitro cell movements are not merely
transitory but they signal the initiation of a
mass lateral migration which is directional
(perpendicular to the long axis of the isolated
notochord) and continuous (up to 72 hours in
culture). A few cells migrate from the cephalic and caudal ends but by far the greatest tissue expansion occurs laterally. By 72 hours
the growth area is nearly circular and has a
diameter approximately t h e length of t h e
original notochord segment.
Most investigators consider the avian notochord a n epithelioid s t r u c t u r e (Hay, ’68;
Carlson, ’73 a, b; Carlson et al., ’74; Lauscher
and Carlson, ’75). As Bancroft and Bellairs
(’76) correctly point out, however, a clearly resolvable lumen is not present and its cells do
not demonstrate close intercellular junctions
near their “apical” surfaces. Therefore, to designate the notochord a s a t r u e epithelium
may be misleading. This is particularly true
in light of the present study, which suggests
that the notochordal “epithelial” histoarchitecture may be a t least partially dependent
upon an intact PNS. Certainly one could not
expect an embryonic epithelium to maintain
all of its morphological characteristics when
removed from its environment, stripped of its
“exoskeleton,” and placed i n culture. The
changes we describe, however, suggest a major departure from a more epithelial structure
following culture with PNS intact (ie, radially arranged cells with close intercellular
junctions and surrounded by basal lamina) to
a more mesenchymal structure following cul-
ture with PNS removed (ie, layers of spreading stellate cells). This is in striking contrast
to other true embryonic epithelia from which
surrounding ECM has been removed (Cohen
and Hay, ’71; Hay and Dodson, ’73) and which
maintain their epithelial morphology in organ culture.
Data in the present study indicate that und e r identical culture conditions t h e PNS
plays a role i n m a i n t a i n i n g tissue shape
while PSM does not. It seems reasonable that
the restriction of notochordal cells and the
concomitant dispersion of somitic cells in vivo
may likewise reflect differing functions of
their respective extracellular encasements.
For example, cellular dispersion may be prevented by physicochemical properties of the
PNS while PSM is incompetent to act in this
way. Alternatively, the PNS may prevent cells
from attaching to a substratum that promotes
migration while the PSM is not capable of
such activity. It must also be pointed out,
however, t h a t notochordal disintegration in
vitro following PNS removal may represent
a n artifact of culture conditions and therefore, the possibility t h a t the notochord may
remain compacted in vivo if the PNS is removed cannot be ruled out. Nevertheless, observations in the present study support our
general hypothesis that the avian notochord
is held passively in an epithelioid configuration by a self-perpetuated accumulation of
matrix (PNS) on its surface. This interpretation is consistent with the concept proposed
by Hay (’68) t h a t suggests t h a t embryonic
epithelia derived from primary mesenchyme
(ie, notochord, somites, lateral mesoderm, etc)
may subsequently disperse to form secondary
mesenchyme. It seems possible that the dispersion and subsequent expansion of notochordal cells described in the present study
may be a morphological manifestation of this
principle in a tissue which, under in vivo conditions, is incapable of such expression.
We gratefully acknowledge the expert technical assistance of Mr. Armando Romero and
Mr. Thomas Demlow in this project. Thanks
are offered to Dr. Allen C. Enders for his critical evaluation of the manuscript.
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