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The development of proline-containing extracellular connective tissue fibrils by chick notochordal epithelium in vitro.

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The Development of Proline-containing Extracellular
Connective Tissue Fibrils by Chick Notochordal
Epithelium In Vitro
'r2
CRISTINA K. LAUSCHER AND EDWARD C. CARLSON
Department of Anatomy, University of Arizona, Tucson, Arizona
ABSTRACT
Notochords were isolated from Hamburger-Hamilton stages 1315 chick embryos by trypsinization and microdissection. These were shown by
electron microscopy to be completely devoid of extracellular materials or mesenchymal contaminants. Cultivation of notochordal isolates was carried out on
a non-collagenous (Falcon Plastic) substratum for 0 to 48 hours. At 12 hours
of in vitro incubation, a discontinuous basal lamina could be demonstrated on
the surface of notochordal cells. This was followed by the appearance of microfibrils of various sizes and other components of the extracellular matrix. By 48
hours of in vitro incubation, the same extracellular materials which surround
the notochord in vivo (notochord sheath) could be demonstated in vitro.
Autoradiographic studies show that tritiated proline is taken up by notochordal
cells and secreted to the extracellular space where label is associated with basal
lamina, microfibrils and ground substance. When cis-hydroxyproline, a known
collagen-specific inhibitor is added to the system, tritiated proline label is located
primarily intracellularly and fewer areas of active fibrillogenesis are noted.
This suggests that ultrastructurally recognizable materials produced by notochordal cells in vitro may be at least partially collagenous. Significantly, these
materials are produced in vivo at the same time (following stage 10) that notochordal tissues actively induce somite differentiation and cartilage formation.
It seems reasonable that a biochemically or ultrastructurally identifiable component of the extracellular matrix may possibly mediate such induction.
Ultrastructural investigations centered
around the developing chick embryo have
demonstrated a network of intertwining
extracellular connective tissue fibrils concentrated on the surface of the notochord
(Duncan, '57; Jurand, '62; Low, '68; Hay,
'68; OConnell and Low, '70; Frederickson
and Low, '71; Carlson, '73a). Significantly,
these microfibrils and accompanying basal
lamina and amorphous materials, which
together comprise the notochord sheath
were present prior to the differentiation of
secondary mesenchymal cells from the
sclerotome (Low, '68; Frederickson and
LOW,'71; Olson and Low, '71). The sheath
has been suspected to be a product of the
notochordal epithelium (Duncan, '57; Low,
'68; Carlson, '73a,b), and recently Carlson
et al. ('74a) have shown that extracellular
connective tissue fibrils are produced by
notochordal epithelial cells in vitro. The
ANAT. REC., 182: 151-168.
sequence of events leading to the formation
of notochordal microfibrils in vitro, however, has not been previously described.
That the notochord may be actively
secretory was supported by biochemical
studies (Linsenmayer et al., '73) which
showed that cultured notochordal cells are
capable of producing non-type I collagen.
More recently, Carlson and Upson ('74)
used similar culture techniques to demonstrate that notochordal cells produce "native" striated collagen fibrils. In addition,
several histochemical investigations have
inferred that mucopolysaccharides may be
produced by the notochord (OConnel and
Low, '70; Ruggeri, '72).
Received Aug. 8, '74. Accepted Dec. 18, '74.
1Aided by a n NIH Institutional Grant for General
Research Support (RR05675) and a grant from the
Southern Arizona Heart Association.
2 Presented from the platform at the Thirteenth Annual Meeting of the American Society for Cell Biology, November 14-17, 1973, Miami Beach, Florida.
151
152
CRISTINA K. LAUSCHER AND EDWARD C. CARLSON
The present study was designed to demonstrate the fine structural appearance of
early fibrillogenesis by notochordal cells in
vitro. Our observations showed that these
cells are actively secretory at 12 hours of
incubation when cultured on a non-collagenous (Falcon Plastic) substratum. Furthermore, our autoradiographic studies
showed that developmental and secretory
patterns may be altered when notochordal
tissues were incubated in the presence of
cis-hydroxyproline, a known collagen specific inhibitor.
MATERIALS AND METHODS
Notochordal isolation and
cultivation
Fertile hens eggs of the White Leghorn
strain were incubated at 37°C in a redwood forced-air incubator for two and one
half days. The resultant embryos were
excised and transferred by means of a
sterile filter paper ring to culture medium
that consisted of 15% fetal bovine serum,
10% chick embryo extract, 50 units/ml
penicillin-streptomycin and 2 units/ml
fungizone in Ham’s F-10 growth medium
(Flow Laboratories). Embryos were staged
according to Hamburger and Hamilton
(’51). Cross-sectional scissor cuts divided
the embryos just caudal to the heart and
rostra1 to the terminal somites. The intervening tissue section from each embryo
was placed in cold (4°C) 1% trypsin for
40 minutes. During trypsinization, notochords were isolated from the adjacent
tissues by gentle flushing through small
bore Pasteur pipettes and final dissociation
by drawn-glass microneedles. Trypsin was
deactivated by transferring isolated notochords to 15% fetal bovine serum (FBS)
followed by final replacement of the FBS
with culture medium. These isolates were
further incubated on 35 mm plastic tissue
culture dishes (Falcon Plastics) 0 to 48
hours in carbon dioxide-flushed incubation
at 37°C (60% relative humidity). Unincubated isolated notochords were fixed immediately and served as controls.
Fixation, embedment and
microscopy
Cultured notochords were fixed one hour
in cold ( 4 “C) paraformaldehyde-glutaral-
dehyde fixative (Karnovsky, ’65) buffered
at pH 7.3 with 0.2 M sodium cacodylate
buffer. Post-fixation in cacodylate-buffered
OsOl lasted one hour and was followed by
dehydration in ethanol. Unincubated isolated control notochords were similarly
fixed and following osmication surrounded
by several drops of 2% Nobel agar which
aided their further manipulation. In addition, propylene oxide was employed as an
intermediate solvent following ethanol dehydration.
All tissues were embedded in an EponAraldite combination (Anderson and Ellis,
’65) and cured at 37°C overnight and 60°C
for two days. One micron thick sections
were cut and stained with toluidine blue
and observed in an Ultraphot I1 (Zeiss
Optics, Inc.) for determination of tissue
orientation and position. Thin sections
were cut with a diamond knife and collected on uncoated 300 mesh copper grids.
These were stained with uranyl acetate
(5% in absolute ethanol) and lead citrate
(Venable and Coggeshall, ’65) and observed in a Philips EM 300 electron microscope at original magnifications of 3,300 to
33,000 diameters.
Autoradiography
Autoradiographic analysis of cultured
notochordal tissues was carried out at the
level of light and electron microscopy.
Tritiated proline (25 ,Ci/ml culture medium) was added for either a 3 or 6-hour
pulse or for a continuous label. In addition, approximately one-half of the cultures received cis-hydroxyproline (40 pg/
ml). These tissues were further cultured
48 hours.
Since incorporated 3H-proline may not
be the only source of radioactivity in autoradiographs, the following procedures
were carried out to ensure that contamination was minimized : ( a ) Pulse-labeled
cultures were rinsed several times with
non-radioactive culture medium containing L-proline prior to further culturing in
the isotope-free medium. ( b ) Continuouslabeled cultures were similarly washed extensively with “cold culture medium and
buffer before the aldehyde fixation. (c)
Cultures which had been grown in isotopefree medium only were prepared for autoradiography to determine the contribution
PROLINE-CONTAINING FIBRILS
153
of the unlabeled tissues to the observed not completed their migration toward the
radioactivity. ( d ) Autoradiography of cul- embryonic axis and hence the perinototure medium-conditioned substratum (Fal- chordal space is nearly cell-free. In light
con plastic) was carried out to show microscopy this space shows only poorlythe extent of label derived from the iso- resolved fibrillar material which intervenes
tope-free culture medium and/or the sub- between the notochord and overlying neustrat um ,
roepithelium. The relatively smooth notoLight microscopic autoradiographs were chordal surface is shown by electron miprepared from one-half micron thick sec- croscopy (fig. 2 ) to be surrounded by a
tions stained with toluidine blue, coated continuous basal lamina which separates
with evaporated carbon and dipped in the epithelium from bundles of 100-200 A
Ilford L4 emulsion. These were exposed microfibrils and other extracellular comfour days at 6°C in plastic light tight slide ponents embedded in an electron-lucent
plate boxes which contained CaS04 des- matrix. These materials comprise the notosicant. The slides were developed in Dektol, chord sheath. Notochordal cells are highly
coverslipped, observed and photographic- differentiated at this stage of development
ally recorded.
and exhibit a complement of organelles
Electron microscopic autoradiography (Ruggeri, ’72) which is consistent with
was carried out on thin sections placed on their known secretory capacity.
1” X 3” glass microscopic slides previously
Notochords dissociated from adjacent
coated with celloidin. These sections were tissues by trypsinization are shown by
stained with uranyl acetate (2.5% in 50% cross-sectional microtomy to retain their
ethanol), coated with a layer of evaporated cylindrical shape (fig. 3 ) but numerous
carbon and dipped in Ilford L4 emulsion cytoplasmic blebs project from their sur(thickness determined by purple interfer- faces. Such blebbing is not uncommon in
ence color) at 40°C. Following four weeks tissues from which the basal lamina has
exposure in total darkness (as above) at been removed (Hay and Dodson, ’73).
6 ” C , sections were developed in Phenidon Electron microscopy (fig. 4) shows that
(Lettre and Paweletz, ’66) for one minute the surface of notochordal isolates is deat 15°C. The celloidin film and attached void of basal lamina, ultrastructurally recthin sections from each glass slide were ognizable extracellular matrix or mesenfloated onto water. Two hundred mesh chymal contaminants. A loss of vacuoles
copper grids were then positioned over the and reduction in dilated intercellular
tissue sections. Grids, sections and celloidin spaces is the only recognizable alteration
film were picked up from the surface of in notochordal morphology. Cellular damthe water with Whatman #31 filter paper. age or cytotoxic effects are not observed.
After drying, the grids were detached from
Following 12 hours of in vitro incuthe filter paper and submerged in isoamyl bation, circumferential notochordal cells
acetate for 3-4 minutes to reduce the thick- adhere to the plastic substratum and the
ness of the celloidin to a light gold inter- tissue begins to form small nodular growth
ference color. Electron microscopic obser- areas. Cellular blebbing continues at the
vation of these thin sections was carried surface of the epithelial tissue but a cenout.
tral cluster of cells remains closely aggregated within the growth area (fig. 5).
OBSERVATIONS
Within the intercellular spaces of these
Stages 13-15 embryos exhibit a cylin- central cells a discontinuous basal lamina
drical notochord (fig. 1) located in the con- is occasionally seen (fig. 6 ) . Rough endonective tissue space ventral to the larger plasmic reticulum, mitochondria and other
developing neural tube. Numerous spheri- cellular organelles are within the range of
cal vacuoles, which are known to be intra- normal variation and the cells appear to be
cellular (Jurand, ’62) and pleomorphic actively secretory.
extracellular spaces are present within the
The notochordal growth area begins to
notochordal epithelium. At this stage and spread outward by 24 hours of in vitro incephalocaudal position, secondary mesen- cubation (fig. 7). The central cell cluster
chymal cells from the sclerotomes have retains its epithelial appearance while
154
CRISTINA K. LAUSCHER AND EDWARD C. CARLSON
basal cells become stellate as they migrate
peripherally along the surface of the culture dish. Intracellular vacuoles and dilated intercellular spaces are scattered
throughout the tissue. Adjacent to lateral
surfaces of notochordal cells extracellular
connective tissue matrix composed of
amorphous material, small (100 A ) microfibrils and discontinuous basal lamina is
frequently seen (fig. 8 ) .
By 36 hours of in vitro incubation, edges
of the nodular growth area expand peripherally and the cellular mound begins to
flatten out (fig. 9 ) . At this time there is an
increased accumulation of extracellular
material at the surface of notochordal cells
(fig. 10). Randomly aligned microfibrils
are frequently seen closely associated with
or embedded in basal lamina material.
Following 48 hours of in vitro incubation the notochordal growth area reaches
0.5-1.0 mm as its perimeter migrates
radially in the plastic culture dish (fig.
11 ). Electron microscopic observation (fig.
12) of intercellular spaces shows widely
dispersed small ( 100 A ) and large (200 A)
microfibrils, some of which exhibit faint
irregular striations (fig. 13). A continuous
basal lamina is closely adherent to the surface of notochordal cells. These materials
are ultrastructurally indistinguishable from
those observed in the in vivo notochord
sheath (fig. 2).
If notochordal cells are incubated 48
hours in the presence of SH-proline, silver
grains are noted over cytoplasm, nuclei
and intercellular spaces with approximately equal density (fig. 14). Grain
counts show that this is an approximately
10-fold increase over background. Electron
microscopic autoradiography of similar
tissue (figs. 15, 16) demonstrates that
label is associated with basal lamina, microfibrils and other extracellular materials,
as well as with rough endoplasmic reticulum and polyribosomes within notochordal
cells. We are unable to detect any differences in labeling patterns when embryos
are pulse labeled ( 3 to 6 hours) or exposed
to the tritium continuously. When isotopefree cultures or culture medium-conditioned substrata are prepared for autoradiography, only occasional random silver
grains are noted. The label in these preparations never exceeds that of background
in tissues to which isotope has been added.
Light microscopic autoradiographic examination of cis-hydroxyproline treated
cultures shows widely dispersed stellate
cells while few clusters of aggregated cells
are seen. There is a markedly decreased
ratio of extracellular to cellular label (fig.
17). Furthermore, there is a reduction in
total silver grains per unit area when compared with untreated controls (fig. 14).
Ultrastructurally, the silver grains are associated with nuclei, rough endoplasmic
reticulum, Golgi zones and other cellular
organelles (fig. 18). Areas of active fibrillogenesis are infrequently observed.
DISCUSSION
In the present study we describe the development of basal lamina, microfibrils and
amorphous materials produced by notochordal epithelial cells in vitro. This developmental process closely resembles the
early secretory activity of a number of embryonic epithelia (Low, '68; Hay and Revel,
'69; Dodson and Hay, '71; Cohen and Hay,
'71; Johnson et al., '74). Nevertheless, we
have shown that isolated notochords exhibit the following features which are
unique to this tissue: ( a ) isolated notochordal cells produce ultrastructurally recognizable components of extracellular connective tissue matrix when grown on a
non-collagenous (Falcon plastic) substratum, ( b ) the earliest morphological manifestation of this activity (basal lamina)
is present within 12 hours of in vitro culture time, ( c ) paradoxically, non-type I
collagen produced by notochordal cells in
vitro (Linsenmayer et al., '73; Miller and
Mathews, '74) seems to be stimulated to
aggregate into large striated collagen fibrils
(Carlson and Upson, '74) when cultured
in the presence of BAPN, a specific inhibitor of covalent intermolecular crosslinking
in tropocollagen (Narayaman, '72).
That the notochord may be capable of
producing extracellular fibrils was first suggested by Duncan ('57). He observed the
heavy concentration of microfibrils surrounding the early chick notochord (notochord sheath) and proposed that these may
be derived from the notochordal surface.
Low ('68) showed that these early fibrils
were present prior to the arrival of differentiated mesenchymal cells. This sug-
PROLINE-CONTAINING FIBRILS
gested that the fibrils were derived from
embryonic epithelia, but left their specific
cellular progenitor open to question.
More recently Carlson ('73a) provided
circumstantial evidence for notochordal
fibrillogenesis in a description of the production of basal lamina, microfibrils and
small, striated collagen fibrils in dilated
intercellular spaces deep within the substance of the notochordal epithelium. It
seemed reasonable that such materials
were formed following notochordal secretion to the interstices. This process was not
uncommon and had been observed in several types of epithelia which secrete proline-rich connective tissue proteins to their
intercellular spaces (Hay, '64; Revel, '65;
Dodson and Hay, '71). In another report
Carlson ('73b) showed that notochordal cells exhibit within their cytoplasm,
periodic, collagen-like fibrillar material in
membrane-bound dense bodies. Similar
dense bodies were seen in chick corneal
epithelium (Trelstad, '71), an epithelium
which is known to secrete collagen (Hay
and Dodson, '73).
The first direct evidence for the production of extracellular connective tissue
fibrils by notochordal epithelium was provided by Carlson et al. ('74a). Isolated
denuded notochords were grown in vitro
and a regrowth of notochordal microfibrils
was observed following 48 and 72 hours
incubation.
The present investigation shows the in
vitro development of notochordal extracellular matrix from 0 to 48 hours and
demonstrates that notochordal fibrillogenesis is not limited to microfibrils but includes other components of the matrix as
well. The process begins with the appearance of a faint discontinuous basal lamina
at 12 hours, followed by small (100 A )
microfibrils and amorphous materials at 24
hours. By 36 hours these microfibrils become larger and more numerous though
still closely associated with the notochordal
basal lamina. Following 48 hours of culture, scattered, larger (200 A) microfibrils,
some of which show faint irregular striations, are present. At this time the extracellular matrix is composed of basal
lamina, microfibrils of various sizes and
flaky amorphous materials embedded in an
electron lucent ground substance, and is
155
remarkably similar to that seen in the in
vivo perinotochordal space. More importantly, however, the developmental sequence by which extracellular materials
are formed, is closely paralleled by other
embryonic epithelia both in vivo (Low, '67,
'68; Hay and Revel, '69; Frederickson
and Low, '71; Carlson, '73a; Johnson et al.,
'74) and in vitro (Dodson and Hay, '71;
Cohen and Hay, '71; Hay and Dodson, '73).
These morphological similarities of development suggests similar cellular and extracellular metabolic processes, but i t is
impossible to argue that either the biochemical mechanism of formation or the
composition of the final extracellular products of various embryonic epithelia are
identical.
At the time that the notochord sheath
is developing in vivo, notochordal cells are
highly differentiated (Ruggeri, '72). They
exhibit a well-established rough endoplasmic reticulum and Golgi zones and numerous polyribosomes. This has led a number
of investigators to attempt to determine the
degree of secretory activity exhibited by
notochordal cells and to elucidate the nature of their products. OConnell and Low
('70) showed that perinotochordal microfibrils developed in a ground substance in
which histochemically demonstrable mucopolysaccharides could be identified. This
accorded with histochemical studies by
Ruggeri ('72) who concluded that notochordal cells secrete chondroitin sulfate A
and/or C. Frederickson and Low ('71)
demonstrated that at least small 100 A
microfibrils were digested by hyaluronidase
and proposed that these early fibrils might
be composed primarily of protein-polysaccharide.
In a study of the secretory products of
isolated notochords in vitro, Linsenmayer
et al. ('73) provided biochemical evidence
for the production of collagen. The collagen was composed of three identical
a-1 chains and although a small amount
of a-2 material (< 10% ) was observed, the
majority of the secretion was determined
to be a non-type I collagen3 This was con3Type I collagen has a chain composition of
(al[I]za2) and is the principal cpllagen of bone, skin
and tendon (Grant and Prockop, 72). Since a2 chains
are peculiar to this collagen type, collagens which
consist of three a1 chams (e.g.. notochordal collagen)
are considered to be non-type I.
156
CRISTINA K. LAUSCHER AND EDWARD C. CARLSON
sistent with the observation of Frederickson and Low (’71) who showed that
some perinotochordal microfibrils (primarily larger 200 A fibrils) were unstriated
but yet were attacked by collagenase.
Hence, a collagen moiety which is not
ultrastructurally demonstrable was postulated. Ruggeri (’72) concurred with this
conclusion and suggested on the basis of
an ultrastructural study of developing
chick notochord in vivo that this tissue
secretes “collagenous microfibrils.”
From these data we know that the notochord is capable of producing an ultrastructurally recognizable extracellular matrix including basal lamina, microfibrils
and other amorphous materials. Collagen
and glycosaminoglycans are also secreted
by this transitory embryonic epithelium.
The precise relationship of the biochemical products to the morphological entities,
however, remains to be elucidated.
In the present study, autoradiographic
techniques are employed to test the effect
of cis-hydroxyproline, a proline analog
known specifically to inhibit cellular extrusion of collagen (Rosenbloom and Prockop,
’71), on notochordal secretion of labeled
material. Radioactive proline is taken up
by cultured notochordal cells and secreted
into the extracellular space where it is
associated with basal lamina, microfibrils
and other extracellular components. A
general intracellular label is also observed
throughout cytoplasm and nuclei. It is unlikely that this non-specificity (figs. 14-16)
is due to free radioactive amino acid. Extensive washing and (in pulsed tissues)
culturing for up to 45 hours in isotopefree medium precludes glutaraldehyde
binding of unincorporated 3H-proline. Furthermore, our control studies showed that
neither culture medium nor the substratum
could be responsible for the observed radioactivity. Therefore, we interpret the observed non-specific labeling pattern to indicate localization of incorporated ’H-proline. Presumably in embryonic tissues, proline is incorporated into numerous proteins
necessary for cellular metabolism and is
not limited to collagen or protein polysaccharides. It is possible that only about 10%
of proline-labeled proteins are collagen
(Johnson et al., ’74).
When cis-hydroxyproline is added to the
system several alterations in morphology
and labeling patterns are noted. Fewer cell
clusters are observed and fibrillogenesis is
limited to the extracellular space adjacent
to these cells. The remainder begin to migrate peripherally, lose their epithelial configuration and become stellate. Second,
light microscopic autoradiographs of these
preparations show a reduced intracellular
label when compared with untreated controls. This could reflect a replacement of
ambient and 3H-proline by the analog
in newly synthesized collagen molecules.
Rosenbloom and Prockop (’71) show that
at a dose of 48 ,g/ml, approximately 10%
of proline residues are replaced by cishydroxyproline. Finally, the ratio of extracellular to cellular label is decreased with
respect to untreated controls. Few silver
grains occupy the extracellular space
while intracellular grains are concentrated
within cisternae of rough endoplasmic reticulum and Golgi zones. Presumably the
inhibitory activity of cis-hydroxyproline
allows collagen to be synthesized but not
extruded (Rosenbloom and Prockop, ’71).
Hence i t is possible that the observable decrease in extracellular label is a direct
effect of such inhibition. It seems reasonable to suggest, therefore, that the prolinecontaining extracellular materials produced by notochordal cells, and reduced in
the presence of cis-hydroxyproline, might
be composed, in part, of collagen.
Great caution must be exercised in interpreting these results, however, since proteochondroitin sulfate (Pal et al., ’66) is
rich in proline (10% of amino acid residues are proline). Most embryonic tissues,
including the notochord, secrete sulfated
glycosaminoglycans. Therefore, it is possible that the proline-containing extracellular matrix produced by notochordal cells
in vitro is not collagenous. In light of the
present investigation, however, this latter
conclusion can only be reached if one
assumes that inhibition of glycosaminoglycans secretion is coupled to inhibition of
collagen extrusion.
Tissue and organ culture studies show
that a collagenous substratum stimulates
the secretion of extracellular matrix by
chick cornea (Dodson and Hay, ’71 Hay
and Dodson, ’73) and neuroepithelium
PROLINE-CONTAINING FIBRILS
(Cohen and Hay, ’71). It is also true that
these same tissues failed to produce such
materials without the advantage of being
cultured on a predominantly collagenous
substratum. The present investigation demonstrates that notochordal epithelial cells
are capable of initiating fibrillogenesis on
a non-collagenous substratum within 12
hcurs after isolation and trypsinization.
Thus the notochord seems to express a developmental autonomy which is uncommon among non-mesenchymal cell types.
It seems significant that in vivo notochordal cells are most active in fibrillogenesis following stage 10 (Ruggeri, ’72)
when the notochord begins its inductive
activity on the sclerotomes of the somites
resulting in the production of cartilagenous
precursors of the bony vertebral column
(Seno and Biiyokozer, ’58; Strudel, ’63;
Holzer, ’64; Lash, ’64; Ellison et al., ’69).
The concomitant initiation of notochordal
secretory activity and the commencement
of somite differentiation and cartilage formation, lead one to speculate that the notochordal epithelium could act as one of the
initiating tissues in a series of subsequent
developmental inductions. This process
might be mediated by a biochemically or
morphologically identifiable component of
the extracellular matrix.
We have recently shown (Carlson et al.,
’74b) that although notochordal cells do
not require a collagenous substratum for
active fibrillogenesis, they are embryologically competent to respond to such stimulation and, indeed, produce much more fibrillar material under these conditions. Therefore, the inductive activity of the notochord
could lead to cytodifferentiation and subsequent secretory activity of other cell
types. The extracellular products of these
non-notochordal tissues could, in turn, promote the further production of notochordal
secretions.
ACKNOWLEDGMENTS
We gratefully acknowledge the excellent
technical assistance of Rosalyn Upson and
Jerrolynn Campbell in this project, Ms.
Campbell also typed the manuscript.
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PLATES
Abbreviations
bl, basal lamina
ECS, extracellular space
IB, interstitial body
ICS, intercellular space
IG, interstitial granule
Id, lipid droplet
N, notochord
NT, neural tube
nuc, nucleus
m, mitochondrion
rer, rough endoplasmic reticulum
TSP, connective tissue space
PLATE
EXPLANATION OF FIGURES
1 Light micrograph of cross-section through stage 14 chick embryo.
The cylindrical notochord ( N ) is located in the connective tissue
space (TSP) ventral to the neural tube ( N T ) . Numerous intracellular
spherical vacuoles are present. At this stage and cephalocaudal position, the perinotochordal space is cell-free. Toluidine blue. X 600.
160
2
Fine structure of chick notochorda1 surface a t stage 14. A continuous
basal lamina ( b l ) surrounds the entire notochord while 100 A to
200 A microfibrils (arrows) are intertwined in the adjacent connective tissue space (TSP). These extracellular materials comprise the
notochord sheath. (ICS) intercellular space. x 35,400.
3
Light micrograph of cross-section through stage 14 isolated trypsinized notochord ( N ) . The surface which is typically smooth exhibits
numerous cytoplasmic blebs. These blebs are not uncommon in epithelial tissues from which basal lamina h a s been removed. Intracellular vacuoles and dilated intercellular spaces are infrequently observed.
Toluidine blue. x 800.
4
Electron micrograph of chick notochord surface similar to that shown
in figure 3. There is a striking absence of basal lamina and other
components of the in vivo notochord sheath in the extracellular
space (ECS). Cellular organelles are morphologically unaltered. Id,
lipid droplet; nuc, nucleus; m, mitochondrion. X 11,600.
PROLINE-CONTAINING FIBRILS
Cristina K. Lauscher and Edward C. Carlson
PLATE 1
PLATE 2
EXPLANATION OF FIGURES
5 Light micrograph of a cross-section through notochordal tissue incubated 12 hours on Falcon plastic. Circumferential cells begin to
loosen from the central cell cluster and adhere to the culture dish.
The growth area loses its cylindrical shape and assumes a nodular
appearance. Toluidine blue. X 350.
6
Notochordal cell surface following 12 hours of in vitro incubation.
Ultrastructurally, a finely filamentous, interrupted basal lamina
(arrow) is adjacent to the cell surface. The flocculent precipitate i n
the extracellular space (ECS ) represents proteinaceous material
derived from the culture medium. m, mitochondrion; rer, rough endoplasmic reticulum. x 26,600.
7 Notochordal tissue cultured 24 hours in vitro shows peripheral migration of cells along the surface of the culture dish. At this culture
time, intracellular vacuoles (arrow) are present. Light micrograph,
Toluidine blue. x 800.
8
Electron micrograph of notochordal cell in tissue similar to that
seen in figure 7. There is a proliferation of basal lamina material
(bl), small 100 A microfibrils (arrows), and amorphous materials in
the extracellular space (ECS). nuc, nucleus. x 22,500.
9
Light micrograph showing that the bulk of the notochordal growth
area flattens outward following 36 hours of in vitro incubation. Toluidine blue. x 240.
10 At 36 hours of i n vitro incubation increased numbers of microfibrils
(arrows) blend imperceptibly with basal lamina material a t the notochordal cell surface. ECS, extracellular space, electron micrograph.
x 30,000.
162
PROLINE-CONTAINING FIBRILS
Cristina K. Lauscher and Edward C. Carlson
PLATE 2
163
PLATE 3
EXPLANATION OF FIGURES
11
At 48 hours of i n vitro incubation, cultured notochordal cells spread
outward on the plastic substratum and form a flattened nodular
growth area 0.5-1.0 m m in diameter. Peripheral cells taper off to
form a monolayer. Light micrograph, Toluidine blue. x 215.
12 Electron micrograph of notochordal cell and adjacent extracellular
space (ECS) a t 48 hours of in vitro incubation. A thickened continuous basal lamina ( b l ) is frequently seen surrounding contiguous
notochordal cells. Large 200 A microfibrils (arrows) show faint
irregular striations and are interspersed with smaller fibrils and
amorphous materials similar to interstitial bodies ( I B ) . The partially
membrane bound dense body (IG) resembles a n interstitial granule.
These extracellular materials closely resemble the components of the
in vivo notochord sheath (fig. 2). x 57,400.
13 Higher magnification of large microfibrils from extracellular space
of notochordal cells incubated 48 hours in vitro. Microfibrils average
240 A i n diameter and occasionally exhibit a faint irregular periodicity the macroperiods of which average approximately 450 A (arrows).
x 91,000.
164
PROLINE-CONTAINING FIBRILS
Cristina K. Lauscher and Edward C. Carlson
PLATE 3
165
PLATE 4
EXPLANATION O F FIGURES
34
Light microscopic autoradiograph of notochordal tissue treated with
3H-proline and incubated 48 hours i n vitro. Silver grains are located
over cytoplasm, nuclei and extracellular space. (ECS). Toulidine
blue. x 1,200.
15
Electron microscopic autoradiograph of notochordal cell surface from
tissue similar to that shown in figure 14. Basal lamina, ( b l ) microfibrils (arrows), and other components of the extracellular matrix
are associated with tritium label. Intracellularly, silver grains are
present over rough endoplasmic reticulum polyribosomes and other
cellular organelles. IB, interstitial body; ECS, extracellular space.
X 28,500.
16 Higher magnification of extracellular matrix produced by notochordal
cells at 48 hours of in vitro incubation. Tritium labeled silver grains
are noted over fragmentary basal lamina material ( b l ) and numerous
microfibrils (arrows) cut in various planes of section. x 73,000.
17 Light microscopic autoradiograph of notochordal tissues cultured 48
hours in the presence of 3H-proline and cis-hydroxyproline. Intracellular label is diminished with respect to untreated control tissues (fig.
14). Moreover, the extracellular to cellular ratio of silver grains is
markedly diminished. ECS, extracellular space. Toluidine blue.
x 1,200
18 Electron microscopic autoradiograph of cis-hydroxyproline treated
notochordal cells from tissue similar to that seen in figure 17. The
majority of silver grains are limited to the cytoplasm and nuclei.
Areas of active fibrillogenesis are infrequently observed. x 5,250.
166
PROLINE-CONTAINING FIBRILS
Cristina K. Lauscher and Edward C. CarIson
PLATE 4
167
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