/. Embryol. exp. Morph. 94, 95-112 (1986)
95
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The distribution of fibronectin, laminin and entactin in
the neurulating rat embryo studied by indirect
immunofluorescence
FIONA TUCKETT AND GILLIAN M. MORRISS-KAY
Department of Human Anatomy, University of Oxford, South Parks Road, Oxford
OX1 3QX, UK
SUMMARY
This paper forms part of our study of the extracellular matrix and its role in the morphogenesis
of the brain during the period of neurulation in the rat embryo. Using indirect immunofluorescence with polyclonal antibodies, we present here a descriptive study of the distribution
of the matrix glycoproteins fibronectin, laminin and entactin.
The observed distribution of the fibronectin matrix implicates it in providing a structural element in several morphologically active sites; in addition our observations support the previously
suggested involvement of fibronectin in the migration of neural crest cells. Entactin was present
only in the basement membranes in conjunction with laminin which was not itself confined to
these regions. Laminin was also identified within the mesenchymal extracellular matrix, and its
general distribution confirms the previously documented role of laminin in maintaining epithelial structure and organization. No patterning in the distribution of these three glycoproteins
could be correlated with the change in shape of the neural epithelium associated with either tube
formation or neuromere morphogenesis.
INTRODUCTION
Extracellular materials play important roles in epithelial morphogenesis and cell
migration, as demonstrated for example by studies on salivary gland morphogenesis (Bernfield, Banerjee, Koda & Rapraeger, 1984) and avian neural crest cell
migration (Newgreen & Thiery, 1980). We have previously investigated the
distribution and morphogenetic functions of hyaluronate and proteoglycans in
neurulation, neural crest cell migration and neuromere formation in the cranial
region of rat embryos. Hyaluronate is essential for the creation of extracellular
spaces around mesenchyme cells, and is particularly localized in the mesenchyme
subjacent to the neural epithelium (Solursh & Morriss, 1977; Morriss & Solursh,
1978a,6). Recent results (Morriss-Kay, Tuckett & Solursh, 1986) suggest that its
major role is related to the control of mesenchymal cell number rather than cell
migration or neuroepithelial morphogenesis. Proteoglycans (particularly chondroitin sulphate proteoglycans) in the neuroepithelial basement membrane play
an essential role in the thickening and curvature of this epithelium during
Key words:fibronectin,laminin, entactin, neural tube, heart, neural crest, extracellular matrix.
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F. TUCKETT AND G. M. MORRISS-KAY
neurulation (Morriss-Kay & Crutch, 1982). Glycosaminoglycans do not appear to
be involved in the development of the cranial neuromeres (Tuckett, 1984).
We now extend these studies of the extracellular matrix to include fibronectin,
laminin and entactin. Fibronectin is a glycoprotein of the extracellular matrix with
the ability to form meshworks; neural crest cells preferentially adhere to it in vitro
(Greenberg, Seppa & Hewitt, 1981); it promotes cell migration, and binds to
collagen, hyaluronate and proteoglycans (Yamada, Hayashi & Akiyama, 1982).
Laminin is a glycoprotein of the basal lamina (Timpl etal. 1979). It mediates
the binding of epithelial cells to type IV procollagen in the basal lamina and
is probably essential for the maintenance of epithelial structure (Terranova,
Rohrbach & Martin, 1980). Entactin is a sulphated glycoprotein; it is mainly found
in basement membranes but at times also contributes to the extracellular matrix of
mesenchymal tissues (Hogan, Taylor, Kurkinen & Couchman, 1982). We describe
here the distribution of these three molecules in the cranial region and heart of rat
embryos during and immediately after the period of cranial neurulation, as
revealed by indirect immunofluorescence. The primary aim was to discover any
correlations between the distribution of these substances and morphogenetic
events which occur within these regions, at this period of embryogenesis. Previous
mammalian studies have been concerned with the stages immediately prior or
subsequent to this period (Leivo, Vaheri, Timpl & Wartiovaara, 1980; Wan,
Tsung-Chieh, Chung & Damjanov, 1984; Sternberg & Kimber, 1986).
MATERIALS AND METHODS
Preparation of specimens
Wistar strain rat embryos were explanted in phosphate-buffered saline (PBS) on either day 9
or day 10 of pregnancy (day of positive vaginal smear = day 0). The extraembryonic membranes
were removed from day-10 embryos; day-9 embryos were left intact within their membranes
(4- to 5-somite stage). The number of somites present was counted before fixation.
The embryos were fixed in modified St Marie's fixative (Sainte-Marie, 1962) according to the
method described by Icardo & Manasek (1983, 1984); the fixation period was either 1-2 h at
4°C, or overnight at -20°C. The embryos were dehydrated at 4°C in two changes of absolute
ethanol followed by two changes of xylene (10 min each change) and allowed to reach room
temperature before embedding in paraffin wax.
The embedded embryos were stored at 4°C until they were sectioned. Serial sections of 10 /jm
thickness were mounted on glass slides and stored at 4°C until they were required for
immunohistochemistry.
Labelling of sections
The sections were hydrated according to the method described by Icardo & Manasek (1983,
1984). The sections were incubated with primary antibody at a 1:50 dilution in PBS. Control
sections were incubated with either preimmune serum or PBS instead of the primary antibody
(no difference was observed in the backgroundfluorescencebetween these two solutions). After
several washes in PBS, the sections were incubated with the secondary antibody at a 1:50
dilution in PBS; the sections were thoroughly washed in PBS before mounting in u.v.-inert
aqueous mountant (Gurr). Both of the incubations were performed in a humid atmosphere, at
Distribution of three glycoproteins in the rat embryo
97
38°C, for 30min. Some sections were double-labelled, i.e. the primary and secondary antibody
incubations were repeated using a second set of non-crossreactive antibodies. The primary
antibodies were: goat anti-rat fibronectin (CP Laboratories); rabbit anti-mouse laminin
(Bethesda Research Laboratories); and rabbit anti-mouse entactin (gift from Brigid Hogan,
NIMR, London). The secondary antibodies used were: FITC-conjugated rabbit anti-goat IgG
(Miles or Sigma); rhodamine-conjugated rabbit anti-goat IgG (Miles); FITC-conjugated goat
anti-rabbit IgG (Miles or Sigma).
The specificity of the two commercially obtained primary antibodies (anti-fibronectin and
anti-laminin) was ascertained by preabsorption with fibronectin (Sigma) or laminin (Bethesda
Research Laboratories) (Fig. 1); in addition antibody specificity was determined by immunoblotting and the purity of the antigens was ascertained by gel electrophoresis (not illustrated
here).
The slides were stored in black plastic bags at 4°C. Sections were viewed with an Olympus
BH2 microscope fitted with a reflected light fluorescence attachment; Fujichrome ASA 400
positivefilmwas used to photograph the sections for reference, and Ilford HP5filmwas used for
publication photographs.
RESULTS
Neural epithelium
Throughout the period of cranial neurulation, the neuroepithelial cells did not
stain for fibronectin, laminin or entactin. A fibronectin-rich and laminin-rich
basement membrane were observed at all stages of development and at all levels
within the embryo. Entactin basement membrane staining was very much weaker:
at the earliest stage studied (4-somite stage), entactin fluorescence was confined
to the forebrain and midbrain regions; however, by the 8-somite stage the
fluorescence had progressed more caudally into the hindbrain, although this was
very much weaker than the staining seen in the 4-somite stage embryos and was
also of a patchy nature. By the 12-somite stage, the entactin basement membrane
staining had disappeared at some forebrain and hindbrain levels, the greatest
fluorescence (although very weak compared with fibronectin and laminin fluorescence) being observed within the midbrain. In embryos with 15-16 somites there
was no epithelial basement membrane staining with entactin antibodies.
With the development of the optic sulci within the expanding forebrain, a
regional difference in the staining of fibronectin and the other two glycoproteins
was observed. The optic sulci bulge outwards into the cranial mesenchyme and
around the bulge, the basement membrane was deficient in both laminin and
entactin fluorescence but stained strongly for fibronectin. Basement membrane
fluorescence for laminin and entactin remained around the neural groove region
and in the region of future forebrain neural fold apposition. This patterning of
glycoprotein distribution around the optic sulci persisted throughout the period of
cranial neurulation and was not dependent on the plane of sectioning (Fig. 2).
Changes in fibronectin staining intensity were also observed at different locations within the neuroepithelial basement membrane. At certain levels within
the midbrain and hindbrain, the lateral margins of the neural epithelium were
deficient in fibronectin fluorescence and this was associated with the presence of
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F . T U C K E T T AND G. M.
MORRISS-KAY
Fig. 1. Coronal sections along the neural tube of 12-somite-stage embryos illustrating
the fluorescence obtained with antisera previously preabsorbed with either fibronectin
or laminin. Bar, 50 fan. (A) Anti-laminin preabsorbed with laminin; no non-specific
fluorescence. (B) Anti-fibronectin preabsorbed with fibronectin; no non-specific
fluorescence. (C) Anti-laminin preabsorbed with fibronectin. FITC-labelled antilaminin fluorescence of the mesenchymal extracellular matrix is punctate whilst within
the neuromere basement the fluorescence is continuous. (D) Anti-fibronectin preabsorbed with laminin. FITC-labelled anti-fibronectin fluorescence within the
mesenchymal extracellular matrix is generally fibrillar; around the neuromeres the
basement membrane fluorescence is continuous.
Fig. 2. Transverse sections through the forebrain/midbrain region of a 10-somitestage embryo (A,B) and a 12-somite-stage embryo (C,D). Bar, 100jum. (A) FITClabelled anti-laminin. Staining is discontinuous around one optic sulcus and is not
present around the other. (B) Rhodamine-labelled anti-fibronectin. (C) FITC-labelled
anti-laminin. Neuroepithelial basement membrane stains intensely at the lateral
margin in the region of the forebrain neuropore and around the neural groove; towards
the evaginating optic sulcus this becomes thinned and discontinuous (arrows). (D)
Rhodamine-labelled anti-fibronectin. Basement membrane staining remains continuous around the optic sulcus. Laterally the mesenchymal matrix fluorescence is punctate, whilst medially around the notochord (n) the fluorescence is more fibrillar in
form.
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F. TUCKETT AND G. M. MORRISS-KAY
neural crest cells in the immediately adjacent mesenchyme (Fig. 3A,C). After
emigration of the neural crest cells to more distal locations, the fibronectin
fluorescence was restored at the lateral margins. In several sections anti-laminin
antibodies demonstrated a similar deficiency in the basement membrane associated with the emergence and emigration of neural crest cells (Fig. 3B). With the
onset of forebrain apposition, the basement membrane of the apposing neural
folds was found to fluoresce more strongly for fibronectin than at earlier stages of
development and compared with other areas of the forebrain; this increase in
fluorescence became even more obvious by the 12-somite stage of development
and persisted after apposition had occurred (Fig. 4).
In coronal sections stained with fibronectin and laminin antibodies, there was no
discontinuity or variation in intensity of fluorescence either within the forebrain,
or more caudally surrounding the neuromeres (Fig. 1C,D).
Surface ectoderm
The surface ectodermal cells did not stain with antibodies to the three glycoproteins but there was a glycoprotein-rich basement membrane. Fibronectin was
present in the basement membrane at all levels within the embryo and at all stages
of development, and the fluorescence was generally strong. Laminin was also
present within the basement membrane although there was a variation in staining
intensity apparent at the later stages of development: at the level of the pharyngeal
arches the fluorescence was patchy and in some sections disappeared altogether.
Entactin fluorescence of the basement membrane was very faint, only just above
the background level and was only discernible in a very few sections in earlysomite-stage embryos. By the 12-somite stage, more entactin was detectable but
this was generally confined to the rostral-most levels of the embryo.
The basement membrane fluorescence was continuous with the basement membrane at the lateral margins of the neural folds, except where neural crest cells
were emerging from the epithelium; at the level of the optic pits, the basement
membranes of surface ectoderm and neural epithelium apparently abutted,
resulting in a localized increase in intensity.
Foregut endoderm
With fibronectin and laminin antibodies some of the strongest fluorescence was
observed in the basement membrane of the gut endoderm; the endodermal cells
themselves did not stain for any of the glycoproteins.The fluorescence for fibronectin and laminin was greatest in the basement membrane of the ventral wall of
the foregut. This fluorescence was continuous with the fluorescence associated
with the dorsal mesocardium; the basement membrane and dorsal mesocardium
were connected by a fibrous reticulum. The intense foregut fluorescence for
fibronectin and laminin persisted throughout the period of cranial neurulation.
Entactin staining in the foregut basement membrane was very weak and at the 4somite stage was confined to a few sections. At later stages more of the foregut was
Distribution of three glycoproteins in the rat embryo
101
entactin-positive, the weak fluorescence being located ventrally, but without a
reticular connection with the adjacent dorsal.mesocardium.
Mesenchymal extracellular matrix and somites
Entactin was never observed within the mesenchymal extracellular matrix. The
distribution and intensity of stain for laminin and fibronectin varied at different
levels of the embryo and at different stages of development.
At the 4-somite stage, there was a punctate staining of the extracellular matrix
with both fibronectin and laminin antibodies, which decreased in a rostrocaudal
direction. The staining obtained for laminin was much less intense than that for
fibronectin and at caudal levels there was no laminin fluorescence. Using fibronectin antibodies it was found that the somatopleuric mesodermal layer was rich in
fibronectin whilst the splanchnopleuric layer was deficient in fibronectin, as were
the more medial intermediate and paraxial mesoderm. By the 8-somite stage of
development, both fibronectin and laminin fluoresced more intensely. At the
forebrain level there was a mesh of fibronectin-rich fibres; this was also found at
more caudal levels associated with the primary mesenchyme of the midbrain and
hindbrain regions; around the notochord the density of the fibres increased to
form a reticulum which connected the notochord with the neural groove (Fig. 5B),
and in later stages extended laterally to surround the dorsal aortae. There was
no fibrillar fluorescence of the neural crest mesenchyme, the punctate staining
persisted in this region. Within the first pharyngeal arch the mesenchymal extracellular matrix displayed punctate fluorescence at the 8-somite stage but by the
10-somite stage this was beginning to be infiltrated by strands of fluorescence,
indicative of the development of a fibrous meshwork within this region.
Antibodies to laminin stained in a punctate manner throughout the period
studied. The intensity of fluorescence increased at the later stages examined,
although there continued to be a rostrocaudal decrease in the staining intensity.
At somitic levels, the somite cells showed no fluorescence for either fibronectin
or laminin but were surrounded by a fibronectin and laminin-rich basement
membrane. In the 4-somite-stage embryos, the more lateral unsegmented mesoderm was fibronectin- and laminin-free although a reticulum of fibronectin fluorescence was seen spreading inwards from the overlying ectoderm and underlying
endoderm. Associated with the increase in staining at somitic levels at later stages
of development, was the appearance of a more widespread fibronectin- and
laminin-rich extracellular matrix. There was no fibrillar material spreading inwards from the ectoderm and endoderm basement membranes in the 8-somitestage embryos or older, although fibronectin fluorescence within the mesenchymal
extracellular matrix was fibrillar. In a 12-somite-stage embryo where the first
somites were beginning to disperse (i.e. sclerotome cells had migrated), fluorescence with laminin antibodies was noticeable on the developing dermamyotome
cells (Fig. 5A), suggesting that a laminin-rich basement membrane was developing
between the dermatome and myotome.
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Distribution of three glycoproteins in the rat embryo
103
Extraembryonic membranes
The extraembryonic membranes were included with the embryos only at the 4somite stage. The amnion and extraembryonic mesoderm stained strongly for
fibronectin, weakly for laminin and were negative for entactin. In contrast, the
extraembryonic endoderm fluoresced strongly for both laminin and entactin but
did not stain for fibronectin.
Heart
Initially at the 4-somite stage when the heart consists of a simple longitudinal
tube, fibronectin was the main glycoprotein to fluoresce although there was some
weak laminin fluorescence associated with the dorsal mesocardium. The dorsal
Fig. 4. Neural epithelium at the anterior neuropore (10-somite stage). At the lateralmost part of the neuroepithelial basement membrane there is an increase in fibronectin
fluorescence. Rhodamine-labelled anti-fibronectin. Bar, 50/im.
Fig. 3. Transverse section of a 12-somite-stage embryo cut immediately anterior to the
optic pits. At the site of emergence of the neural crest cells, at the lateral margin of the
neural epithelium, basement membrane fluorescence is discontinuous. Bar, 50jum.
(A) Rhodamine-labelled anti-fibronectin. (B) FITC-labelled anti-laminin. (C) Rhodamine-labelled anti-fibronectin. High-power view showing the strands of fibronectin
fluorescence which remain at the site of crest cell emergence. Neural crest cells
(arrowed) are not stained.
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F. TUCKETT AND G. M. MORRISS-KAY
mesocardium and the endocardium fluoresced with fibronectin antibodies. There
was no fluorescence of the splanchnopleuric mesoderm. With further development
of the embryo, the heart tube forms a loop within the pericardial cavity. The
endocardium and pericardium stained strongly for fibronectin and laminin, and
only weakly for entactin. Even after the breakdown of the dorsal mesocardium, a
strong fluorescence to all three glycoproteins persisted in the region between the
Fig. 5. (A) FITC-labelled anti-laminin. Transverse section at the level of the first
somite (12-somite stage). Sclerotome cells have migrated away from the somite leaving
the dermamyotome; fluorescence can be seen in the region of the dermatome
basement membrane. Bar, 50 ^m. (B) FITC-labelled anti-fibronectin. Section through
the midbrain (12-somite stage). Fluorescence around the notochord (n), neural groove
(g), foregut (/) and dorsal aortae (a) is morefibrillarthan in more lateral regions. Bar,
Distribution of three glycoproteins in the rat embryo
ventral gut endoderm and the heart tube. Between the splanchnopleuric mesoderm of the pericardial cavity (the myoepicardial mantle) and the endocardium, a
cell-free space was traversed by fibrils of fluorescence. The fibrils of fibronectin
and laminin were more numerous, longer and fluoresced more strongly in the
truncus and rostral end of the bulbus cordis (Fig. 6A,B); entactin antigenicity
also developed with time within this reticulum; at the proximal end of the bulbus
cordis (right ventricle) and the left ventricle there was only a very narrow cell-free
space with a few fibrillar strands traversing it. At the most advanced stage of
development studied here (15- to 16-somite stage), generally the fluorescence
within the reticulum was weaker although all three glycoproteins were expressed;
the fibronectin fibrils fluoresced much less strongly in these later-stage embryos.
Within the developing myocardium there was punctate fibronectin fluorescence
and fibrils of laminin fluorescence, laminin being the stronger. Within the walls of
the developing ventricles, myocardial trabeculation was associated with an initial
increase in fluorescence of both laminin and fibronectin,. and the onset of entactin
antigenicity within the myocardium. A peak in fibronectin fluorescence associated
with myocardial trabeculation was observed around the 12-somite stage (Fig. 6C),
being weaker here at the 15- to 16-somite-stage.
DISCUSSION
We have described here the distribution of three glycoproteins of the cell surface
and extracellular matrix (fibronectin, laminin and entactin) in rat embryos during
the period of cranial neurulation, as revealed by indirect immunofluorescence.
The results are summarized in Tables 1-3.
The fixation and embedding protocol was based on the method described by
Icardo & Manasek (1983, 1984). Duband & Thiery (1982) found that SainteMarie's fixative and paraffin embedding, both of which were used in this study,
decreased the antigenicity of fibronectin. We obtained a very strong fluorescence
for both fibronectin and laminin, suggesting that the antigenicity of both was good
in our sections. The fluorescence obtained with entactin antibodies was in general
weaker, and was confined to basement membranes. This may reflect a masking of
antigenic determinants but we do not believe this to be true; evidence for the
validity of our results for entactin comes from the very bright fluorescence which
was observed in the parietal endoderm. Reichert's membrane, the thick basement
membrane of the parietal endoderm, is known from other studies to contain
entactin (for references see Hogan, Barlow & Kurkinen, 1984).
We found laminin to be a component of almost all basement membranes,
whereas entactin was not always present. Entactin was never found in the absence
of laminin. These observations are consistent with evidence that entactin forms a
stable non-covalent complex with laminin (Carlin, Jaffe, Bender & Chang, 1981;
Hogan et al. 1982). Fibronectin was present in all basement membranes at all
stages.
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F . T U C K E T T AND G. M.
MORRISS-KAY
Fig. 6. Transverse sections through the heart of a 12-somite-stage embryo: (A,B) at
the level of the truncus and bulbus cordis; (C) at the level of the ventricular
trabeculated myocardium, g, foregut; a, dorsal aorta; b, bulbus cordis; t, truncus. Bar,
100 jum. (A) FITC-labelled anti-laminin. (B) Rhodamine-labelled anti-fibronectin.
Note the brighter fluorescence within the reticulum compared with (A). (C) FITClabelled anti-fibronectin. Within the trabeculated myocardium there is an increase in
fibronectin fluorescence which reaches a peak in intensity at this stage of development.
Compare with (B) which is of a more rostral level and displays much less myocardial
fluorescence.
Distribution of three glycoproteins in the rat embryo
107
In association with formation and further development of the optic sulcus,
basement membrane immunoreactivity was incomplete. Elsewhere in the neural
epithelium all three glycoproteins were present, but at the two optic sulci both
Table 1. Summary of the general distribution of fibronectin
Somite stage
8
12
16
Basement membranes:
neural epithelium
surface epithelium
foregut endoderm
Extracellular matrix:
forebrain level
midbrain level
hindbrain level
somitic level
Heart:
dorsal mesocardium
endocardium
myocardium
reticulum
Extraembryonic
membranes:
amnion
mesoderm
endoderm
Reichert's membrane
Table 2. Summary of the general distribution oflaminin
Somite stage
8
Basement membranes:
neural epithelium
surface epithelium
foregut endoderm
Extracellular matrix:
forebrain level
midbrain level
hindbrain level
somitic level
Heart:
dorsal mesocardium
endocardium
myocardium
reticulum
Extraembryonic
membranes:
amnion
mesoderm
endoderm
Reichert's membrane
12
16
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F. TUCKETT AND G. M. MORRISS-KAY
laminin and entactin were missing. Basement membrane modification may therefore be implicated in formation and, or, maintenance of the sharp angle of the
sulcus. Morphologically, this bend in the neural epithelium differs from neuroepithelial curvature associated with neural tube formation in that the cells are
clearly wedge-shaped with very broad basal surfaces. Cytochalasin D had no effect
on optic sulcus shape suggesting that this structure is not microfilament-dependent
(Morriss-Kay & Tuckett, 1985). Basement membrane modification has also been
observed in relation to cleft formation during branching of the mammary and
salivary glands (Bernfield, Banerjee, Koda & Rapraeger, 1984): type IV collagen
and chondroitin sulphate proteoglycan were lost, while laminin and other components remained. Laminin is considered to be essential for normal epithelial
structure and organization (Terranova etal. 1980), whilst type IV collagen may
also be essential since it is present in all basement membranes (Timpl & Martin,
1982). It may be that the loss of one or other of them enables bending to occur by
modifying the relationship between the basal epithelial surface and the basement
membrane.
We were unable to identify any variation in the distribution of fibronectin,
laminin or entactin in relation to neuromere formation. Similarly, we have been
unable to correlate the distribution of glycosaminoglycans (using alcian blue
histochemistry) with neuromeres (Tuckett, 1984). It seems likely by analogy with
other systems that a tissue interaction is involved; however, either this occurs at an
early stage, determining the neuromeric pattern well in advance of neuromere
Table 3. Summary of the general distribution of entactin
Somite stage
8
Basement membranes:
neural epithelium
surface epithelium
foregut endoderm
Extracellular matrix:
forebrain level
midbrain level
hindbrain level
somitic level
Heart:
dorsal mesocardium
endocardium
myocardium
reticulum
Extraembryonic
membranes:
amnion
mesoderm
endoderm
Reichert's membrane
±
-
+
+
-
-
-
-
±
±
±
—
—
++
++
12
16
Distribution of three glycoproteins in the rat embryo
morphogenesis, or the molecules involved in the extracellular component of the
interaction have yet to be identified.
Coinciding with the onset of neural crest cell migration from the midbrain and
hindbrain regions, basement membrane fluorescence was lost from the lateral
neural epithelium. Once the crest cells had migrated away, basement membrane
fluorescence was restored. This sequence of loss and restoration of the basement
membrane supports the view that neural crest cell emigration is quantal, as
suggested by fibronectin studies in the chick (Newgreen & Thiery, 1980) and by
scanning electron microscopy in the rat embryo (Tan & Morriss-Kay, 1985). In the
chick embryo, Newgreen & Thiery (1980) observed differences in neural-crestrelated fibronectin distribution at different axial levels during crest cell migration:
pioneer crest cells at cranial and sacral levels synthesized fibronectin while crest
cells from cervical to lumbar axial levels did not. We also observed differences
in neural-crest-associated fibronectin at different axial levels within the cranial
region: rostral to the somites there was punctate fluorescence between the neural
crest cells, suggesting that they secrete fibronectin; there was no pattern of
fluorescence which could be correlated with crest cell migration pathways at
somitic levels, suggesting that neural crest cells do not secrete fibronectin at more
caudal levels.
Of the three matrix glycoproteins we have studied here fibronectin is the one
which is generally associated with cell migration during embryonic development.
However recent evidence has led to the hypothesis that fibronectin and laminin
may play reciprocal roles in controlling cell movement during the development of
the peripheral and central nervous systems respectively. There are three lines of
evidence to support this hypothesis: (i) fibronectin and not laminin has been
identified along neural crest cell migration pathways prior to the differentiation of
the peripheral nervous system (reviewed by Le Douarin, 1984); (ii) in vitro it has
been demonstrated that both central and peripheral neurones extend on a lamininbound substratum, whereas only peripheral neurones extend on a fibronectinbound substratum (Rogers etal. 1983); (iii) in vivo, Leisi (1985) found transient
expression of laminin immunoreactivity within the neural epithelium which
coincided with periods of neuronal migration; the earliest stage at which laminin
was described occurred at a similar stage of development to the one studied here.
However the findings of Leisi are in direct conflict with our observations and of
others (Bignami, Chi & Dahl, 1984; Wan etal. 1984; Le Douarin, 1984): laminin
immunoreactivity was confined to the basement membranes and the mesenchymal
extracellular matrix; thus we conclude that during the period of cranial neurulation, laminin has no specific morphogenetic role.
Apposition of the lateral edges of the neural epithelium to form a closed
tube was associated with an increase in basement membrane fibronectin fluorescence in the forebrain region but not elsewhere. Closure of the anterior neuropore occurs separately from closure of the midbrain/hindbrain neuropore, and
fusion of the apposing epithelia is not an immediate consequence of apposition
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F. TUCKETT AND G. M. MORRISS-KAY
(Morriss & New, 1979 and unpublished observations). Adhesion of the apposing
apical surfaces may set up tensions requiring that the adhering epithelia are
strongly bonded to their basement membrane, and through it to the underlying
mesenchyme. An increase in the fibronectin content of the basement membrane
could bring this about. Alternatively (or in addition) the increase in fibronectin may be related to the increase in cell-cell contacts (Chen etal. 1978;
Furcht, Mosher & Wendelschafer-Crabb, 1978) or to changes in cell alignment
and position (Yamada, Olden & Pastan, 1978; Zetter, Martin, Birdwell &
Gospodarowicz, 1978) which occur during the fusion process.
Around the notochord a fibronectin-rich reticulum stretched upwards to the
neural groove basement membrane and laterally towards the cranial mesenchyme
or somites. The mammalian notochord is a flimsy structure compared with that
of avian and amphibian embryos (Morriss-Kay, 1981); this reticulum may be
important in communication between the notochord and adjacent cells, e.g for
maintenance of neural groove shape, or it may provide extra structural support. In
addition it is suitably placed for playing a role in the guidance of sclerotome cells in
their migration towards the notochord from the somites, as previously described
in amphibian embryos (Lofberg etal. 1978), and providing a substrate for their
proliferation.
In the heart, the distribution of fibronectin was similar to that reported by
Icardo & Manasek (1983) in chick embryos. The extracellular matrix (cardiac
jelly) of the reticulum has also been shown to be rich in glycosaminoglycans and
collagen, whose temporal and spatial pattern of development suggests that they
are synthesized and secreted by the endocardium (Markwald & Adams-Smith,
1972; Manasek etal. 1973; Johnson, Manasek, Vinson & Seyer, 1974; Markwald,
Fitzharris & Adams-Smith, 1975; Manasek, 1976). Our observations of a generally
low level of myocardial but high endocardial and reticular fluorescence suggest
that the same is true for fibronectin, laminin and entactin in the mammalian heart.
The fibrillar pattern of laminin fluorescence in the myocardium at the latest stages
examined probably indicates the onset of development of a laminin-rich basement
membrane secreted by and enclosing the myocardial cells. The pattern of fluorescence in the dorsal mesocardium at early stages suggest that all three glycoproteins are involved in maintenance and change of the position of the heart in
relation to the foregut until the dorsal mesocardium breaks down.
The results of this study are consistent with previous reports on the distribution
of fibronectin suggesting roles in neural crest cell migration and as a supportive
element in the reticulum of the heart. In addition they suggest that it has a
supportive role where it forms a reticulum around the notochord, and a role in
epithelial fusion during closure of the anterior neuropore. The role of entactin is
not clear; its appearance and disappearance from different epithelial basement
membranes was not obviously related to any consistent morphogenetic change;
the only exception to this was the disappearance of both laminin and entactin from
the optic sulcus basement membrane. Laminin was present in all other basement
membranes, confirming its essential role in epithelial structure and organization.
Distribution of three glycoproteins in the rat embryo
111
We wish to thank Martin Barker for technical assistance and Tony Barclay for photographic
assistance; Brigid Hogan for the entactin antibody; Penny Thomas for the gel electrophoresis
and immunoblotting; and the MRC for financial support.
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(Accepted 20 December 1985)