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Immunolocalization of basal lamina components during development of chick otic and optic primordia.

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THE ANATOMICAL RECORD 235:443-452 (1993)
lmmunolocalization of Basal Lamina Components During
Development of Chick Otic and Optic Primordia
S. ROBERT HILFER AND GWENDALYN J. RANDOLPH
Department of Biology, Temple University, Philadelphia, Pennsylvania 19122
ABSTRACT
Immunolocalization of laminin, fibronectin, and type IV
collagen was examined during early morphogenetic shape changes of the
avian inner ear and eye. The ear was studied from formation of the otic
placode to invagination of the otic pit and the eye from the optic vesicle
stage to formation of an optic cup. Distribution and intensity of immunoreactivity were compared in the two organ primordia and in adjacent epithelial layers. Laminin formed a continuous layer at the basal surface of the
otic ectoderm and adjacent neural tube at all stages. The basal surfaces of
the optic and lens epithelia also were continuously covered with laminin
throughout development. The otic placode became attached to the neural
ectoderm through a single layer of fibronectin and collagen IV between the
layers of laminin. The ring-like attachment between the edges of the optic
cup and lens primordium had the same structure. In addition, the central
regions of the optic and lens primordia were attached by fibrils containing
type IV collagen, whereas finer strands containing fibronectin and laminin
also connected the otic epithelium and neural tube. The results are discussed in terms of models of invagination for the two primordia.
0 1993 Wiley-Liss, Inc.
Key words: Otic placode, Optic vesicle, Organogenesis, Laminin, Fibronectin, Type IV collagen
Invagination of epithelial sheets to form pit-like
structures occurs during organogenesis of many embryonic primordia. The mechanisms that control invagination have been identified as comprising both intracellular and extracellular forces. Much attention
has been paid to the role of the cytoskeleton in causing
bending of epithelial sheets (reviewed in Hilfer and
Searls, 1986; Trinkaus, 1984). An extensive literature
also exists on the involvement of extracellular matrix
in organ formation. For example, interference with collagen, proteoglycan, and glycoprotein synthesis inhibits branching of salivary, lung, and kidney primordia
(Klein et al., 1989; Nakanishi et al., 1986; Spooner and
Faubion, 1980; Spooner et al., 1985; Thompson and
Spooner, 1983) and invagination of optic (Yang and
Hilfer, 1982; Gerchman et al., 1991) and otic (Rausch
and Hilfer, 1988; Gerchman, et al., 1991) primordia.
The involvement of extracellular macromolecules in
organotypic shape changes appears to be through their
linkage with the cell surface. Integral membrane proteins, such as integrins (reviewed in Ginsberg et al.,
1990) and the heparan sulfate proteoglycan syndecan
(Hayashi et al., 19871, act as receptors principally for
N-linked glycoproteins, including fibronectin and laminin but also for collagen, the major structural component of the basal lamina. Epithelial links t o the basal
lamina and extended extracellular matrix may initiate
shape changes or stabilize them once they have occurred. If such molecules have a role in the process of
shape change, differences most likely exist in their
0 1993 WILEY-LISS. INC.
presence, distribution, concentration, or molecular
form a t critical developmental stages.
In this study, the distributions of three major components of basal laminae are compared in primordia of
the eye and inner ear. Since invagination of these organ primordia is controlled differently, it was expected
that differences would exist in the temporal compositions of their basal laminae. In both primordia, invagination is inhibited by tunicamycin (Yang and Hilfer,
1982; Rausch and Hilfer, 1988), a drug that inhibits
N-linked glycosylation (Mahoney and Duskin, 1979).
However, the optic primordium can be stimulated to
form precociously or inhibited from invaginating by
agents that affect cytoskeletal function (Hilfer et al.,
1981; Brady and Hilfer, 1982; Maloney and Wakely,
1982), while the otic primordium is unresponsive to
these treatments (Hilfer et al., 1989). Fluorescent immunolocalization techniques were used to study temporal and regional differences in distribution of laminin, fibronectin, and type IV collagen. The similarities
in developmental distribution outweigh the differences
between the basal laminae of these two primordia.
Some differences do exist in the connections between
Received April 28, 1992; accepted August 13, 1992.
Gwendalyn J. Randolph is now at the Department of Pathology,
State University of New York at Stony Brook, Stony Brook, NY
11794.
444
S.R. HILFER AND G.J. RANDOLPH
Figs. 1-4
445
OTIC AND OPTIC BASAL LAMINA
basal laminae of these organs and the basal laminae of
adjacent epithelial layers.
MATERIALS AND METHODS
Preparation of Specimens
Fertilized white leghorn eggs (Truslow Farms,
Chestertown, MD) were incubated a t 37°C in a humidified commercial egg incubator. Embryos ranging from
stages 10 through 15 (Hamburger and Hamilton, 1951)
were removed from the yolk, transferred to 0.1 M phosphate-buffered saline (PBS), pH 7.2, containing 1.0 mM
calcium and magnesium as chloride salts, and cut just
caudal to the otic placodes/vesicles. The embryos were
fixed for 20 min at room temperature in freshly prepared 3.0% paraformaldehyde in PBS containing Ca2+
and Mg2+.Fixed embryos were embedded in acrylamide (Johnson and Blanks, 1984) to provide support for
the delicate structure of these young embryos. Fixed
embryos were placed on a pad of polymerized acrylam-
Fig. 1. Fluorescence micrographs of chick embryos at the level of
otic primordia, treated with antilaminin and fluorescein isothiocyanate (F1TC)-labeled secondary antibodies. Bar = 100 pm. a: At stage
10, laminin surrounds the neural tube (NT) and forms a continuous
layer under the ectoderm, including the otic primordium (OP). Intensity of fluorescence is greater between neural tube and otic placode
than in regions where they are not adjacent. b: At stage 11, the distribution is much the same as earlier. This thinner section allows
laminin to be visualized as two lines between neural tube and otic
primordium. c: At stage 14, the difference in intensity of laminin
fluorescence between medial and lateral regions of the otic pit is not
as distinct as earlier. The most dorsal region still reacts more intensely than the rest of the surface (arrowhead). d: By stage 15, the
otic pit has separated from the surface of the neural tube, and differences in fluorescent intensity are no longer visible. At all stages,
laminin immunoreactivity within the mesenchyme is punctate.
Fig. 2. Phase-contrast micrographs of the same frozen sections embedded in acrylamide as in Figure 1. Bar = 100 pm. a: The primordium (OP) forms a slight depression along the neural tube (NT) a t
stage 10. b: By stage 11, the otic primordium is folded (arrow) between the surface adjacent to the neural tube (NT) and the surface
adjacent to somitomeric mesoderm. c: At stage 14,the ventral otic pit
has separated partially from the neural tube (NT); mesenchymal cells
(M) are in the gap. d By stage 15, a layer of mesenchymal cells lies
between the otic pit (OP) and the neural tube (NT). Convergence of
dorsal and ventral folds partially closes the pit.
Fig. 3.Fluorescence micrographs of chick embryos at the level of
otic primordia, treated with antifibronectin and FITC-labeled secondary antibodies. Bar = 100 pm. a: At stage 11, the neural tube (NT),
otic primordium (OP), and pharynx (P) are surrounded by a layer of
fibronectin, which appears to merge where the otic and neural primordia are apposed. The mesenchyme also is immunofluorescent
(arrow). b At stage 13 the distribution of fluorescence has not
changed. The gap (arrowhead) is a sectioning artefact. NT, neural
tube. c: At stage 14, fibronectin immunoreactivity in the ventral separation of the apposed region shows as two lines, and adjacent mesenchyme is more intensely fluorescent than at earlier stages. NT,
neural tube. d By stage 15, the intensity of fluorescence is diminished along the basal surface of the otic pit (OP).
Fig. 4. Higher magnifications of the ventral apposition between the
otic primordium and neural tube. Bars = 10 pm. a: Distribution of
laminin in the region of the otic fold at stage 11 (enlargement of Fig.
lb). Cells a t the fold are elongated (arrowhead) and maintain contact
with the surface of the neural tube (NT).Some fine strands of laminin
(arrows) bridge the gap between the two laminin layers. b Between
the two separating layers at stage 14, the strands of laminin seen in
the gap at stage 11are not visible. NT, neural tube. c: Distribution of
fibronectin at the basal surface of the fold at stage 13. Strands of
fibronectin stretch between the two cell layers (arrow) and surround
mesenchyme cells (M). d The same region a t stage 14 contains mesenchyme cells surrounded by a fibronectin-containing matrix.
ide and surrounded by acrylamide monomer, which
was polymerized by addition of 10% ammonium persulfate at room temperature. The acrylamide blocks were
trimmed, surrounded by OCT Compound (Miles Laboratory, Naperville, IL), and quick frozen in liquid nitrogen for sectioning. Sections of 10-15 p,m were prepared at -26°C and mounted on gelatin-coated slides.
Antibodies
Monoclonal mouse antibodies against avian laminin
(Bayne et al., 1984) and fibronectin (Gardner and Fambrough, 1983) were obtained from the Developmental
Studies Hybridoma Bank (maintained by the Department of Pharmacology and Molecular Sciences, Johns
Hopkins University School of Medicine, Baltimore,
MD, and the Department of Biology, University of
Iowa, Iowa City, IA, under contract N01-HD-6-2915
from the NICHD).
Immunolocalization of laminin and fibronectin also
was studied using a polyclonal rabbit antimouse laminin antibody kindly provided by Dr. H.K. Kleinman
and a polyclonal rabbit antimouse fibronectin antibody
kindly furnished by Dr. J.M. Chen. Polyclonal rabbit
antimouse collagen IV antibody was the gift of Dr. C.
Little. Whole-molecule antirabbit IgG antibodies conjugated with fluorescein isothiocyanate (FITC; Atlantic Antibodies, Scarborough, ME) or antimouse IgG
conjugated with tetramethylrhodamine isothiocyanate
(RITC, Cappel, West Chester, PA) were used as secondary antibodies.
lmrnunofluorescence
The lowest dilutions were determined at which antilaminin (full strength, monoclonal; 1:3,200 polyclonal),
anticollagen IV (1:200), and antifibronectin (full
strength, monoclonal; 1:lOO polyclonal) antibodies
could be used and still provide a bright fluorescent
signal. Fluorescently labeled secondary antibodies
were diluted 1:lOO relative to their concentrations a t
purchase for use in immunostaining. Dilutions of all
antibodies were made with PBS. Slides were incubated
at 20°C for 15 min in PBS containing 0.2% bovine serum albumin (BSA) to block nonspecific binding. The
slides were incubated with primary antibody in humid
chambers at 20°C overnight, washed with PBS containing 0.2% BSA, and incubated for 1 h r at 20°C with
secondary antibodies. Finally, slides were washed in
PBS and mounted with Crystal/Mount (Biomeda, Foster City, CA). Immunostained slides were examined
using a Leitz (Rockleigh, NJ) Ortholux I1 or Nikon
(Melville, NY) Optiphot microscope equipped for epifluorescence and were photographed using Kodak TRIX Pan film.
Digestions
Several methods were used to test masking as a
cause for absence or low intensity of immunoreactivity.
Before antibody staining, sections were digested with
0.1% bovine testicular hyaluronidase (Sigma) in 0.1 M
acetate buffer, pH 5, at 37°C for 1hr, to remove hyaluronate and chondroitin sulfates (Meier and Hay, 1973).
To evaluate whether collagen IV immunoreactivity
could be enhanced by degradation of heparan sulfates,
Borenfreund's nitrous acid treatment (Cifonelli, 1968)
was used, as described by Kosher and Searls (1973).
446
S.R. HILFER AND G.J. RANDOLPH
Fig. 5. Fluorescence micrographs of frozen sections of chick embryos
embedded in acrylamide, treated with anticollagen type IV antibody
and FITC secondary antibody after mild proteolysis. Bar = 100 pm.
Collagen type IV fluorescence forms a continuous layer at the basal
surfaces of the otic primordium and neural tube a t stage 11 (a), stage
12 + (b),and stage 14 (cf.
Fig. 6. Phase-contrast micrographs of the same sections and at the
same magnifications as in Figure 5.Bar = 100 pm. a: Section through
an otic primordium at stage 11. The placode has started to fold parallel to the neural tube. b: At stage 12+, the initial fold is well
established, and additional folds are forming dorsal (DF) and ventral
(VF) to it. c: By stage 14,continued ventral folding (VF) has shaped
Mild proteolytic digestion just prior to antibody processing (Finley and Petrusz, 1985) was tested as a
means of amplifying the intensity of antibody immunoreactivity. Slides pretreated with PBS for 15 min a t
4°C were incubated with 0.025% trypsin (Worthington,
Freehold, NJ) for 1 min at 4°C and subsequently
washed with 0.025% soybean trypsin inhibitor (Worthington). Control slides were treated with a 1:1 mixture of trypsin and soybean trypsin inhibitor.
the primordium into a deep pit, and the dorsal fold (DF) is closing the
opening.
Fig. 7.Higher magnifications of the distribution of collagen IV in
otic primordia. Bars = 10 pm. a: The upper region of the section
shown in Figure 5a. At stage 11, the apposition point near the dorsal
margin of the otic primordium is highly immunofluorescent. b: The
ventral margin of the apposition between otic primordium and neural
tube in the same section. Fibrils containing collagen IV span the gap
between the two basal laminae. c: At stage 14,where mesenchyme
separates the two epithelial layers ventrally, collagen is present in
only a few, fine fibrils (arrow), which span the gap.
Control Studies
Nonspecific immunoreactivity of the secondary antibody was assessed by substituting PBS plus BSA, hybridoma cell media, nonimmune serum, or acetate
buffer (for the testicular hyaluronidase digestions) for
the primary antibody in the staining procedure. No
immunoreactivity was detected. Specificity of the antimouse laminin antibody was surveyed by incubating
the antilaminin antibody with laminin-impregnated
OTIC AND OPTIC BASAL LAMINA
nitrocellulose paper (purified laminin was a gift from
Dr. J.M. Chen) or with 5% condensed milk. Solutions of
antilaminin antibody preabsorbed with laminin did not
react with the sections, whereas preabsorption with
milk had no effect on immunoreactivity. To ascertain
the relative differences in immunoreactivity between
paraformaldehyde-fixed sections and sections fixed under milder conditions, several embryos were placed in
OCT directly after removal from the yolk and immediately frozen in liquid nitrogen-cooled isopentane. After
sectioning, the slide were fixed in acetone a t -20°C for
2-10 min and allowed to air dry in a cold chamber
before reacting with antibodies.
RESULTS
General Considerations
In this study, frozen sections of chick optic and otic
primordia between stages 10 and 15 were treated with
antibodies raised to fibronectin, laminin, and type IV
collagen, Nine embryos were examined for each stage
and antibody type. The photomicrographs shown in the
figures have been selected as typical of the composite
observations. When more than one source of antibody
against a single basal lamina component was used (see
Materials and Methods), the same results were obtained with each source.
Frozen sections of embryos fixed in 3.0% paraformaldehyde for 20-25 min resulted in immunoreactivity of
the same intensity as sections of embryos fixed with
acetone when the antilaminin and antifibronectin antibodies were used. Sections of embryos fixed in 3.0%
paraformaldehyde and reacted with the collagen type
IV antibody resulted in immunofluorescence of very
low intensity in basal laminae. Digestions of sections
with bovine testicular hyaluronidase or nitrous acid
did not enhance reactivity to anticollagen type IV.
However, a mild trypsin digestion, performed on sections prior to antibody application, consistently uncovered bright collagen IV immunoreactivity without degradation of the tissue, when the embryos were fixed
with paraformaldehyde for less than 30 min. Trypsin
digestions partially decreased laminin immunof luorescence and resulted in complete loss of fibronectin localization. Frozen sections of unfixed embryos, which
were subsequently fixed with acetone, were immunofluorescent for collagen type IV without resorting to
trypsin digestions or other manipulations. Acetone fixation could not be used routinely in this study because
of poor morphological preservation of the tissue.
Ofic Primordium
From stage 10 to stage 12, the otic epithelium becomes thicker and forms a fold parallel to the longitudinal axis of the embryo (Figs. la,b, 2a,b). By stage 14,
the otic primordium forms a pit (Figs. l c , 2c), followed
by narrowing of the opening a t later stages (Figs. Id,
2d). Laminin immunofluorescence (Fig. 1) is bright
and spatially relatively uniform from stage 10 through
stage 15. From stage 10 to stage 13, laminin localizes to
distinct parallel lines along the length of the otic primordium and appears slightly brighter adjacent to the
neural tube than adjacent to the underlying somitomere (fig. la,b). The basal surface of the neural tube is
uniformly immunoreactive but also appears brighter
447
adjacent to the otic primordium than in the ventral
region. At stage 14, the otic epithelium begins to separate from the neural tube (Fig. 2c), and the brighter
immunofluorescence is lost in the region of separation
(Fig. lc). By stage 15, the two epithelia are separated
by mesenchymal cells (Fig. 2d). Laminin localization is
uniformly along the entire basal surface of the otic pit
and is brighter than along the neural tube (Fig. Id).
The pattern of fibronectin localization is similar to
that of laminin from stage 10 to stage 14 (Fig. 3a-c). At
earlier stages, fibronectin forms a solid band between
the basal surfaces of the otic and neural epithelia, but
two separate layers are visible at stage 14 (Fig. 3 4 . The
intensity of fibronectin reactivity along the otic pit and
neural tube basal laminae is sharply reduced by late
stage 15 (Fig. 3d). The same sections display bright
fibronectin reactivity in the pharyngeal region, indicating that poor antibody coverage of the sections cannot be the cause of the observed reduction in intensity.
Antifibronectin reactivity at stage 15 is not enhanced
by treatment of sections with bovine testicular hyaluronidase, which digests hyaluronate and chondroitin
sulfate (Meier and Hay, 1973). These proteoglycan
components presumably could deter antibody accessibility to fibronectin due to their intimate interaction
with fibronectin in vivo (Jilek and Hormann, 1979;
Perkins et al., 1979).
Comparison of laminin and fibronectin immunolocalization at higher magnification shows a difference in
their distributions. From stage 10 to stage 13, when the
medial otic placode is in close contact with the neural
tube, laminin (Fig. 4a) appears as a distinct line at
each epithelial basal surface, with fine cross connections between the two layers. By stage 14, the two layers of laminin show no cross connections (Fig. 2b). Fibronectin fills the gap between the laminin layers, and
cross connections are not visible until the otic and neural layers separate at stage 14 (Fig. 412).At that time,
cross connections that contain fibronectin become visible at the edge of the fused region, where the otic
epithelium is deflected over somitomeric mesoderm.
Thread-like connections no longer are seen where mesenchymal cells separate the two epithelial layers. Instead, fibronectin forms a fine meshwork surrounding
the invading cells (Fig. 4d).
From establishment of the placode to formation of
the otic pit (Figs. 5, 6 ) , immunolocalization of collagen
IV follows the pattern of laminin. Anticollagen antibody binds with uniform intensity along the entire
basal length of the otic epithelium and neural tube
from before stage 10 (Fig. 5a) to after stage 14 (Fig. 5c).
Collagen IV immunofluorescence (Fig. 7a) merges
where the two basal laminae are in close contact dorsally and forms cross connections ventrally (Fig. 7b).
Upon mesenchymal invasion, little immunoreactivity
is found in the space surrounding the mesenchymal
cells (Fig. 7c). As described above, inconsistent localization occurs with anticollagen antibodies except after
mild trypsinization. This procedure reduces laminin
and eliminates fibronectin localization. Digestion of
chondroitin sulfate or heparan sulfate does not improve
collagen immunoreactivity, suggesting that fibronectin rather than proteoglycans occupies the major binding sites on collagen IV in this primordium. This is in
contrast to cartilage formation, where proteoglycan
448
S.R. HILFER AND G.J. RANDOLPH
Figs. 8-12.
449
OTIC AND OPTIC BASAL LAMINA
masking of types I and I1 collagen has been demonstrated (von der Mark et al., 1976).
Optic Prirnordiurn
The optic primordium was investigated from the period prior to formation of the lens placode at stage 13 to
the establishment of a deep optic cup at stage 15 (Figs.
9, 11, 14). Laminin is distributed over the entire basal
surface of the optic primordium and adjacent ectoderm
(Fig. 8). However, at all stages, the lateral one-half of
Fig. 8. Immunofluorescence of frozen sections through the optic region of chick embryos, treated with antilaminin followed by FITClabeled secondary antibody. Bar = 100 pm. a: At stage 13,the basal
laminae of presumptive lens ectoderm and lateral optic vesicle (OV)
fluoresce more intensely than the medial optic surface and neural
tube (NT). Little if any laminin is visible between the two layers. b
At stage 14, a similar distribution is apparent even in this thick
section. c: At stage 15,laminin is distributed along the basal surfaces
of both sides of the optic cup and around the lens vesicle. Little if any
immunoreactivity is detectable between the basal laminae of retinal
disc and lens, but these two basal laminae fluoresce more intensely
than those of the future retinal pigment epithelium, and outer lens
surface, and the future cornea.
Fig. 9. Phase-contrast micrographs of the same sections as in Figure
8. Bar = 100 pm. a: At stage 13, the pseudostratified cells of the
lateral (RD) and medial (P) walls of the optic vesicle are the same
height. The overlying ectoderm (E) has not formed a lens placode. b:
In this thick section of a stage 14 primordium, the invaginated retinal
disc (RD) can be recognized as thicker than the medial future retinal
pigment epithelium (PI. The adjacent ectoderm has formed a thickened lens pit (L). c: At stage 15, the retinal disc is folded inward
against the medial wall, and the lens has formed a vesicle (LV) and
separated from the retinal and ectodermal surfaces. The surface ectoderm is the future cornea (CO).
Fig. 10. Immunofluorescence of optic primordia after treatment
with antifibronectin and FITC-labeled secondary antibody. Bar = 100
pm. a: At stage 13 + , fibronectin appears as separate, continuous
layers along the basal surfaces of the retinal disc and lens placode. A
small amount of immunoreactivity in the form of particles or fine
filaments is between the layers. The basal lamina of the future retinal
pigment epithelium is less intensely fluorescent. The mesenchyme
also has slight fluorescence. b At stage 14,the same pattern is seen
with additional fluorescence between mesenchyme cells. c: At stage
15, the lens surface is less immunoreactive than the point of retinal
disciectodermal apposition. The central region of the retinal disc,
where it separates from the lens, shows weak fluorescence (arrowhead). The future retinal pigment epithelium shows uneven reactivity.
Fig. 11. Phase-contrast micrographs of the sections in Figure 10.
Bar = 100 pm. a: At stage 13 + , a lens placode (LP) has formed, and
the retinal disc is recognizable by its elongated cells. b: This section is
from the edge of the optic primordium of a stage 14 embryo. The
shallow optic cup and adjacent lens are visible. c: This section, from a
slightly younger stage 15 embryo than that shown in Figure 8c, was
covered with some displaced acrylamide. The lens and retinal disc are
closely apposed except at their centers.
Fig. 12. Higher magnifications of selected regions of optic primordia. Bar = 10 pm. a: The ventral region of a stage 13 + optic primordium. Laminin is distributed along the retinal disc (RD) and ectoderma1 surfaces but not between. b The dorsal fusion point of the same
optic primordium. Laminin is present as two unconnected layers. RD,
retinal disc. c: Enlargement of the ventral region of the retinal disc
(RD) and lens placode shown in Figure 10a. The ventral fusion is
shown (arrowhead). At the center of the retinal disc and lens, fibronectin is present as particles between the separated basal laminae as
well as within these layers. d Enlargement of the dorsal junction of
retinal disc, lens vesicle, and cornea shown in Figure 1Oc. A sheet of
fibronectin-containing material connects the lens vesicle and margin
of the retinal disc (arrow). A single, highly fluorescent band of fibronectin lies between the margin of the cup and the apposed ectoderm.
the primordium (the future neural retina or retinal
disc) stains more intensely than the medial surface (the
future retinal pigment epithelium). The basal laminae
of the retinal disc and lens placode become fused a t the
margins just prior to the initiation of invagination
(Johnston et al., 1979). Laminin occurs a t two distinct
lines through this peripheral band, with no indication
of joining (Fig. 12a,b). This separation differs from the
otic primordium, in which fine strands traversed the
laminin layers of otic and neural basal laminae (Fig.
1).
Immunolocalization of fibronectin also shows greater
intensity in basal laminae of the retinal disc and lens
placode than of the future retinal pigment epithelium
(Fig. 10). As early as stage 13+ , fibronectin forms a
single fluorescent line between the retinal disc and the
dorsal and ventral margins of the newly formed lens
placode (Figs. 10a, 12c).By stage 15, a subtle reduction
occurs in fibronectin intensity, with only the margin of
the optic cup remaining as brightly reactive as the lateral basal laminae a t earlier stages (Figs. lOc, 12d).
This position represents the point of fusion of the ectodermal and neuroectodermal basal laminae. A thin and
weakly fluorescent connection exists between the region of fused basal laminae, the surface ectoderm, and
the lens vesicle (Figs. lOc, 12d). Fibronectin immunofluorescence is weakest where the lens is separated
from the center of the retinal disc (Fig. 1Oc). Crossconnecting fibers of laminin or fibronectin are not seen
at the center of the retinal disc in the thinnest sections
in which ectodermal and retinal disc basal laminae can
be resolved as separate layers (Figs. 8,10,12a-c);however, some particles of fibronectin localize between the
two layers. This distribution contrasts with the localization of these constituents in the otic primordium.
The distribution of collagen type IV is similar to that
of laminin and fibronectin only in that the basal lamina of the future retinal pigment epithelium is less
reactive than that of the retinal disc and lens primordium (Fig. 13a,b). These differences in intensity remain until late in stage 14, when collagen type IV immunofluorescence of the central retinal disc is reduced
to that of the future retinal pigment epithelium, while
the lens vesicle continues to react more intensely (Fig.
14c). Collagen IV localizes as a single band in the fused
basal laminae at the optic/lens primordia margins from
stage 13+ to stage 15, while collagen type IV immunofluorescence forms two parallel bands between the
rest of the retinal disc and lens primordium (Fig. 15a).
Fibrillar connections that contain type IV collagen become visible between the retinal disc and lens beginning with formation of the lens placode and increase in
number during invagination. By stage 15, an extensive
fibrillar meshwork of collagen type IV is formed between the optic cup and the lens pit in the central region (Figs. 14c, 15b).
DISCUSSION
This study shows that laminin, fibronectin, and type
IV collagen exist as continuous sheets a t the basal surfaces of the avian otic primordium when it first forms.
All three extracellular macromolecules also are
present along basal surfaces of the optic primordium
and lens epithelium even before the initiation of invagination. The data extend an earlier study on chick
450
S.R. HILFER AND G.J. RANDOLPH
Fig. 13. Immunofluorescence of optic primordia after mild proteolysis and treatment with anticollagen IV and FITC-labeled secondary
antibody. Bar = 100 pm. At all stages, the ectodermalioptic interface
fluoresces more intensely than other parts of the preparation. a: At
stage 12, type IV collagen localizes as separate layers a t the basal
surfaces of both the ectoderm and optic vesicle. The ventral region of
apposed layers reacts more intensely than the rest of the primordium.
b: In contrast, a single intense band of collagen fluorescence is seen
between the retinal disc and lens placode at stage 13 + . c: At stage
14 + , the margins of the optic cup and the gap between lens and
retinal disc are intensely immunoreactive.
otic development (Richardson et al., 1987) and show
that the distribution of these basal laminar components in the chick optic primordium is similar to that of
the mouse (Svoboda and O’Shea, 1987; Haloui et al.,
1988).This distribution of the three components is consistent with the observation that all these embryonic
structures have complete basal laminae.
The distinctive patterns of invagination of the otic
placode and optic vesicle suggest that points of folding
may be established by differential expression of extracellular macromolecules. These differences could be
qualitative or quantitative. Qualitatively, a hinge
point could be determined either by availability of a
unique adhesive component or by removal of a component that prevents adhesion. Alternatively, folding
could occur either as a result of increased adhesiveness
at a hinge point or as decreased adhesiveness along a
fold. Qualitative differences were not found in the distribution of laminin, fibronectin, and type IV collagen
but may exist in other extracellular components that
have not been examined so far. A more subtle qualitative difference could occur as in the control of laminin
A chain expression in the developing kidney (Klein et
al., 1988; Ekblom et al., 1990). It is possible that similar control mechanisms operate in other organ primordia, including the eye and inner ear.
Several types of quantitative differences do exist in
the distributions of these three extracellular components in otic and optic primordia. The lateral surface of
Fig. 14. Phase-contrast micrographs of the sections shown in Figure
13. Bar = 100 pm. a: Optic vesicle at stage 12, cut through the optic
stalk (0s).
b: Horizontal section of a stage 13 optic vesicle. c: Nearmidline section of a stage 14 + optic cup and lens pit.
+
Fig. 15. High magnifications of collagen IV fluorescence. Bars = 10
pm. a: At stage 12 immunoreactivity is more intense a t the ectoderma1 surface than at the optic vesicleioptic stalk surface, but the basal
laminae form distinct layers. Some collagen type IV-containing material lies between the two basal laminae. b: At stage 14 +, the space
between the central optic cup (RD)and lens is filled with a meshwork
of collagen type IV-containing fibrils and sheets.
the optic primordium is more immunoreactive for laminin, fibronectin, and collagen type IV than the medial
surface, and the otic primordium-neural tube interface
is somewhat more immunoreactive than otic epithelium adjacent to somitomeric mesoderm. These differences as well as decreased fibronectin immunoreactivity after completion of invagination are difficult to
relate to the process of invagination. The observed
points of fusion and cross connections between the
basal laminae of each primordium and adjacent cell
layers, rather than differences in concentration, are
more likely to play a role in invagination. The otic
epithelium is closely apposed to the adjacent neural
tube, based on immunolocalization of both fibronectin
and collagen in this region prior to invasion of mesenchyme. Similarly, basal laminae are fused at the margins of the retinal disc and lens placode, based on immunolocalization of collagen type 1V and fibronectin in
this study and earlier silver staining studies (Johnston
et al., 1979). These points could act to stabilize cell
layers and prevent them from bending while active or
passive movements in other parts of the primordia
cause the invagination.
There are several interesting differences as well as
similarities in the distribution of these three macromolecules in the otic and optic primordia. In the chick,
both primordia form so that neural crest cells do not
penetrate between the ectoderm and neural ectoderm
(Anderson and Meier, 1981). In both, fibronectin is a
451
OTIC AND OPTIC BASAL LAMINA
significant component of the gap between the cell layers even in the absence of mesenchymal cells. Both
primordia make contacts with adjacent epithelia by fusion of basal laminae. In both, fibronectin and type IV
collagen localize to the fused region, but laminin forms
distinct layers a t each epithelial surface. However, the
shapes of the fused zones differ in the two primordia;
the entire medial region of the otic placode is bound to
the neural tube, while the retinal disc and lens placode
are attached only a t their margins. Cross connections
external to the fused regions also differ. The otic placode has only a few cross connections with the neural
tube at the margin of the fused region, whereas the
retinal disc and lens are connected by a n extensive
series of fibrils within the confines of the marginal attachment zone. Otic cross connections consist of all
three components, whereas cross connections between
lens and retinal disc consist primarily of type IV collagen.
These differences in organization of the basal laminae in the two primordia may reflect differences in the
methods by which folding occurs. Preliminary studies
support the conclusion that glycoproteins play a role in
invagination of both primordia. Treatment of otic
(Rausch and Hilfer, 1988; Hilfer et al., unpublished)
and optic (Yang and Hilfer, 1982) primordia with tunicamycin inhibits invagination. Injection of antilaminin antibody has a similar effect (Visconti et al., unpublished). However, the mechanisms that control
folding appear to be entirely different. Otic invagination is not affected by agonists or antagonists of Ca2+
transport (Hilfer et al., 1989), but inhibition of proteoglycan synthesis or injection of enzymes that degrade
glycosaminoglycans prevents or delays folding (Gerchman et al., 1991, unpublished). Optic cup formation,
in contrast is dependent on adenosine triphosphate
(ATP), Ca2' , and calmodulin (Brady and Hilfer, 1982).
In both, however, anchorages to adjacent cell layers
appear to play important roles in folding.
The data from this study are consistent with the following models for formation of the otic pit and optic
cup. Formation of the otic vesicle begins when the medial region of the otic placode becomes held to the adjacent neural tube by fusion of the outer surfaces of
their apposed basal laminae. Increase in volume of the
somitomeric mesoderm ventral to the lateral region of
the otic placode causes the primordium to fold along a
hinge point adjacent to the ventral margin of the neural tube. Ventral migrations of neural crest cranial and
caudal to the placode cause additional folding (Meier,
1978), which results in the box-like shape of the otic pit
(Hilfer et al., 1989). Completion of folding to produce
a n otic vesicle results from detachment of the otic epithelium from the neural tube by invasion of mesenchymal cells. In contrast, formation of the optic cup
begins with fusion of the basal lamina of the optic vesicle a t the margin of the retinal disc with the basal
lamina at the margin of the newly formed lens placode.
This ring acts to prevent expansion in diameter of the
optic primordium during invagination. Folding is initiated by a combination of constriction of cell apices at
the region of fused basal laminae and expansion of cell
apices within the central region of the retinal disc
(Hilfer and Hilfer, 1983). Formation of a lens vesicle
and vitreous cavity depends on release of the central
region of the retinal disc from the lens epithelium and
possibly a shift in position of the fused basal laminae to
the new margin of the expanding optic cup. These two
models raise a number of questions about the ways in
which morphogenetically active epithelial layers produce and interact with their surface coats and extracellular matrices. Answers to these questions are
necessary for a n understanding of the way in which
organogenesis is controlled.
ACKNOWLEDGMENTS
This study was supported by NSF grant
DCB8812287 and a grant from the Deafness Research
Foundation. The technical assistance of Joyce W.
Brown is greatly appreciated. We especially thank Drs.
Edward Gruberg, Joel Sheffield, and Jinq-mai Chen for
generously allowing the use of their equipment and for
their excellent technical advice.
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