The optic vesicle develops as an evagination of the cephalic neural folds. We have examined the early development of the optic vesicle in Swiss Webster mice using correlated transmission electron microscopy (TEM), scanning electron microscopy (SEM), light microscopic (LM) measurements of cell shape changes, immunohistochemical localization of basal lamina (BL) components (type IV collagen, laminin and heparan sulphate proteoglycan (HSPG)) and ultrastructural analysis of the BL.

Like the neuroepithelium in other regions, the low columnar cells of the neural plate in the future optic vesicle region become high columnar, then wedge shaped following constriction of the cell apices to form the C-shaped vesicle. In this region, the cells elongate 2 times their initial height before the neural tube closes, then shorten 20 % as the vesicle is completed. Cell apices decrease in width by about one half during vesicle formation. Deposition of BL components was initially even, with type IV collagen and laminin reduced in deposition in regions of outpouching. At later stages the linear, even distribution of all four components was re-established. Ultrastructural analysis confirmed the BL discontinuity and re-establishment and correlated the observed cell shaping alterations with apparent increases in the number of microtubules (during elongation) and microfilaments (during apical constriction). The number of apical intercellular junctions also appeared to increase in number during optic vesicle formation, possibly providing stability and coordination to the évagination process.

The mammalian eye primordial tissue develops from the neuroepithelial cells in the headfold region as an évagination, the optic pit. As the cephalic neural folds elevate, the optic primordium evaginates further to form a sulcus, the early optic vesicle (EOV). When the neural folds fuse and the anterior neuropore closes, the optic vesicle is fully formed, termed here late optic vesicle (LOV). The optic vesicle then invaginates upon itself to form the optic cup. We have chosen to study the development of the early eye primordium because, as the tissue changes from a flat plate of cells to become spherical, there is a dramatic cell shape change. We have documented these cell shape changes using several techniques including scanning electron microscopy (SEM), morphometrical analysis and transmission electron microscopy (TEM).

Much of the information available on the early development of the mammalian eye is derived from paraffin serial sections of early human embryos (Barber, 1955; Mann, 1969; Duke-Elder, 1963; Dekaban, 1963; O’Rahilly, 1966) and experiments designed to study later events such as the inductive relationship between the optic cup and lens (Geeraets, 1976; Schook, 1978; Schluter, 1978). Although Kaufman (1979) commented on the temporal correlation between cephalic neural tube closure and optic vesicle formation in the mouse embryo, he primarily described neural tube formation. Optic vesicle formation in the quail (Thorogood, Bee & von der Mark, 1986) and chick embryos (Camatini & Ranzi, 1976; Hilfer, Brady & Yang, 1981) has been described, but quantitative data are lacking.

The mechanism responsible for producing the neuroepithelial cell shaping changes has been the subject of many studies. One of the predominant hypotheses states that the cytoskeleton (microtubules and microfilaments) may produce the observed cell shape changes. This hypothesis has been tested by using agents that selectively inhibit microtubules (Wilson, 1970; Berry & Shelanski, 1972; Pearce & Zwaan, 1970; Karfunkel, 1971, 1972, 1974; Wessells, Spooner, Ash, Bradley, Luduena, Taylor, Wrenn & Yamada, 1971; Messier, 1978) or microfilaments (Linville & Shepard, 1972; Morriss-Kay, 1981; Morriss-Kay & Tuckett, 1985). Vincristine sulphate (VS) was previously employed to inhibit microtubule elongation during optic vesicle development (Svoboda & O’Shea, 1984). The optic vesicles did not form normally in the presence of VS in vivo or in embryos cultured during optic vesicle development; however, the neuroepithelial cells did elongate, indicating that a mechanism other than microtubule elongation may control cell shape. The whole-embryo culture system has also been utilized to study the role of microfilaments during neurulation. Rat embryos exposed to low doses of a microfilament disrupting drug, cytochalasin B or D (CB or CD) for 1 h, then allowed to recover in culture, illustrated that microfilaments were necessary for neural fold elevation, apposition and curvature formation (Morriss-Kay & Tuckett, 1985). Although the development of the optic vesicle was not directly examined, these studies show that disrupting one of the cytoskeletal elements was sufficient to cause optic vesicle and neural tube defects.

Another hypothesis is that extracellular matrix (ECM) or surrounding mesenchymal cells may contribute to tissue and cell shaping changes (Bernfield & Wessells, 1970). Supporting evidence for the ECM theory is derived from the way epithelial cells react to ECM in culture. Epithelial cells become cuboidal then columnar and express specific proteins when cultured on ECM. Mouse mammary epithelial cells cultured on plastic stay flat but become columnar on floating collagen gels (Emerman & Pitelka, 1977). These cells also increase mammary-specific protein synthesis when cultured on basement membrane gels or floating collagen gels (Lee, Lee, Kaetzel, Parry & Bissell, 1985). Rat hepatocytes also respond to ECM by changing cell shape and increasing hepatocytespecific proteins (Sattler, Michalopoulous, Sattler & Pitot, 1978; Michalopoulos & Pitot, 1975).

The two theories may be linked because the changes in cytoskeleton may be influenced by ECM as shown in the corneal epithelial model. Corneal epithelial cells isolated without a basal lamina (BL) extend cell processes (blebs), which contain unorganized actin filaments (Sugrue & Hay, 1981). After culturing the corneal epithelium on solid ECM such as frozen-killed lens capsules (Meier & Hay, 1974) or in the presence of soluble ECM (Sugrue & Hay, 1981) the cells reorganize the actin cytoskeleton and increase collagen synthesis. The corneal epithelia increase collagen synthesis only when cultured with ECM molecules which reorganize the actin cytoskeleton (Sugrue & Hay, 1981). If the actin cytoskeleton is disrupted by cytochalasin D, the epithelia do not increase collagen production in the presence of exogenous ECM (Svoboda & Hay, 1987). There is additional evidence in cultured fibroblasts that ECM molecules, particularly fibronectin, coalign with the actin cytoskeleton (Hynes & Destree, 1978).

Although the embryonic neuroepithelial cells have not been used in these types of experiments, the presence of actin filaments in the base of neuroepithelial cells (Sadler, Greenberg, Coughlin & Lessard, 1982) and subjacent BL containing hyaluronate (Morriss & Solursh, 1978), fibronectin (Newgreen & Theiry, 1980; Sternberg & Kimber, 1986), laminin, entactin (Sternberg & Kimber, 1986; Tuckett & Morriss-Kay, 1986) and type IV collagen (Thorogood et al. 1986) suggest a similar organizational structure. Tuckett & Morriss-Kay (1986) reported that as the optic primordium evaginates the BL became deficient in laminin and entactin but reacted strongly with antifibronectin antibodies. However, Thorogood et al. (1986) reported the presence of laminin and type IV collagen as an unbroken line around the entire optic vesicle of the H&H stage-15 quail embryo. In addition, these researchers found type II collagen around the developing optic vesicle of the quail embryo (Thorogood et al. 1986). We extend these studies by examining the distribution of type IV collagen, laminin, fibronectin and HSPG at earlier and later stages of optic vesicle development. In addition, the immunofluoresce data are correlated with TEM analysis of the BL in corresponding areas. This information is important in establishing baseline data for further experiments, such as comparison of normal development to that of mutant animals like the Small eye (Sey) described recently (Hogan, Horsburg, Cohen, Hetherington, Fisher & Lyon, 1986) and experimentally induced malformations of the optic vesicle and lens.

Electron microscopy

Female Swiss-Webster mice (Charles River Laboratories) were bred with males of the same strain (1st day is day of vaginal plug) and sacrificed at intervals from the 8th to the 10th day of gestation. Embryos (n = 250) were removed from the decidua and chorion, examined, staged and fixed in a solution of 1 % glutaraldehyde in 0·1 m-sodium cacodylate buffer (pH 7·4, 320mOsmol) for 2h at room temperature. The embryos were then stored in 0·1 m-sodium cacodylate buffer with 5 % sucrose (pH 7·4, 327 mOsmol) at 4°C prior to further processing. Embryos to be examined by TEM and LM were postfixed in 1 % osmium tetroxide in 0·1 m-sodium cacodylate buffer containing 0·15 M-sucrose for 30min, followed by dehydration through graded al-cohols and infiltration with Araldite 502. Thick and thin sections were cut. Thin sections were double stained with uranyl acetate and lead citrate, viewed and photographed using either a Philips 201 or a JEOL 100S microscope.

SEM was employed to examine topographical and cell surface changes during optic vesicle formation. Embryos to be examined using SEM were fixed and stored as described for TEM, then dehydrated, critical-point dried in freon, sputter coated with gold, viewed and photographed in an E-TECH scanning electron microscope. Additional embryos were critical-point dried and fractured through the developing optic vesicle, so that the lateral cell surface could be examined directly.

Immunocytochemistry

For correlative immunocytochemical studies of the basement membrane, similarly staged CD-I (Charles River Laboratories) mouse embryos were lightly fixed in a solution of 10 % phosphate-buffered formalin for 1 h at room temperature. They were then dehydrated and infiltrated in polyester wax (BDH) to 90 % (Kusakabe, Sukakura, Nishizuka, Sano & Matsukage, 1984). Serial 6–8 μm cryostat sections were cut and collected on poly-lysine-coated glass slides. Slides and blocks were stored at —70°C prior to additional processing.

Slides were rehydrated, treated with normal goat serum (1:20), washed in PBS, then exposed to the primary antibody for 2h at room temperature in a moist chamber. Controls were exposed to PBS or preimmune serum in place of the primary antibody. Sections were then washed, exposed to the second antibody (goat anti-rabbit IgG conjugated to FITC) diluted 1:50 for 30min. Slides were coverslipped with glycerol containing phenylenediamine and examined using a Leitz Dialux Orthoplan microscope equipped for epifluorescence. Photographs were taken using Kodak 2475 recording film.

Laminin, type IV collagen and HSPG were derived from EHS tumour and antibodies were raised in rabbits. All were affinity purified and exhibited no cross reactivity on ELISA. Sources and optimal dilutions of the antibodies were as follows:-laminin (1:50, Dr J. P. McCoy, University of Michigan); HSPG (1:20, Dr J. Hassell, National Institute of Dental Research); type IV collagen (1:100, Dr H. Furthmayr, Yale University). Antibodies to human serum fibronectin were obtained from Cappel laboratories and were used at a dilution of 1:10.

Morphometrical analysis

A Zeiss compound microscope equipped with a camera-lucida drawing tube projecting onto a Zeiss MOP III digitizer was utilized to analyse the toluidine-blue-stained semithin sections. Each vesicle was treated as an independent unit. After a camera-lucida drawing of the region was completed, the mean and standard deviation for each cell dimension was recorded. Cell dimensions that were recorded included apical width, basal width, apical to basal length, apical to the top of the interphase nucleus length and the length of the interphase nucleus. Interphase nuclei were identified by location within the cell and morphological appearance at the LM level. Metaphase nuclei were distinguished because of their location at the luminal surface and characteristic morphology. Cells containing them were excluded from all digital measurements. To ensure accurate measurements and to decrease variability in the system, the magnification was checked with a 100μm graticule before each vesicle was measured.

Since the optic vesicle varied in shape throughout the period studied, the digital data were compiled on sections taken through the centre of the neural fold region destined to become the vesicle in the early stages and through the centre of the sulcus at the EOV and LOV stages. The overall shape of the vesicle was known from the SEM data and the LM sections that corresponded to the centre of the vesicle region were chosen for analysis. It was necessary that the cells were cut at the same angle, therefore, the vesicles were cut in the same plane. The plane of section for each stage is illustrated in the camera-lucida drawings (Fig. 1). Only one section was measured from each optic vesicle, eliminating the possibility of duplicating data. Six or seven optic vesicles were measured for each datum point (see Table 1). The fused optic stalk was excluded as well as portions of the vesicle that were not cut in cross section. The number of cells in interphase or mitosis are recorded in Table 2.

Table 1.

Optic vesicle cell measurements

Optic vesicle cell measurements
Optic vesicle cell measurements
Table 2.

Total number of cells measured including mitotic cells

Total number of cells measured including mitotic cells
Total number of cells measured including mitotic cells
Fig. 1.

(A) Camera-lucida drawings of the neuroepithelium in the optic region from four stages of development. The drawings represent (1) the 1- to 2-somite stage, (2) neural fold (4 – 6 somites) stage, (3) early optic vesicle (9 – 11 somites) stage and (4) late optic vesicle (18 – 20 somites) stage of development. The arrowheads point to the evaginating comer zone. The lateral wall zone (lw), and fusion zone (f) are also labelled in the late optic vesicle stage diagram. Bar, 50 μ m. (B) The average cell shape from each developmental stage is represented in this graph. The graph illustrates the data listed in Table 1.

Fig. 1.

(A) Camera-lucida drawings of the neuroepithelium in the optic region from four stages of development. The drawings represent (1) the 1- to 2-somite stage, (2) neural fold (4 – 6 somites) stage, (3) early optic vesicle (9 – 11 somites) stage and (4) late optic vesicle (18 – 20 somites) stage of development. The arrowheads point to the evaginating comer zone. The lateral wall zone (lw), and fusion zone (f) are also labelled in the late optic vesicle stage diagram. Bar, 50 μ m. (B) The average cell shape from each developmental stage is represented in this graph. The graph illustrates the data listed in Table 1.

The mean and variance of each cell dimension were measured from the cells of each vesicle using a microcomputer. Average cell dimensions were compared using Student’s r-test. Statistical significance was considered to be a probability of less than or equal to 0·01.

The optic vesicle develops as an evagination of the prosencephalon prior to neural tube closure. A flat plate of cells becomes a spherical structure during the period encompassing the early 1- to 2-somite to the 20- to 25-somite stages of development. The cells progress from a low columnar shape (1- to 2-somite stage) to the wedge shape characteristic of the mature optic vesicle on the 10th day of gestation (Fig. 1).

To facilitate discussion, stages exemplifying the formation of the optic vesicle have been chosen to illustrate the alterations of this region. These stages are described by the somite stage and degree of optic vesicle maturation (1) early neural fold stage, 1 – 2 somites, (2) late neural fold stage, 4 – 6 somites, (3) early optic vesicle stage (EOV) 9 – 11 somites and (4) late optic vesicle stage (LOV) 18 – 20 somites. These stages are illustrated in camera-lucida drawings (Fig. 1) and scanning micrographs (Figs 2, 3). The early and late optic vesicle regions have been further subdivided based on cell and vesicle shape or the future destination of the cells (Svoboda & O’Shea, 1984). The deepest point of evagination of the sulcus at the EOV stage where the cells are extremely wedge shaped has been termed the corner zone (Fig. 1A). Later, at the LOV stage there are two corner zones separated by the cells forming the lateral wall of the vesicle. The optic stalk forms from the area of neuroepithelial cells which are medial to the corner zones. We have termed this area the fusion zone (Fig. 1A).

Fig. 2.

Scanning electron photomicrographs of embryos at the 1- to 2-somite (A,B) and 4- to 6-somite (C,D) stages of development. The scale bars are equal to 50 μ m. (A) A frontal view of a 1- to 2-somite staged embryo illustrating the earliest indication of optic pit formation (arrow). (B) A transverse fracture through the future optic region (arrow) of the early neural fold. The neuroepithelial cells (ne) are columnar in shape and in close contact with the underlying mesenchyme (m). (C) A frontal view of an embryo at the neural plate stage (4 – 6 somites). The neural folds have elevated considerably and the optic pit is forming (arrow). The heart (h) is located below the prosencephalon at this developmental stage. (D) A fracture through the neural fold stage optic pit showing that the cells are becoming wedge shaped (arrow) and are in tight contact with the surface ectoderm. Bar, 50 μ m.

Fig. 2.

Scanning electron photomicrographs of embryos at the 1- to 2-somite (A,B) and 4- to 6-somite (C,D) stages of development. The scale bars are equal to 50 μ m. (A) A frontal view of a 1- to 2-somite staged embryo illustrating the earliest indication of optic pit formation (arrow). (B) A transverse fracture through the future optic region (arrow) of the early neural fold. The neuroepithelial cells (ne) are columnar in shape and in close contact with the underlying mesenchyme (m). (C) A frontal view of an embryo at the neural plate stage (4 – 6 somites). The neural folds have elevated considerably and the optic pit is forming (arrow). The heart (h) is located below the prosencephalon at this developmental stage. (D) A fracture through the neural fold stage optic pit showing that the cells are becoming wedge shaped (arrow) and are in tight contact with the surface ectoderm. Bar, 50 μ m.

Fig. 3.

Scanning electron photomicrographs of embryos at the early (A,B) and late (C,D) optic vesicle stages. Bar, 100μ m. (A) The optic vesicles are becoming more evaginated (arrow) as the neural folds approach each other in this embryo at the early optic vesicle stage. The embryo is partially turned at this stage and has 9 – 11 somites. (B) A cross-sectional view of the optic vesicle showing the wedge-shaped cells in the corner zone (arrow). (C) After the anterior neural folds close, the optic eminence (arrow) represents the position of the optic vesicle in this 18- to 20-somite embryo. (D) A fracture through the late optic vesicle shows the relationship of the three tissue layers, neuroepithelium (n), mesenchyme (m), and surface ectoderm (s), as well as the shape of the vesicle structure.

Fig. 3.

Scanning electron photomicrographs of embryos at the early (A,B) and late (C,D) optic vesicle stages. Bar, 100μ m. (A) The optic vesicles are becoming more evaginated (arrow) as the neural folds approach each other in this embryo at the early optic vesicle stage. The embryo is partially turned at this stage and has 9 – 11 somites. (B) A cross-sectional view of the optic vesicle showing the wedge-shaped cells in the corner zone (arrow). (C) After the anterior neural folds close, the optic eminence (arrow) represents the position of the optic vesicle in this 18- to 20-somite embryo. (D) A fracture through the late optic vesicle shows the relationship of the three tissue layers, neuroepithelium (n), mesenchyme (m), and surface ectoderm (s), as well as the shape of the vesicle structure.

Cell shaping changes during optic vesicle formation

Two methods were used to determine the change in cell shape during the development of the optic vesicle. Embryos from the four representative stages were examined by SEM; the whole embryo was used to see the overall shape of the vesicle as it was forming. The shape of individual cells was examined by fracturing the embryos after critical-point drying. Breaking the embryos after drying causes the fracture plane to go between cells, so that the cell shape can be easily seen. The cells were also measured directly from plastic semithin sections to measure cell shapes.

Scanning electron microscopy

Embryos isolated at the 1- to 2-somite stage of development are characterized by a dorsal concave flexure (unturned stage), a large allantoic bud and wide headfolds. A frontal SEM view shows the neural folds have not completely elevated and no demarcation between the future optic pit region and the surrounding neuroepithelium can be seen (Fig. 2). The neuroepithelial cells in the headfold region are low columnar (Fig. 2), and the apical and basal surfaces are approximately the same width.

As the cephalic neural folds elevate, the optic primordium begins to evaginate to form the optic pit (Fig. 2). Fractures through the optic primordium show that the cells have increased in length and become high columnar (Fig. 2). The embryo has developed 4 – 6 somites but is still ‘unturned’.

Just prior to anterior neuropore closure and the turning of the embryo to adopt the fetal position, the optic vesicle region becomes deeply evaginated (EOV stage) (Fig. 3). Fractures through the early vesicle show that the cells in the corner zone are wedge shaped and cytoplasmic protrusions are visible along the luminal surface of the neuroepithelium (Fig. 3). The chick embryo does not appear to have an equivalent stage of optic vesicle development (Hilfer et al. 1981).

As the anterior neural folds close and the embryo completes the turning process and assumes a fetal position (18 – 20 somites), the optic vesicle outpockets further. In transverse section the mature vesicle (LOV stage) appears as a tight ‘C’ shape. Two fusion zones are narrowing to form the future optic stalk. There are two corner zones separated by a lateral wall. The lateral wall is the region of the optic vesicle that changes polarity and invaginates to form the optic cup later in development.

As the optic vesicle enlarges, it bulges and approaches the lateral surface ectoderm, the optic eminence (Fig. 3). Fractures through the optic vesicle show the cells have formed a well-organized pseudostratified epithelium. The three zones of the vesicle can be clearly seen (Fig. 3).

Morphometrical analysis of cell shape changes

The morphometry employed for this paper is simple and reproducible. Embryos were embedded in plastic and sectioned so that the vesicle was cut through the centre (see Materials and methods for details). Camera-lucida drawings were made of each stage (Fig. 1A), and measurements of the apical, basal, apical to basal, apical to the top of the nucleus and the nuclear lengths (Table 1, Fig. IB) were made. A schematic drawing based on average cell measurements from the morphometric data is presented in Fig. IB. The number of cells measured and the mitotic index are recorded in Table 2.

The early neural fold stage neuroepithelial cells are low columnar (Fig. 2; Table 1). The nuclei are located in the basal half of the cell. The apical width was smaller than the basal width of the cells.

Between 1- to 2-somite and 4- to 6-somite stages (neural fold stages), the cells nearly doubled in length (Fig. 1; Table 1). The cells in the centre of the neuroepithelium appear to be nearly rectangular, with the apical width slightly larger than the basal width (Table 1). The nuclei are located farther from the apical surface during interphase at the 4- to 6-somite stage (Table 1), compared to the 1- to 2-somite neuroepithelium and are more centrally located. The cells in the centre of the optic pit are triangular in shape.

The typical cell at the EOV stage has increased in length 2 times and decreased its apical width by since the neural plate stage. The nuclei are located in the basal half of the interphase cells and the terminal bars are prominent at the apical pole. The overall appearance of the optic vesicle in cross section is a semicircle (Figs 1A, 3), with one corner zone and two fusion zones.

After the neural folds close and the vesicle matures, the cells decrease in average length. They are still more elongated than at the neural fold stages, but shorter than at the EOV. The apical and basal widths have continued to decrease, but the length and relative position of the nucleus is unchanged from the EOV stage (Table 1).

Ultrastructural analysis

The cytoplasm contains numerous mitochondria, rough endoplasmic reticulum, a well-developed Golgi apparatus, free ribosomes, lipid droplets, coated pits, with some microvilli located at the luminal surface and occasional microtubules oriented perpendicular to the luminal surface. Junctional complexes are located in the apical region of the cells, intercellular canaliculi are also seen in the middle regions of the lateral borders between cells. Junctional complexes are not prominent in the basal cell area and there is not a highly developed basal actin mat. The BL appears patchy in some areas (see BL result section).

As the cells elongate to become neural folds, microtubules appear more numerous in the cytoplasm and are usually located lateral to the nuclei. The intercellular space has decreased as the cells become more tightly apposed. An occasional rounded mitotic cell and numerous microvilli are present at the luminal surface.

When the optic vesicle evaginates and the cells become more wedge shaped, microfilament bundles appear near the apical plasma membrane associated with tight junctions. Microtubules are similar to those seen in neural-fold-stage epithelial cells. Cytoplasmic protrusions, portions of cytoplasm that may have been squeezed into the lumen by apical constriction of the cells, as described by Freeman (1972), are present. Some of the extracellular material contains degenerating midbodies, which have also been described at the luminal surface of the neuroepithelium of the chick embryo (Bancroft & Bellairs, 1975).

Basal lamina maturation

During the period of optic vesicle formation, the distribution of BL components (HSPG, laminin, fibronectin and type IV collagen), changes considerably. At the 1- to 2-somite stage of development, the basal surface of the neuroepithelium in the future forebrain region stains intensely and uniformly for all four components. The BL appears as a continuous sheet along the basal surface of the neuroepithelial cells. With elevation of the neural folds and initial invagination of the optic pit, there are regional differences in the deposition of these components. Near the midline, deposition of all four BL proteins is particularly dense; progressing laterally staining became less intense. In the corner zone area of the EOV the BL appears as a continuous layer beneath the neuroepithelial cells (Fig. 5A). At the lateral border of the neuroepithelium, in the region of neural crest cell migration, the staining for type IV collagen and laminin is considerably less intense and often appears broken. HSPG and fibronectin are densely deposited at the base of the neuroepithelium and in the subjacent mesenchyme into which the neural crest cell migrate (data not shown).

At the EOV stage, there also are regional variations in the deposition of these basal lamina components (Fig. 4). All four are present near the midline and in the basal lamina of the surface ectoderm. However, unlike the uniform linear deposition of HSPG and fibronectin, the deposition of type IV collagen and laminin become patchy at the lateral margin of the forming optic vesicle. Laminin is either very sparsely deposited in this region or is absent in all embryos examined (Fig. 4). Ultrastructurally, the BL also appears patchy in the area where laminin and type IV staining is light (Fig. 5B), whereas nearer the midline in the fusion zone the BL is well formed (Fig. 5C).

Fig. 4.

Transverse sections through the developing forebrain region in 9- to 11-somite stage embryos illustrating the patterns of immunoreactivity associated with the neuroepithelial basement membrane (BM) of the EOV. (A) Type IV collagen distribution. BM of the surface ectoderm and neuroepithelium are stained, except in the most lateral region of the optic vesicle where deposition is patchy (arrowed). (B) Distribution of laminin. Laminin is associated with BL of neuroepithelium and surface ectoderm but is lost hear the lateral margin of the forming optic vesicle (arrowed). (C) Immunofluorescence localization of HSPG. Note the uniform, dense deposition of HSPG in the neuroepithelial BM and in association with blood vessel BM located in the mesenchyme. (D) Deposition of fibronectin in the neuroepithelial BM and mesenchyme surrounding the developing optic vesicle, ne, neuroepithelium; m, mesenchyme; se, surface ectoderm. Bar, 100 μ m.

Fig. 4.

Transverse sections through the developing forebrain region in 9- to 11-somite stage embryos illustrating the patterns of immunoreactivity associated with the neuroepithelial basement membrane (BM) of the EOV. (A) Type IV collagen distribution. BM of the surface ectoderm and neuroepithelium are stained, except in the most lateral region of the optic vesicle where deposition is patchy (arrowed). (B) Distribution of laminin. Laminin is associated with BL of neuroepithelium and surface ectoderm but is lost hear the lateral margin of the forming optic vesicle (arrowed). (C) Immunofluorescence localization of HSPG. Note the uniform, dense deposition of HSPG in the neuroepithelial BM and in association with blood vessel BM located in the mesenchyme. (D) Deposition of fibronectin in the neuroepithelial BM and mesenchyme surrounding the developing optic vesicle, ne, neuroepithelium; m, mesenchyme; se, surface ectoderm. Bar, 100 μ m.

Fig. 5.

Electron micrographs of the basal lamina (BL) of embryos at the 4- to 6-somite (A), and 9- to 11-somite, EOV (B,C) stages of development. (A) The BL of the NF epithelial cells is continuous, even between cell gaps (arrows). The basal portion of the cell contains ribosomes, but no actin cortical mat. (B) The most lateral portion of an EOV-stage vesicle is the corner zone. The immunohistochemical data suggests that type IV collagen and laminin are sparse in this area (Fig. 4A,B). This micrograph shows that the BL structure itself also has areas of thinning, (arrow) and the cell appears to be extending a cell process (cp) into the area without a continuous BL. (C) The BL is continuous (arrows) near the midline in the area that will become the optic stalk, the fusion zone. Bar, 1 μ m.

Fig. 5.

Electron micrographs of the basal lamina (BL) of embryos at the 4- to 6-somite (A), and 9- to 11-somite, EOV (B,C) stages of development. (A) The BL of the NF epithelial cells is continuous, even between cell gaps (arrows). The basal portion of the cell contains ribosomes, but no actin cortical mat. (B) The most lateral portion of an EOV-stage vesicle is the corner zone. The immunohistochemical data suggests that type IV collagen and laminin are sparse in this area (Fig. 4A,B). This micrograph shows that the BL structure itself also has areas of thinning, (arrow) and the cell appears to be extending a cell process (cp) into the area without a continuous BL. (C) The BL is continuous (arrows) near the midline in the area that will become the optic stalk, the fusion zone. Bar, 1 μ m.

At the LOV stage, the continuity of the BL is re-established and all four components form a continuous boundary around the vesicle. There are no regional differences in deposition in the neuroepithelial BL, but HSPG and fibronectin are more densely deposited in the space between the optic vesicle and the future lens placode (Fig. 6). TEM examination of the basal surface of the LOV showed that the BL is continuous and has some fibrillar material in the intervening extracellular space (Fig. 7A,B).

Fig. 6.

Transverse section through the optic vesicle in embryos of 18 – 20 somites. (A) Type IV collagen is now uniformly distributed around the optic vesicle and is associated with surface ectoderm and blood vessel BM in the region. (B) Laminin is also found in surface ectoderm and optic vesicle BL. (C) HSPG is localized to BM of the optic vesicle, surface ectoderm and blood vessels, with some mesenchymal staining as well. Note the particularly dense deposition between surface ectoderm and neuroepithelium (arrow). (D) Like HSPG, fibronectin at this stage is localized to BM and mesenchyme, ne, neuroepithelium; se, surface ectoderm; m, mesenchyme. Bars, 100 μ m.

Fig. 6.

Transverse section through the optic vesicle in embryos of 18 – 20 somites. (A) Type IV collagen is now uniformly distributed around the optic vesicle and is associated with surface ectoderm and blood vessel BM in the region. (B) Laminin is also found in surface ectoderm and optic vesicle BL. (C) HSPG is localized to BM of the optic vesicle, surface ectoderm and blood vessels, with some mesenchymal staining as well. Note the particularly dense deposition between surface ectoderm and neuroepithelium (arrow). (D) Like HSPG, fibronectin at this stage is localized to BM and mesenchyme, ne, neuroepithelium; se, surface ectoderm; m, mesenchyme. Bars, 100 μ m.

Fig. 7.

Electron micrographs of the basal surface from 18- to 20-somite stage optic vesicle cells. (A) The BL in the corner zone area of an LOV-stage embryo is a continuous layer beneath the neuroepithelial cells. Fibrils are seen associated with the BL. (B) The BL in the lateral wall area is also continuous and fibrillar substance is also seen near the basal cell surface. Bar, 1 μ m.

Fig. 7.

Electron micrographs of the basal surface from 18- to 20-somite stage optic vesicle cells. (A) The BL in the corner zone area of an LOV-stage embryo is a continuous layer beneath the neuroepithelial cells. Fibrils are seen associated with the BL. (B) The BL in the lateral wall area is also continuous and fibrillar substance is also seen near the basal cell surface. Bar, 1 μ m.

Neuroepithelial cell shaping alterations

The morphogenetic changes of an invaginating neuroepithelial structure are documented in the present study. The cell shape changes are directly measured and ultrastructural changes documented. We show that during the formation of the optic vesicle, the neuroepithelial cells more than double in length, then shorten as the neural tube closes. At the same time the cells narrow at both the apical and basal ends. The interphase nucleus remains relatively constant in size and cellular position throughout the period of optic vesicle formation.

The mechanisms involved in the production of cell shaping changes may be solely intracellular (produced by the cytoskeletal elements) or solely extracellular (changes that result from either the neuroepithelial BL or from the subjacent mesenchymal ECM). Alternatively, because of the transmembrane association between cytoskeletal elements, particularly F-actin via talin and vinculin with fibronectin (Tamkum, DeSimone, Fonda, Patel, Horwitz & Hynes, 1986), cell shaping changes may result from the cooperative interplay of ECM and cytoskeleton.

Evidence that cytoskeletal elements are required for normal optic vesicle formation comes from our previous studies that indicate that interference with microtubules (Svoboda & O’Shea, 1984) or microfilaments (but see Morriss-Kay & Tuckett, 1985) disrupt optic vesicle development. Like cell shape alterations involved in the morphogenesis of other epithelial organs (Bernfield & Wessells, 1972), numerous investigators have described that concomitant with cellular elongation, microtubules become apparent in the neuroepithelial cytoplasm and with narrowing of the cellular apices the microfilament bands become more prominent in that region (Burnside, 1973; Karfunkel, 1974; Camatini & Ranzi, 1976). Whether these changes actively produce the observed shape changes or whether they are a passive result of forces generated elsewhere remains a matter of controversy.

In addition to the tubule/filament hypothesis that has been proposed to explain changes in cell shape involved in neurulation (cf. Karfunkel, 1974) other mechanisms have been suggested to account for the observed changes. Using lanthanum as a tracer, Karfunkel, Hoffman, Phillips & Black (1978) demonstrated that there are changes in cell adhesiveness during neurulation and optic cup formation in Xenopus and Ambystoma embryos and suggested that increased adhesion between the neuroepithelial cells may produce the observed cell shape changes. Similarly, Beebe, Feagans, Blanchette-Mackie & Nau (1979) in studies on chick lens development, have suggested that changes in cell shapes may result from alterations in cell volume. With increasing volume, the nucleus is displaced basally, resulting in the wedge-shaped cells. Contrary to Beebe et al. (1979), Odell, Oster & Burnside (1981) proposed that epithelial cell shaping changes were dependent upon a constant cell volume, a microfilament network at the apical pole of the cells and intracellular connections which would cause a wave motion of contraction and deformation.

The first appearance of microtubules in the optic vesicle of the mouse embryo occurs prior to cell elongation, although a few randomly oriented microtubules can be seen at earlier stages (1 – 2 somites). It has been suggested that microtubules may produce this elongation because of the temporal correlation between the appearance of microtubules in other areas of the neuroepithelium and cell elongation (Waddington & Perry, 1966). Microtubules were also suggested to function as a structural support (Waddington & Perry, 1966), in transport of cytoplasmic material (Lofberg, 1974; Burnside, 1973) and participation in nuclear migration during cell division (Messier, 1978). As discussed in the introduction, studies using microtubule inhibitors can cause neural tube and optic vesicle defects.

Since microfilaments have been shown to be functionally similar to muscle actin (Ishikawa, Bischoff & Holtzer, 1969), it has been suggested that they may function as an intracellular contractile apparatus (Wessells et al. 1971; Allison, 1973; Goldman, 1975; Wuerker & Palay, 1969). In many developmental processes that require changes in cell shape, microfilaments have been demonstrated in the cells involved (cf. Wessells et al. 1971, for review). During the development of the mouse optic vesicle, the microfilaments appear juxtaluminally during the time that the cells are becoming wedge shaped (EOV). However, short treatment of rat embryos at the 9- to 10-somite stage with a microfilament-disrupting drug, CD, does not cause abnormal optic vesicle formation, possibly because of the stage of development at the time of exposure to CD (Morriss-Kay & Tuckett, 1985).

Quantification of neuroepithelial cell shape alterations

A number of methods for measuring changes in regional topography and neuroepithelial cell shape have been devised. In one early attempt, Burnside & Jacobson (1968) reported that in the newt embryo, as apical surface area decreased, the thickness of the neuroepithelium increased. Hilfer, Brady & Yang (1981) measured regional changes in neuroepithelial size and areas through the chick optic vesicle using camera-lucida drawings. This technique has pointed out areas of expansion, but the individual cell shape alterations that produced the regional changes were not measured.

Schoenwolf & Franks (1984) have determined the number of cells of four basic shapes: spherical, spindle, flask and inverted flask in the chick neuro-epithelium during neurulation. This technique has isolated important regional patterns and has identified areas of ‘active’ morphogenetic bending; however, measurements of individual cells were not carried out. Brun & Garson (1983) have similarly determined the pattern of cell shaping changes during neurulation in the salamander and have reported an increase in the number of wedge-shaped cells in constriction zones. The pattern of events was complicated in Ambystoma embryos, because unlike chick and rodent embryos, the neuroepithelium is stratified, making the interpretation of measurements of only the superficial cells somewhat difficult.

While the amount of cell elongation, constriction or volume change may ultimately reflect species differences involved in the differing morphologies of the cephalic neural tube, morphometric studies of cell shape are of importance both because they demonstrate the degree of shape changes and also because they form the basis for the analysis of mutant eye development or teratogen-induced cell shape alterations.

The role of the neuroepithelial BL in optic vesicle formation

Like the BL of other developing epithelia, the neuroepithelial BL contains hyaluronate, fibronectin, entactin, laminin and type IV collagen (Morriss & Solursh, 1978; Newgreen & Thiery, 1980; Sternberg & Kimber, 1986; Tuckett & Morriss-Kay, 1986; Thorogood et al. 1986). This is the first report of HSPG in the neuroepithelial BL. In addition, the quail embryo appears to have type II collagen associated with the basal surface of neuroepithelium and surface ectoderm cells during early development (Thorogood et al. 1986). We also observed fibrillar substance, which may have been collagen surrounding the optic vesicle (see Fig. 7). Although the type II staining was closely associated with the BL of the quail, electron immunohistochemical localization needs to be completed. In addition, it would be interesting to see if type II collagen is also distributed similarly in the mammalian embryo.

The basal lamina may serve a structural role during neurulation, forming a stable base along which morphogenetic cell shaping changes can occur. In addition to providing a stable base, it seems likely that the presence of highly hydrated glycosaminoglycans in the BL may regulate contact between neuroepithelial cells and subjacent mesenchyme, while interactions between ECM and cytoskeleton may determine not only neuroepithelial shape but also patterns of protein synthetic activity.

Localized alterations in the BL composition might determine not only regional patterns of proliferation, but also might be expected to produce the outpouching of the optic vesicle, a critical event in determining not only the shape of the telencephalon but also of the developing facial region. Similarly, changes in the type or quantity of BL components may also play a role in the reversal of polarity and thickening of the lateral wall just prior to its invagination to form the optic cup.

As in the formation of other epithelial organs (c.f. mammary or salivary gland), deposition of the more structural BL component, type IV collagen, was patchy and uneven in areas of active cell shape change, while laminin was less dramatically affected. Interestingly, after its final form was determined, the even deposition of these two components was re-established.

Current investigations are in progress to extend these studies to the ultrastructural level and to examine the role of the neuroepithelial BL in producing regional characteristics of the developing nervous system.

The authors are grateful for technical assistance from Marilee Webb, secretarial assistance from Roberta Anderson and financial support from NIH grant HD 07097, AR-R23-35878, NS21108.

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