Light microscope observations on actin distribution ... - Development

J. Embryol. exp. Morph. 78, 23-32 (1983)

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Printed in Great Britain © The Company of Biologists Limited 1983

Light microscope observations on actin distribution during morphogenetic movements in the chick embryo* By DAVID OSTROVSKY 1 , JOSEPH W. SANGER 2 AND JAMES W. LASH 2 ' 3 From the Department of Anatomy, University of Pennsylvania and Department of Biology, Millersville University of Pennsylvania, Pennsylvania

SUMMARY

The cellular distribution of actin during two morphogenetic processes in the chick embryo has been observed, using a high-resolution fluorescent technique, with heavy meromyosin as a probe. These cytoskeletal elements have been implicated in all cell and tissue movements in the embryo. It is now commonly accepted that microfilaments are necessary to provide the motive force for morphogenesis. Two morphogenetic movements in the early embryo have been studied at the light microscope level. During somitogenesis, the mesenchymal segmental plate becomes transformed into a meristic series of transient epithelial vesicles. Again, actin distribution is diffuse and random before the morphogenetic event. During epithelialization, actin becomes prominent in the apical regions of the epithelial cells. Cells in the somitic epithelial vesicles, the core cells, appear to be passive participants in this process, and consequently show no distinct cellular localization of actin.

INTRODUCTION

There have been many reports implicating microfilaments in morphogenetic processes. Baker & Schroeder (1967) described a role for microfilaments in amphibian morphogenesis. Burnside (1971) also noted their role in amphibian neurulation. Cloney (1966) and Lash, Cloney & Minor (1973), in their studies on ascidian metamorphosis, showed that microfilaments provided the motive force for epithelial contraction during tail resorption, and that this process could be reversibly inhibited with cytochalasin B. There have since been many reports analysing the role of these cytoskeletal elements in cell and tissue movements. It is now widely accepted that microfilaments are indeed actin and myosin, and provide the mechanism for cell and tissue movements (cf. Jacobson & Ebendal, * Supported by NIH grants GM 25653 and HL 15835 (JWS) and HD 00380 and HD 15985 (JWL). 1 Author's address: Department of Biology, Millersville University of Pennsylvania, Millersville, Pennsylvania 17551, U.S.A. 2

Authors' address: Department of Anatomy, School of Medicine/G3, University of Pennsylvania, Philadelphia, Pennsylvania 19104, U.S.A. 3 Reprint requests.

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D. OSTROVSKY, J. W. SANGER AND J. W. LASH

1978; Burgess & Schroeder, 1979; Sanger & Sanger, 1979; Pepe, Sanger & Nachmias, 1979). With some notable exceptions (e.g. Sadler, Greenberg, Coughlin & Lessard, 1982), actin distribution in cells and tissues during morphogenesis has been analysed using transmission electron microscopy. Fluorescent heavy meromyosin can be used in a one-step procedure to identify actin in cells and tissues on a light-microscope level (Sanger, 1975a,b,c). Using this technique, we have observed with the light microscope the cellular distribution of actin during two morphogenetic processes in the chick embryo; neurulation and somitogenesis.

MATERIALS AND METHODS

Embryos Stage-8 (Hamburger & Hamilton, 1951) White Leghorn chick embryos were used for studies on neurulation, and HH Stage 12 for studies on somitogenesis. Histological preparation These procedures were modified from those of Sainte-Marie (1962) and Franklin & Martin (1980). The embryos were removed from the yolk, pinned to the bottom of a paraffin-coated dish, and covered with phosphate-buffered saline (PBS) at 4°C. The region to be analysed was marked with carbon particles (Fig. 1). After marking, the PBS was removed and replaced with 3-5 % formaldehyde (made up fresh from paraformaldehyde) in PBS. Embryos to be treated with cytochalasin B (Sigma, St. Louis, Mo.) were also removed from the yolk and

Fig. 1. A whole mount of a HH stage-12 chick embryo, with two carbon marks (arrows) delimiting the area to be analysed for somitogenesis. Asterisks mark the area analysed for neurulation. Segmental plate (a), forming somites (b), and nascent somites (c) are indicated. Micrometer markings for measurements can be seen across the bottom of the photograph. X1800.

Actin distribution during chick morphogenetic movements

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placed with the drug (lOjUg/ml) for l h at room temperature (20-22 °C) before fixation. The embryo was fixed at room temperature for l h . After the initial fixation, the embryos were removed to a small plastic dish containing PBS, and the carbon-marked area isolated with dissecting knives. From this point, the PBS was deaerated to prevent small bubbles from attaching to the embryo fragments. Immediately after isolation, the embryo fragments were transferred to 100 % methanol (4°C) and placed in a freezer at — 20 °C. The methanol was changed every 20min, and the total fixation time was l h . The tissues were then rehydrated through a series of ethanol solutions at 4°C into PBS, also at 4°C. After three 90min rinses in PBS (room temperature), the tissues were exposed to the heavy meromyosin probe for 18 h at 4°C. The heavy meromyosin was labelled (Sanger, 1975a,b) with lissamine rhodamine B sulphonyl chloride (Molecular Probes, Inc., Junction City, Oregon). After this treatment, the tissue was thoroughly rinsed with PBS (three washes, 60min each) and dehydrated through an ethanol series before embedding in Araldite. Tissues were sectioned at 1 /im using an LKB microtome III. The plastic sections were transferred to a 3" x 1" microscope slide with a wire loop and the slide set upon a 100 °C hotplate for 5 min. The sections were covered with Histoclad (Clay-Adams, New York) and a No. 1 thickness coverslip. Observations were made using a Zeiss IV Fl epifluorescence condenser equipped with rhodamine filters. Photographs were taken using Kodak Tri-X (ASA 400)filmwhich was then developed using Kodak HC-110 developer. RESULTS

Somitogenesis Sections, were examined from three different regions of the stage-12 embryo; the segmental plate, somites in the process of forming from the segmental plate, and nascent somites showing the organization of an epithelial vesicle. A cross section of the segmental plate is shown in Fig. 2. There is no detectable localization of actin at this stage of development. All cells exhibit a diffuse pattern of actin staining. In some cells there is an asymmetric distribution which is due to the absence of actin in the region of the nucleus. As the somite is forming, an indication of the future epithelial vesicle can be seen (Fig. 3). Epithelialization is more advanced in the ventrolateral portion of the somite (Fig. 3), but will soon encompass the entire somite (Fig. 4). During somite formation there is both an increase in cell number as the result of proliferation, and a rearrangement of the cells from a loose mesenchyme (cf. Fig. 2) into an epithelial vesicle (cf. Figs 3 and 4). There is no developmental correlation to account for the first occurrence of epithelialization in the ventrolateral portion of the forming somite. When the somite is an epithelial vesicle (Fig. 3), there is a prominent localization of actin in the basal portion of the epithelial cells of the ventral and medial somite wall (Fig. 4). This is the region of the somite that will soon migrate in a

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D . OSTROVSKY, J. W. SANGER AND J. W. LASH

Fig. 2. A cross section through the segmental plate. There is no obvious tissue localization of actin. In Figs 2-9, actin localization is shown as white, against a grey or black background. Neural tube (n), epithelium (ep), and endoderm (en) designate tissue orientation. X1800. Fig. 3. A cross section of a somite in the process of segmentation and epithelialization. The most prominent actin localization is marked by the arrows. Neural tube (n). X1800.


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Fig. 4. A cross section of a nascent somite. The somite is now an epithelial vesicle, containing core cells (c). Actin localization is most prominent in the apical portion of the epithelioid somite cells (arrows). Neural tube (n). X1800. ventromedial direction as the mesenchymal sclerotome. The epithelial wall is

prominently stratified at this stage. The cells in the centre of the epithelial somite (Figs 3 and 4) are the 'core cells', and show no actin localization. These cells are thought to contribute to the future sclerotome of the older somite. Neurulation Neurulation was studied at the level of the sinus rhomboidalis through the region of the nascent somites (asterisks in Fig. 1). This enabled us to obtain different stages in the sequence of neurulation (Figs 5-9). In the region of the sinus rhomboidalis the neural epithelium is just beginning to fold, and in the region of the nascent somite, neurulation is completed. In all stages of neurulation there was an increasing concentration of actin in the apical regions of the neuroepithehum (Figs 5-9). During neurulation the apical surface (Fig. 5) becomes the luminal surface (Fig. 9), and there is a marked concentration of actin in the apical portion of these cells. Actin localization in the basal portion of the neuroepithehum is sparse in the early stages (Fig. 5), and barely detectable during the final stages of neural tube formation (Figs 7 and 9). This is a severe disruption of the actin localization pattern after treatment with

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6 Fig. 5. A cross section of the neuroepithelium at the beginning of neurulation. Whereas actin is distributed diffusely throughout the epithelium, there is a noticeable concentration at the apical surface (arrows). X1800. Fig. 6. A later, V-shaped, stage in neurulation. The basal portion of the neuroepithelium is almost devoid of actin, whereas the apical portion of the cells show pronounced actin localization (arrows). X1800.


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8 Fig. 7. A cross section of a closure stage of neurulation where the neural tube is almost formed. Pronounced actin localization is evident in the apical regions of the neuroepithelial cells (arrows). Somites (s), Notochord (n). X1800. Fig. 8. A comparable stage of neurulation as shown in Fig. 7, except this embryo had been treated with cytochalasin B. Actin localization is completely disrupted, and the process of tissue folding evident in Fig. 7 shows signs of reversal as the epithelium tends to flatten. Tissue disruption is also evident in the region of the somites and notochord. X1800. 2

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D. OSTROVSKY, J. W. SANGER AND J. W. LASH

n

9 Fig. 9. A cross section of a completed neural tube (n). Some actin localization is still observed in the apical regions of the neuroepithelial cells. There is, however, a much greater concentration of actin in the apical portion (arrow) of the epithelial somite cells in the nascent somite. A higher magnification of this nascent somite is illustrated in Fig. 4. xllOO.

cytochalasin B (Fig. 8). Some residual localization remains in the apical region of the cells (Fig. 8), and the folding neural tube begins to unfold (cf. Figs 7 and 8).

DISCUSSION

The patterns of actin localization during neurulation and somitogenesis are analogous to that observed in other epithelial systems where morphogenetic processes are accompanied by actin localization and tissue contraction at the tissue interface which is becoming concave (Lash et al. 1973; Sadler et al. 1982; Vakaet & Vanroelen, 1982). The process of epithelialization during somitogenesis is poorly understood. Actin localization alone cannot be readily understood in terms of the mechanism of somite formation; it is, however, a significant factor in the process. It is interesting that the dorsolateral aspect of the forming somite appears to start epithelialization after the medial and ventral aspect. The dorsomedial surface of the somite will maintain cell-to-cell contact for a longer period of time than the ventromedial surface. It is the most dorsal portion (dermatome) that will migrate to the epithelium to form the dermis of


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the skin whereas the cells just beneath it (the myotome) will remain in close association as they differentiate into muscle. The relation between actin distribution and these subsequent differentiative events is unknown. It may be significant that the core cells, which appear to be passive participants in the process of somitogenesis, never show indications of actin localization. The pattern of actin localization in the neuroepithelium is also in agreement with the observations that localization and cellular contraction occur in the region of the epithelium that is changing its shape to become a concave structure (cf. Karfunkel, 1972; Bancroft & Bellairs, 1975; Nagle & Lee, 1980). This process has been described by Sadler et al. (1982) in the mouse embryo, and we have extended their observations to obtain more direct evidence of actin involvement. Many reports have indicated that actin localization is disrupted and morphogenetic processes cease in the presence of cytochalasin B (Karfunkel, 1972; Lash etal. 1973; Chernoff & Lash, 1981). As Chernoff & Lash (1981) have shown this to be true for the forming somite, we see a similar phenomenon during neurulation. Similar observations in chick embryo neurulation were made by Karfunkel (1972). Cytochalasin B disrupts the actin pattern of localization in the neuroepithelium, and the actin becomes randomly dispersed. Subsequently the process of neurulation not only ceases, but the neuroepithelium was seen to flatten again. Thus, the pattern of actin localization during morphogenetic movements involving the bending of epithelial sheets or the meristic segmentation of segmental plate mesenchyme can be readily visualized in the light microscope using a high-resolution technique for actin distribution. With a carefully selected series of different developmental stages and a three-dimensional computer-assisted reconstruction, this technique has the potential for giving useful information concerning the mechanisms of these processes. This type of information is not

readily available using the procedure of transmission electron microscopy, the most-commonly used method at present.

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R. M. & MARTIN, M. T. (1981). Pre-embedding immunohistochemistry as an approach to high resolution antigen localization at the light microscope level. Histochem. 72, 173-190. HAMBURGER, V. & HAMILTON, H. L. (1951). A series of normal stages in the development of the chick embryo. /. Morph. 88, 49-92. JACOBSON, C.-O. & EBENDAL (1978). Formshaping movements in neurogenesis. Zoon 6, 257 pp. Almqvist & Wiksell, Uppsala. KARFUNKEL, P. (1972). The activity of microtubules and microfilaments in neurulation in the chick. /. exp. Zool. 181, 289-302. LASH, J. W., CLONEY, R. A. & MINOR, R. R. (1973). The effect of cytochalasin B upon tail resorption and metamorphosis in ten species of ascidians. Biol. Bull. 145, 360-372. NAGLE, R. G. & LEE, H.-Y. (1980). Studies on the mechanism of neurulation in the chick: Micro filament-mediated changes in cell shape during uplifting of neural folds. /. exp. Zool. 213, 391-398. PEPE, F. A., SANGER, J. W. & NACHMIAS, V. T. (1979). Motility and Cell Function, 310 pp. New York: Academic Press. SADLER, T. W., GREENBERG, D., COUGHLIN, P. & LESSARD, J. L. (1982). Actin distribution patterns in the mouse neural tube during neurulation. Science 215,172-174. SAINTE-MARIE, G. (1962). A paraffin embedding technique for studies employing immunofluorescence. Histochem. Cytochem. 10, 250-256. SANGER, J. W. (1975a). Changing patterns of actin localization during cell division. Proc. natn. Acad. ScL, U.S.A. 72, 1913-1916. SANGER, J. W. (19756). Intracellular localization of actin with fluorescently labelled heavy meromyosin. Cell Tissue Res. 161, 431-444. SANGER, J. W. (1975c). The presence of actin during chromosomal movement. Proc. natn. Acad. ScL, U.S.A. 72, 2451-2455. SANGER, J. W. & SANGER, J. M. (1979). The cytoskeleton and cell division. In The Cytoskeleton in Normal and Pathological Processes, Methods and Achievements in Experimental Pathology, Vol. 8, pp. 110-142. Basel: Karger. VAKAET, L. & VANROELEN, C. H. (1982). Localization of microfilament bundles in the upper layer of the primitive-streak-stage chick blastoderm. J. Embryol. exp. Morph. 67, 59-70. FRANKLIN,

{Accepted 17 June 1983)