Modulation of cytoskeletal organization during insect follicle cell morphogenesis

TISSUE 019X6

& CELL Longman

1986 18 (5) 741-752 Group

UK

Ltd

A. J. WATSON* and E. HUEBNERt

MODULATION OF CYTOSKELETAL ORGANIZATION DURING INSECT FOLLICLE CELL MORPHOGENESIS Keywords:

Folliclc

ABSTRACT. changes

in lateral

involved

in

orientation skeletal cylindrical

of

patent

m hoth

follicle area

dynamics

shrinkage. cplthelium

in

follicle

ccl1

addition

to

junctional

don.

address:

of Zoology, Ontario.

:Author

Cell

Canada to

Science

University N6A

whom

Laboratones,

of Western

Depart-

Ontario,

Lon-

5B7. correspondence

should

be

addressed Recewed

48

12 May

juvcmle

mlcrofdament

folhclc apical

Promment follicle

is present

microfilament ccllr.

The

hormone for

the

cyto-

beneath

lateral

the

cells.

In

of microtuhands

arc not

vitcllogemc

in the suhplasmalemmal microvilli.

vitellogenesi\ stimulated

formatlon

the

follicle

distribution

cells and the apical during

cytoskelcton.

A well-developed

vitellogemc

a dispcraed

are

abundance,

revealed

stage\.

hand of microfilaments cells

relative

muoscopy

c~ten~wc

elements

(NA’K’] of

The

changes

cmpha\ize a

patent

a third

ATPaw folhcle

ccl1 ccl1

Rhodnius

The Rhodnius prolixus follicular epithelium undergoes a programmed sequence of morphological alterations during oogensis (Huebner and Injeyan, 19Sl). These changes are a significant component of follicle cell morphogenesis. Cell shape and cell to cell contacts vary from tall columnar, tightly apposed cells during pre-vitellogenesis to decreased cell height, irregular shape and reduced cell to cell contact during vitellogenesis (Huebner and Anderson, 1972; Huebner and Injeyan, 1981; Watson, 1984). This transformation from a tightly packed epithelium to one with large extracellular spaces is known as ‘patency’ and provides the pathway for extraovarian yolk precursors to reach the oocyte (Davey *Present

follicle

modulation.

Introduction

ment

planes. vitcllogenic

to adjacent

in lateral

the

microtubules and

cells contain

a prominent

connecting

arrangement

and

orientated

or apical

contain

in

to post-wtcllogenic

and cross-sectional

and in the projectlons

and

clcctron

involves

Cytoskeletal

investigating

microtuhule

prc-vitellogcmc

prc-vitellogcnic

component.

cell

mlcrotuhulcs

oogcnesis

cpithelium.

hy

transmlarion

longitudinally

vitellogenic

longitudmal

in cytoskelctal

and

non-patent

cells do howcvcr

follicle

tuhuhn.

Rlrodnrus

a patent

ah assessed

pre-follicular of

the

lateral

in the

casentlal

the

vitellogeneais.

during

creating

change

of

from

arrangement

plasmalemma

abundant

cell shape.

immunofluorcscence

organization

oogencais.

morphogenesis

ccl1 shape

and

contrast

cytoskcleton, cell

follicle

this

Antl-tuhuhn

hulcs

cells.

Follicle

1986.

141

and Huebner, 1974; Davey, 19X1; Telfer ef al., 1982). Cytoskeletal elements play important roles in morphogenetic processes linked to cell shape modification (Cohen, 1979a. b). The Rho&us follicle provides an ideal system to analyse the events and control of epithelial cell shape during the follicle differentiation, overall epithelium permeability and epithelial morphogenesis. Only two studies (Huebner, 1976; Abu-Hakima and Davey , 1977~) have considered the role of the follicle cell cytoskeleton in mediating the cell shape changes associated with ‘patency’. Although the presence of microtubules and microfilaments in Rhodnius follicle cells is established (Huebner and Anderson, 1970; Huebner, 1976; AbuHakima and Davey, 1977c), a detailed analysis of their developmental changes and involvement in follicle cell morphogenesis and differentiation has not been done. Studies investigating cytoskeletal mediated cell shape control usually utilize tissue culture cell or isolated cell systems rather

WATSON

742

than intact tissues (Osborn and Weber, 1977; Wolosewick and Porter, 1979; Albertini et al., 1984). Difficulties in the application of the specialized methods required for cytoskeletal isolation of intact tissues are principally responsible. With the recent development of methods adapting immunocytochemical localization of cytoskeletal proteins to paraffin and plastic embedded tissue sections (Bussolati er al., 1980; Rodning et al., 1980; Kramer, 1981; Hogan and Smith, 1982), it is now possible to reassess and extend our knowledge of the role of the follicle cell cytoskeleton during the cell shape changes associated with follicle cell differentiation and ‘patency’. Through the integration of anti-tubulin immunofluorescence methods with normal microscopy, this transmission electron paper will characterize the relative abundance, orientation and dynamics of the follicle cell microtubule and microfilament cytoskeleton within all follicle cell developmental stages. In particular the variation in cytoskeletal organization between the patent, lateral vitellogenic follicle cells and the nonpatent, apical vitellogenic follicle cells is presented. Materials and Methods Animals were maintained at 27°C in high established using routinely humidity methods (Huebner and Anderson, 1972). Only mated adult females were used, and these animals were fed regularly at appropriate intervals to obtain ovarioles at all specific stages of oogenesis. Prior to fixation, the ovaries were dissected and separated into the seven individual ovarioles by desheathing each ovariole in Rhodnius saline (Maddrell, 1969). Transmission electron microscopy

Ovarioles were pre-fixed 1 hr at 4°C in either a modified Karnovsky’s fixative (Huebner and Anderson, 1972) or a modification of Luftig et al. (1977), microtubule stabilization fixative, containing 3.0% glutaraldehyde in PHEM buffer (Schliwa and Van Blerkom, 1981), pH 6.9, 1 mM GTP. Several fixations also included the addition of 0.5% w/v tannic acid (TA) and 0.05% w/v Saponin (Maupin and Pollard, 1983). Following a buffer wash, ovarioles were

AND HUEBNER

post-fixed for 15 min in 0.5% w/v osmium tetroxide (0~0~) in 0.1 M sodium cacodylate buffer, pH 7.2 (Maupin-Szamier and Pollard, 1978). Following rapid dehydration in a cold ethanol series, the ovarioles were infiltrated and embedded in an EponAraldite mixture (Anderson and Ellis, 1965). Semithin and thin sections were cut with a Sorvall MT2-B ultramicrotome. Semithin sections were stained in 1.0% toluidine blue in 1.0% borax. Thin sections were routinely stained and examined in an AEI6B or 801s electron microscope at 60 kV. Immunofluorescence methods Fixations

For paraffin embedding, ovarioles were fixed in 3.0% paraformaldehyde, 0.5% glutaraldehyde in PHEM buffer (Schliwa and Van Blerkom, 1981) pH 6.9+1 PM GTP for 1 hr, followed by ethanol dehydration, and the routinely processed for paraffin embedding in Tissue Prep 56.5”C”* (Humason, 1979). Ovarioles embedded in EponAraldite (processed as already described) (Anderson and Ellis, 1965), were fixed with l,O% paraformaldehyde in PHEM buffer (Schliwa and Van Blerkom, 1981), pH 6.9++,~M GTP for 1 hr. Paraffin sections (5-10 pm) were cut with a Jung rotary microtome. The sections were mounted on albumin-coated glass slides, deparaffinized in toluene, hydrated and placed in Dulbeccos phosphate-buffered saline (PBS). Plastic sections (l-2 pm) were cut with a Sorvall Porter-Blum MT2-B ultramicrotome and heat fixed on glass slides. The embedding media was removed by exposure to saturated NaOH in absolute ethanol for 30 min (Lane and Europa, 1965; Bussolati et al., 1980). The slides were then washed in 100% ethanol and hydrated to PBS. Antisera

The primary (1’) antibodies used were: (1) rabbit anti-tubulin against chicken embryo brain tubulin (Miles Laboratories), 1: 10 dilution; (2) rabbit anti-tubulin against sea urchin tubulin (gift from Dr. Keiji Fujiwara, Department of Anatomy, Harvard Medical School), 1: 50 dilution. The secon*‘%ademark

Fisher Scientific

FOLLICLE CELL MORPHOGENESIS

dary antibody for all experiments was a fluorescein conjugated goat anti-rabbit IgG (Miles Laboratories) 1: 16 dilution. Immunofluorescence

tests

Routine indirect immunofluorescence procedures were applied (Weber and GroschelStewart, 1974; Osborn and Weber, 1982), as follows; tissue sections were first incubated for 1 hr at 37°C in a 1.0% w/v bovine serum albumin (BSA) type V, (Sigma Chemical Co.) in PBS to minimize non-specific staining. The sections were then incubated for 1 hr at 37°C with one of the 1” antibodies, followed by extensive washings in PBS. The sections were finally incubated in the 2°C antibody for an additional hour. Following this step the sections were washed in PBS, mounted with 50% glycerol in PBS, coversliped and examined with a Zeiss photomicroscope II equipped with epi-fluorescence optics. Fluorescence light micrographs were taken on Kodak Tri-X Pan 135 film at IS0 1000, and developed in Acufine developer (Acufine Inc.).

143

cells, although microfilaments are present at the basal cell end, seen in cross-section next to the cross-sectioned microtubules (Fig. 1). Previtellogenic cells

The columnar, tightly apposed. previtellogenic follicle cell epithelium shows a bright anti-tubulin fluorescence pattern restricted to the lateral cell edges (Fig. 4). Longitudinal sections particularly those near or grazing the cell membrane reveal abundant bands of longitudinally orientated microtubules (Fig. 3). The microtubules are concentrated in the middle or nuclear region of the cells (Figs 4, 5). Cross-sections in the apical and basal area of previtellogenic cells contain fewer microtubules (Fig. 6). The results suggest that the longitudinally arranged microtubules encircle the cell circumference in the form of a cylinder. Microfilament bands are not abundant within pre-vitellogenic cells, however a subplasmalemmal band, parallel to the basal lamina is present in the basal area (Fig. 7).

Control

Vitellogenic cells

Usual controls were conducted to assess the background fluorescence imparted to the tissue from the fixation and embedding methods, plus the non-specific binding of the antibodies (Osborn and Weber, 1982). A check of the tubulin labelling in the microtubule rich trophic core and trophic cords (Huebner and Anderson, 1970) was also conducted as an in situ positive control.

The pre-vitellogenic follicle cells, remain tightly apposed until vitellogenesis is initiated. With the onset of vitellogenesis and the gradual formation of the extracellular spaces (patency), dramatic changes in the microtubular and microfilamentous organization of the lateral fohicle cells occurs. Anti-tubulin immunofluorescence of midvitellogenic follicles shows that the apical follicle cells display a distinctly different fluorescence distribution from the lateral follicle cells (Fig. 8). Bright longitudinal bands of fluorescence along the lateral cell edges (similar to that observed within the pre-vitellogenic follicle cells), are present within the columnar, tightly apposed apical follicle cells (Fig. 8). The patent lateral follicle cells do not show a bright linearly orientated fluorescence within their cytoplasm. Instead, the fluorescence is confined to discrete, thin, anastamosing strands throughout the cell cytoplasm (Figs 8, 11, 12). The use of thinner plastic sections greatly increases the resolution and clearly shows this pattern of anti-tubulin fluorescence (Fig. II). Longitudinal cell sections of apical vitellogenic follicle cells reveal dense bands of long microtubules extending the

Results Prefollicular cells

Anti-tubulin immunofluorescence of the pre-follicular cells revealed a bright band of fluorescence along the lateral cell edges and a second band at the basal end of the cells resting on the basal lamina. Transmission electon microscopy verified this fluorescence pattern coincides with the locations of microtubules (Figs 1, 2). Along the lateral cell edges groups of short longitudinally orientated microtubules are present (Fig. 2). Microtubules orientated at right angles to the lateral microtubules are arranged in the basal ends of those cells resting on the basal lamina (Fig. 1). Microfilamentous regions are not prominent within these

744

WATSON AND HUEBNER

entire cell length (Fig. 10). The orientation in the apical cells remains similar to what was observed in the pre-vitellogenic follicle cells. Examination of a variety of section

planes through the apical follicle cells substantiates this pattern if distribution (Fig. 9). Longitudinal sections of the cell cortex dramatically reveal the abundance of long

Expianation of figures

Abbreviations used in figures: AP. apical vitellogenic follicle cells; B, basal lamina; LF, lateral vitellogenic follicle cell; M, mitochondria; MF, microfilaments; MT, microtubules; NU, nucleus; 0, oocyte, P, plasma membrane; PF, prefollicular tissue. Fig. 1. Shows the cross-sectional profiles of microtules (arrows) with associated microfilaments concentrated at the basal ends of pre-follicular cells adjacent to the basal lamina. x 136,ooO. Fig. 2. Longitudinal profiles of microtubules arc isolated along the lateral cell edges of the pre-follicular cells. X70,000. Fig. 3. This section of the cell cortex reveals the dense array of longitudinally orientated microtubules just under the plasmalemma of pre-vitellogenic follicle cells. ~37,000. Fig. 4. Fluorescent micrograph of an Epon-Araldite embedded tissue section shows the bright lateral lines of anti-tubulin fluorescence observed in pre-vitellogenic follicle cells. X1000. Fig. 5. Microtubules (arrow) are organized along the lateral edges of the nuclear area of pre-vitellogenic follicle cells. ~32,000. Fig 6. Microtubules become less numerous in the poles of these cells, as shown by this cross-section through the basal end of a pre-vitellogenic cell. ~20,000. Fig. 7. A microfilamentous area is present, parallel to the basal lamina in a pre-vitellogenic follicle cell. Tannic acid fixation. x45$00. Fig. 8. Anti-tubulin immunofluorescence of a Spm paraffin section, comparing fluorescence pattern between adjacent lateral and apical vitellogenic follicle cells. x700.

the

Fig. 9. Cross-section with large numbers of microtubules (arrows) orientated longitudinally in the apical vitellogenic follicle cells. ~28,000. Fig. 10. This TEM displays the length and close opposition of the prominent microtubules within the apical vitellogenic follicle cells. x 160,000. Fig. 11. This Epon-Araldite section shows a delicate irregular pattern of fluorescence in the patent lateral follicle cells. x750. Fig. 12. Anti-tubulin immunofluorescence, revealing the overall diffuse, fluorescent pattern observed within patent lateral follicle cells as seen in a paraffin section. X900. Fig. 13. Numerous microtubules (arrows) in both longitudinal and cross-section profile are observed in this longitudinal TEM section of a patent lateral follicle cell. x25,OCO. Fig. 14. The exclusion of ribosomes and other cell organelles by the subplasmalemmal of microfilaments is revealed in this TEM. ~32.0GO. Fig. 15. The anti-tubulin lateral follicle cells. X900.

immunoflourescent

band

pattern of a paraffin cross-section of patent

Fig. 16. This cross-section TEM show microtubule (arrows) distribution within the patent lateral cells. Note there are both cross-section and longitudinal profiles of microtubules. x30.000

WATSON

microtubules (Fig. 10). These dense accumulations of microtubules in the apical follicle cells are greater than those of any other follicle cell stage or substage. They are also the longest microtubules observed at any follicle cell stage. The combination of their length and large numbers makes them the predominant cytoplasmic feature of the apical follicle cells. The microtubules of the apical vitellogenic follicle cells, as in pre-vitellogenic cells, are concentrated in the nuclear region of the cell. Although some of the microtubules extend into the basal and apical ends of these cells, their arrangement becomes less regular. This accounts for the diffuse and irregular antitubulin fluorescence seen in apical and basal areas of the cells (Fig. 8). The patent lateral follicle cells contain numerous microtubules (Figs 13, 16). However, in sharp contrast with the apical vitellogenic cells. these microtubules are shorter and not parallel, but rather irregularly arranged so various microtubule section planes are seen in any cell section (Figs 17, 18). The immunofluorescence staining pattern confirms this (Figs 11, 12, 15). The lateral (unlike apical) vitellogenic follicle cells also contain extensive microfilament arrays. These microfilaments are organized into a subplasmalemmal band as clearly revealed in the tannic acid-Saponin fixation (Fig. 14). Microfilaments are also concentrated within the lateral arms connecting adjacent patent cells (Fig. 21). As the extracellular spaces enlarge during late vitellogenesis these arms become more ex-

Figs 17. IX. The patency Fig.

19.

The

fluorescence Fig.

irregular

IS depicted

20.

A few

Discussion The formation of a patent follicular epithelium is an essential developmental event during oogenesis within many insect and vertebrate species (Anderson, 1969; Perry and Gilbert, 1979; Dumont, 1972; Telfer et al., 1982; Abraham et al., 1984). This process provides a pathway for yolk proteins synthesized at extraovarian sites to reach the oolemma for pinocytic incorporation. The associated cell shape changes in the lateral follicle cells are developmentally

orientation

anti-tubulin

of muotuhules

magnification

follicle

are found

(arrows)

electron

immunofluorescence

the post-vitellogenic microtubules

IIUEBNEK

tensive with microtubules becoming a common constitutent as well. The apical projections of the patent lateral follicle cells, which maintain oocyte-follicle cell contact during vitellogenesis also contain dense microfilament bands (Fig. 22). Following vitellogenesis the lateral cells increase their cell volume and form a tightly packed epithelium. Anti-tubulin immunofluorescence results show a diffuse fluorescence around the lateral and basal cell extremities (Fig. 19). The few microtubules found in these cells are parallel to the basal lamina whereas some are localized along the lateral cell edges beneath the plasmalemma (Fig. 20). Clearly, there is an alteration in the number and orientation of the microtubules during this last phase of follicle cell morphogenesis. Microfilamentous areas are not prominent with only a few areas present along the lateral cell edges.

in these higher

within

AND

pattern cells.

in hasal

in lateral

reveals

follicle

cells during

x IIO,(NK), ~350,000.

micrographs. a

basal

dtstribution

of

X%0.

ends of the post-vitcllogenic

folhclc

cells.

the

neighhouring

X6O.OCa. Fig. patent Fig.

21.

Dense

lateral 22.

XhS,OOO.

array

follicle

The

of microfilaments

cells.

within

lateral

arms

connecting

X35.000.

microfilaments

wthin

a apical

projection

of

a patent

lateral

follicle

cell.

WATSON

regulated and arise due to the integration of at least three separate cellular processes. These included: (1) [Na+K+] ATPase induced follicle cell shrinkage. mediated by juvenile hormone (JH) (Abu-Hakima and Davey, 1975, 1977a, 1979; Huebner and Injeyan, 1980; Ilenchuk and Davey, 1983); (2) disassembly and reorganization of the septate and gap junctions along the lateral follicle cell membranes (Huebner and Injeyan, 1981); and (3) cytoskeletal reorganization within the lateral follicle cells correlated with the onset of vitellogenesis. Of these three cellular processes, the cytoskeletal involvement is the least studied. A cytoskeletal role in patency was initially suggested by Huebner (1976) and Abu-Hakima and Davey (1977b), since cytoskeletal disruptors, cytochalasin B and colchicine inhibited the juvenile hormone (JH) induced patency response. Huebner and Anderson (1970, 1972) and AbuHakima and Davey (1977~) described the ultrastructural presence of microtubules and microfilaments in Rhodnius follicle cells. No study has compared the changes in arrangement and abundance of these cytoskeletal elements in follicle cell differentiation during oogenesis in detail, and also utilized immunofluorescence to provide a clear view of the overall changes. The immunofluorescence and TEM results both substantiate that the initiation of patency is correlated with a microtubular reorganization in the lateral follicle cells. This is reflected in a change from the highly organized cylindrically arranged microtubules of the pre-vitellogenic cells to the random pattern observed with the midvitellogenic, lateral follicle cells. Huebner and Anderson (1972) first reported that the vitellogenic apical, follicle cells remain columnar and tightly apposed during patency. The results from our study suggest that the maintenance of a well-organized cylindrically orientated microtubular cytoskelton precludes any possible cell shape change. The vitellogenic, lateral follicle cells decrease in cell height at the onset of patency (Abu-Hakima and Davey, 1977a). This decrease in height is correlated with the reorganization of microtubules in the lateral follicle cells reported here. The formation of a random distribution of microtubules

AND HLJEI~h’L‘Ef<

would greatly decrease the resistance an organized cylindrical arrangement of microtubules would produce against a change in cell height or volume. The microtubules arranged randomly could still support the new cell shape but at the same time would not inhibit any inward movement of the cell boundaries associated with cell shrinkage. The overall process of cell shrinkage by [Na’K+) ATPase mediated pumping of fluid out of the cell (Davey. 1981) would therefore be restrained until the microtubular cytoskeleton becomes rearranged. The conclusion that microtubules play a key role in the dynamics of lateral follicle cell shape in Rhodnius is supported by extensive literature in other eukaryote systems which demonstrate that microtubules maintain and modulate cell shape (Burnside, 1973; Barrett and Dawson, 1974; Tucker and Meats, 1976; Beckerle and Porter, 1983; Frixione, 1983; Albertini et al., 1984; Roos et al., 1984). The formation of prominent microfilamentous bundles within the patent lateral follicle cells. indicates they also contribute to the modulation of cell shape and in particular the formation of the lateral connections between neighbouring lateral follicle cells. Microfilaments are often transitory ceil structures as exemplified by the formation of the contractile ring or cleavage furrow of mitotic cells (Fujiwara rt al. lY78; Schroeder, 1981). Microfilaments can also form the main cytoskeletal constituents of very stable cell structures such as the intestinal brush border (Mooseker. 1976; Hirokawa et a/., 1982) or the sterocilia of the cochlear hair cells (Tilney and Saunders, 1983: Tilney et al., 1Y83). The production of a subplasmalemmal band of microfilaments within the lateral follicle cells could function similar to a contractile ring by producing an inward contractile force that contributes to the formation of the irregular cell shape. The scarcity of dense microfilamentous arrays within the pre-vitellogenic and apical vitellogenic follicle cells shows they are not essential for the maintenance of the columnar shape. The juvenile hormone (JH) mediated cell shape changes of the lateral follicle cells in Rhodnius involve a more complex interaction of cellular processes than generally assumed. JH either directly or indirectly

t’O1 I.I(‘L

.i,

F: (‘FCL.1. MOKPliOtiENESIS

coordinates the rearrangement of the microtubular and microfilamentous elements of the lateral follicle cell cytoskeleton; the disassembly and rearrangement of cell junctions (I Iuebner and Injeyan, 1981) and finally [ Na’ K-1 ATPase cell shrinkage (Davey. IYXI). The details of the dynamics of the follicle cell cytoskeleton during oogenesis, elucidated 111this paper, add to our understanding of the overall process of follicle cell ditferentiation. These results have also verified that the Nl&ni~s follicular epithelium

provides a suitable model system to \tud? the role of cy,toskeletal organization in the modulation ot cell shape in a differentiating tissue. Acknowledgements We thank Dr K. FuJiwara for kindl! \upplying the sea urchin anti-tubulin and W. Sigurdson and G. Kelly for helpful comments. Research was supported I>! NSERC grants to E.H. and an NSF.KC‘ Post-graduate Scholarship to A.W

752

WATSON

AND HUEBNEK

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Follicular modulation during oocyte development in an insect: formation and modification of septatc and gap junctions. Drvl. Biol.. 83, 101-I 13. Humason. G. L. 1979. Animal Tissue Techniques. 4th edn.. 661 pp. W. H. Freeman, San Francisco. Ilenchuk, T. T. and Davey. K. G. 1983. Juvenile hormone increases ouabain-binding capacity of microsomal prepartions from vitellogenic follicle cells. Can. J. Biochem. CeU BioL. 61, 826-831. Kramer, J. M. 1981 Immunofluorexent localization of PGK-I and PGK-2 coenzymcs with specific cells of the mouse testis. L)ev/. Biol., 87, 30-36. Lane. B. P. and Europa, D. L. 1965. Differential staining of ultrathin sections of Epon embedded tissues for light mwoxopy. J. Hittochern. C:vtochrm.. 13, 579. Luftig. R B.. McMillan. P. W.. Wcathcrbcc. J. A. and Weihing. R. R. lY77. lncrcased visualization of microtubules by an improved fixation proccdurc. J. Hurochrm. Cytochem. 25, 175-187. Maddrell, S. H. P. 1969. Secretion by the Malpighian tub&s of Rhodnm. 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