- Email: [email protected]
Microtubules during formation of the micropylar canal in Drosophila melanogaster
Cell Biology
International
Reports,
Vol. 8, No. 4, April
1984
317
MICROTUBULES DURING FORMATION OF THE MICROPYLAR CANAL IN DROSOPHILAMELANOGASTER LUKAS H. MARGARITIS Department
of Biology,
University
of Athens,Greece
ABSTRACT The micropylar apparatus in ~ro.sophiZa meZanogaster contains a 0,8um wide canal through which sperm entry occurs.During oogenesis the canal is formed by deposition of extracellular material (chorion) around long cellular processes filled with microtubules. INTRODUCTION Cell processes reportedly occur by means of cytoplasmic filament elongation; these include actin (as found in the stereocilia of the hair cells-Hirokawa and Tilney 1982, or during ameboid movement of many cell types-Taylor et al, 1980) or microtubules (as found in the filopodia of Protozoa-Travis and Allen, 1981). In most of the cases the cell processes are utilized for contour stability or for cell movement, In this report it is shown that microtubules may participate in the formation of the micropylar canal in the eggs of the Dipteran fi.meZanogaster. Each egg is the end product of a follicle which consists of the oocyte, 15 highly polyploid nurse cells and of about 1100 follicular epithelial cells; these cells have been subgrouped into at least ten categories (Margaritis et al, 1980). One of these categories called "border cells"(King 1970) is involved in the formation of the micropylar apparatus after stage lOB, as described by King and associates (Cummings and King, 1969) and by Margaritis (1980,1984). MATERIALS AND METHODS Oregon R flies of D.meZanogaster removed for ultrastructural analysis et al, 1979, 1980) except that PIPES fixatives and washing solutions. Thin orientations to insure perpendicular "border cell" processes.
were etherized and the ovaries as described elsewhere (Margaritis buffer at pH 7,4 was used in all sections were cut at various and longitudinal views of the
RESULTS AND DISCUSSION Mature follicles consist of the oocyte and the surrounding egg-shell layers which differentiate into regionally specific structures (Fig.1). The anterior pole of the egg contains the micropylar cone (Fig.2) which exhibits a narrow canal (w0,8um in diameter) (Fig.3):the whole micropylar complex is seen to consist of an (inner) "vitelline membrane protrusion" and an (outer) "hollow endochorionic cylinder"containing the canal. Longitudinal sections of follicles at late developmental stages, reveal that the canal is filled with cellular process(es) (Fig.4) deriving from the "border cells". High magnification views of the process[es) in crosssections and in longitudinal sections reveal the existence of microtubule bundles closely packed within them (Figures 5 and 6).The diameter of the 0309-1651/84!040317-05:$03.00/O
Q 1984 Academic
Press Inc. (London)
Ltd.
Cell Biology
318
total
cellular
process(es)
International is
roughly
Reports,
Vol. 8, No. 4, April
1984
0,8um.
The involvement of cytoskeletal elements in the modulation of the extracellular matrix is a phenomenon observed in several systems, such as in the collagenous stroma of the cornea (see Hay, 1983). However, details of the actual interaction taking place are largely unknown at present. This report suggests that the presence of microtubules directs the formation of a canal passing through an extracellular matrix(chorion)(Fig.7). Although it appears that the canal is formed mechanically (due to exclusion of the secreted chorion) there is evidence that microtubule polymerization follows an initial and the oocyte via a step of interaction between the "border cells" "special mass" of extracellular material (manuscript in preparation) (see also Fig.7); therefore by subsequent secretion, chorion is accumulating around the elongating process(es). The net result is then the formation of a canal wide enough (hO,8um) to allow the entry of the sperm (having a diameter of 0,6um- Hildreth and Lucchessi 1963) into the oocyte. Functionally,it is plausible to assume that the presence of microtubules resists tension applied by the secreted chorion so that a canal of certain dimensions is formed. The important of these findings lies in the fact that they provide additional information on the participation of microtubules to the supramolecular organization of secreted material. However, this report leaves behind for further investigation several important questions, such as, a) what is the actual "signal" which directs spatially and temporally the cellular process(es), b) which are the organizing centers of the microtubule polymerization, c) how many follicular cells are involved in the process(es). These questions are under investigation in the author's laboratory utilizing wild-type and mutants of D.meZanogaster. ACKNOWLEDGEMENTS This Research
work has been supported Foundation
by a grant
from
the National
Hellenic
REFERENCES
Cummings,M.R., and King,R.C.(1969) The cytology of the viteZZogenic stages during oogenesis in D.me2anogaster.I. Genera2 staging characteristics.Zeitschrift fur ZelZforschung 118,482-492 Hay,E.D.(1983) CeZZ and extraceZZuZar matrix.Their organization and mutual dependence. In:J.R.McIntosh led). Modern Cell BioZogy,vol 2, "SpatiaZ organization of eukaryotic celZs"pp.508-548. A.R.Liss, New York. HiZdreth,P.E., and Lucchessi,J.C.(1963) FertiZization in Drosophila. I. Evidence for the regular occurence of monospernty.Deve1opmentaZ BioZogy 6,262-278. King,R.C.(1970) Ovarian development in Drosophila melanogas-ter. Academic Press,New York. Margaritis,L.H.f1980) Choriogenesis in Drosophila meknogaster. A model system for the study of ceZZuZar differentiation. Mono,graph, Athens University. /4argaritis,L.H.(1984) Structure and Physiology of the egg-shell. In: G.A.Kerkut and L.I.GiZbert feds).Conp?rehensive Insect Physiology,
Cell Biology
International
Reports,
Vol. 8, No. 4, April
319
1984
Biochemistry and Pharmacology,~ol.l,ch7.Pergamon Press,Oxford. Margaritis,L.H.,E'etri,W.H.,and Wynrzn,A.R.f1979) Structural and image analysis of a crystalline layer from dipteran egg-shell. Geli! Biology International Reports 3,61-67. Margaritis,L.H.,Kafatos,F.C., and Petri,W.H.(1980) The egg-shell of D.melanogaster. I.Fine structure of the Layers and regions of the wild-type egg-shell.Journal of Cell Science 43,1-35. Taylor, D.L., Wang,Y.-L., and Reynolds,G.(1980) Contractile basis of ameboid movement. VII Distribution of f7,uoreseently 1abeZed aetin in living anaebas. Journal of Cell Bioloa 86, 590-598 Travis,J.L., and Allen,R.D. 11981) Studies on the rrwtility of the Foraminifera. I. Ultrastructure of the retieulopodial network of Allogromia laticollaris (Arnold). Journal of Cell Biology 90, 211-221 Received
:
13th February
Accepted:
1984,
22nd March
1984.
Figure 1. Whole mount view of mature egg, with fluorescence optics; the various egg-shell regions are visible due to endogenous fluorescence (at 360mu) to surround the oocyte (OC).C:collar, M:micropylar complex, RA:respiratory appendages (only one of the two appendages is visible). Bar is lOOurn. Figure 2. Scanning electron mature egg showing the (arrow). Bar is lOurn.
micrograph micropylar
of the anterior pole from a protrusion with the canal
Figure 3. Whole mount view (phase contrast optics) of the anterior pole showing the (inner) "vitelline membrane protrusion" (Mv), and the (outer) "hollow endochorionic cylinder"(Mc) of the micropyle containing the canal (arrow). Bar is 50um.
'3 :I,
"a
320
Cell Biology
international
Reports,
Vol. 8, No. 4, April
1984
Figure 4. Longitudinal thin section through the micropylar apparatus of a late developmental stage. The nearly complete "hollow endochorionic cylinder" (MC) is surrounded by the "border cells" on top (bc) and by other follicular cells at the sides. A border cell process(es) (arrow) is seen within the canal. Bar is 5um. Figure 5. Cross-section developmental stage numerous microtubules
through the micropylar apparatus of a late showing the border cell process(es) containing (arrows). Bar is 0,Zum.
Cell Biology
International
Reports,
Vol. 8, No. 4, April
1984
321
Fig.6 High magnification view of the "border cell" process shown in fig.4.Parts of two cells (bcl, bc2) are seen to be filled with closely packed microtubules (arrows). Bar is 0,2um. Fig.? Schematic representation depicting the data presented in this report and elsewhere (Margaritis et al,l980,Margaritis,l984) concerning the arrangement of the follicular cells during the last step in micropyle morphogenesis. bc:border cells containing microtubules,en:endochorion,fc:follicle cells,Mc:outer part of micropyle, Mv:inner part of micropyle, oc: oocyte,PM:oocyte plasma membrane, VM:vitelline membrane.
International
Reports,
Vol. 8, No. 4, April
1984
317
MICROTUBULES DURING FORMATION OF THE MICROPYLAR CANAL IN DROSOPHILAMELANOGASTER LUKAS H. MARGARITIS Department
of Biology,
University
of Athens,Greece
ABSTRACT The micropylar apparatus in ~ro.sophiZa meZanogaster contains a 0,8um wide canal through which sperm entry occurs.During oogenesis the canal is formed by deposition of extracellular material (chorion) around long cellular processes filled with microtubules. INTRODUCTION Cell processes reportedly occur by means of cytoplasmic filament elongation; these include actin (as found in the stereocilia of the hair cells-Hirokawa and Tilney 1982, or during ameboid movement of many cell types-Taylor et al, 1980) or microtubules (as found in the filopodia of Protozoa-Travis and Allen, 1981). In most of the cases the cell processes are utilized for contour stability or for cell movement, In this report it is shown that microtubules may participate in the formation of the micropylar canal in the eggs of the Dipteran fi.meZanogaster. Each egg is the end product of a follicle which consists of the oocyte, 15 highly polyploid nurse cells and of about 1100 follicular epithelial cells; these cells have been subgrouped into at least ten categories (Margaritis et al, 1980). One of these categories called "border cells"(King 1970) is involved in the formation of the micropylar apparatus after stage lOB, as described by King and associates (Cummings and King, 1969) and by Margaritis (1980,1984). MATERIALS AND METHODS Oregon R flies of D.meZanogaster removed for ultrastructural analysis et al, 1979, 1980) except that PIPES fixatives and washing solutions. Thin orientations to insure perpendicular "border cell" processes.
were etherized and the ovaries as described elsewhere (Margaritis buffer at pH 7,4 was used in all sections were cut at various and longitudinal views of the
RESULTS AND DISCUSSION Mature follicles consist of the oocyte and the surrounding egg-shell layers which differentiate into regionally specific structures (Fig.1). The anterior pole of the egg contains the micropylar cone (Fig.2) which exhibits a narrow canal (w0,8um in diameter) (Fig.3):the whole micropylar complex is seen to consist of an (inner) "vitelline membrane protrusion" and an (outer) "hollow endochorionic cylinder"containing the canal. Longitudinal sections of follicles at late developmental stages, reveal that the canal is filled with cellular process(es) (Fig.4) deriving from the "border cells". High magnification views of the process[es) in crosssections and in longitudinal sections reveal the existence of microtubule bundles closely packed within them (Figures 5 and 6).The diameter of the 0309-1651/84!040317-05:$03.00/O
Q 1984 Academic
Press Inc. (London)
Ltd.
Cell Biology
318
total
cellular
process(es)
International is
roughly
Reports,
Vol. 8, No. 4, April
1984
0,8um.
The involvement of cytoskeletal elements in the modulation of the extracellular matrix is a phenomenon observed in several systems, such as in the collagenous stroma of the cornea (see Hay, 1983). However, details of the actual interaction taking place are largely unknown at present. This report suggests that the presence of microtubules directs the formation of a canal passing through an extracellular matrix(chorion)(Fig.7). Although it appears that the canal is formed mechanically (due to exclusion of the secreted chorion) there is evidence that microtubule polymerization follows an initial and the oocyte via a step of interaction between the "border cells" "special mass" of extracellular material (manuscript in preparation) (see also Fig.7); therefore by subsequent secretion, chorion is accumulating around the elongating process(es). The net result is then the formation of a canal wide enough (hO,8um) to allow the entry of the sperm (having a diameter of 0,6um- Hildreth and Lucchessi 1963) into the oocyte. Functionally,it is plausible to assume that the presence of microtubules resists tension applied by the secreted chorion so that a canal of certain dimensions is formed. The important of these findings lies in the fact that they provide additional information on the participation of microtubules to the supramolecular organization of secreted material. However, this report leaves behind for further investigation several important questions, such as, a) what is the actual "signal" which directs spatially and temporally the cellular process(es), b) which are the organizing centers of the microtubule polymerization, c) how many follicular cells are involved in the process(es). These questions are under investigation in the author's laboratory utilizing wild-type and mutants of D.meZanogaster. ACKNOWLEDGEMENTS This Research
work has been supported Foundation
by a grant
from
the National
Hellenic
REFERENCES
Cummings,M.R., and King,R.C.(1969) The cytology of the viteZZogenic stages during oogenesis in D.me2anogaster.I. Genera2 staging characteristics.Zeitschrift fur ZelZforschung 118,482-492 Hay,E.D.(1983) CeZZ and extraceZZuZar matrix.Their organization and mutual dependence. In:J.R.McIntosh led). Modern Cell BioZogy,vol 2, "SpatiaZ organization of eukaryotic celZs"pp.508-548. A.R.Liss, New York. HiZdreth,P.E., and Lucchessi,J.C.(1963) FertiZization in Drosophila. I. Evidence for the regular occurence of monospernty.Deve1opmentaZ BioZogy 6,262-278. King,R.C.(1970) Ovarian development in Drosophila melanogas-ter. Academic Press,New York. Margaritis,L.H.f1980) Choriogenesis in Drosophila meknogaster. A model system for the study of ceZZuZar differentiation. Mono,graph, Athens University. /4argaritis,L.H.(1984) Structure and Physiology of the egg-shell. In: G.A.Kerkut and L.I.GiZbert feds).Conp?rehensive Insect Physiology,
Cell Biology
International
Reports,
Vol. 8, No. 4, April
319
1984
Biochemistry and Pharmacology,~ol.l,ch7.Pergamon Press,Oxford. Margaritis,L.H.,E'etri,W.H.,and Wynrzn,A.R.f1979) Structural and image analysis of a crystalline layer from dipteran egg-shell. Geli! Biology International Reports 3,61-67. Margaritis,L.H.,Kafatos,F.C., and Petri,W.H.(1980) The egg-shell of D.melanogaster. I.Fine structure of the Layers and regions of the wild-type egg-shell.Journal of Cell Science 43,1-35. Taylor, D.L., Wang,Y.-L., and Reynolds,G.(1980) Contractile basis of ameboid movement. VII Distribution of f7,uoreseently 1abeZed aetin in living anaebas. Journal of Cell Bioloa 86, 590-598 Travis,J.L., and Allen,R.D. 11981) Studies on the rrwtility of the Foraminifera. I. Ultrastructure of the retieulopodial network of Allogromia laticollaris (Arnold). Journal of Cell Biology 90, 211-221 Received
:
13th February
Accepted:
1984,
22nd March
1984.
Figure 1. Whole mount view of mature egg, with fluorescence optics; the various egg-shell regions are visible due to endogenous fluorescence (at 360mu) to surround the oocyte (OC).C:collar, M:micropylar complex, RA:respiratory appendages (only one of the two appendages is visible). Bar is lOOurn. Figure 2. Scanning electron mature egg showing the (arrow). Bar is lOurn.
micrograph micropylar
of the anterior pole from a protrusion with the canal
Figure 3. Whole mount view (phase contrast optics) of the anterior pole showing the (inner) "vitelline membrane protrusion" (Mv), and the (outer) "hollow endochorionic cylinder"(Mc) of the micropyle containing the canal (arrow). Bar is 50um.
'3 :I,
"a
320
Cell Biology
international
Reports,
Vol. 8, No. 4, April
1984
Figure 4. Longitudinal thin section through the micropylar apparatus of a late developmental stage. The nearly complete "hollow endochorionic cylinder" (MC) is surrounded by the "border cells" on top (bc) and by other follicular cells at the sides. A border cell process(es) (arrow) is seen within the canal. Bar is 5um. Figure 5. Cross-section developmental stage numerous microtubules
through the micropylar apparatus of a late showing the border cell process(es) containing (arrows). Bar is 0,Zum.
Cell Biology
International
Reports,
Vol. 8, No. 4, April
1984
321
Fig.6 High magnification view of the "border cell" process shown in fig.4.Parts of two cells (bcl, bc2) are seen to be filled with closely packed microtubules (arrows). Bar is 0,2um. Fig.? Schematic representation depicting the data presented in this report and elsewhere (Margaritis et al,l980,Margaritis,l984) concerning the arrangement of the follicular cells during the last step in micropyle morphogenesis. bc:border cells containing microtubules,en:endochorion,fc:follicle cells,Mc:outer part of micropyle, Mv:inner part of micropyle, oc: oocyte,PM:oocyte plasma membrane, VM:vitelline membrane.