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The role of cell division in gastrulation of Fundulus heteroclitus
B.rperirnenfal
THE
Cell
Research
ROLE
20, 277-2S2
OF CELL
(1960)
27’7
DIVISION
FUNDULUS
IN GASTRULATION
OF
HETEROCLITUS R. G. KESSEL
Department Woods
of Zoology, Ho/r, Muss.,
State University of Iowa, Iowa City, Marine and Department of Anatomy, Bowman Gray Winston-Salem, N.C., U.S.A. Received
July
Biological Laboratory. School q/’ Xfrdicinr.
15, 1959
T- E,T,T:OST _
gastrulation illustrates very well the process of epiboly; that process of morphogenctic movement \\-hich has been studied by embryologists in many difTerent organisms since it \vas first described in 1835 by van Raer [13]. The observations of van Raer on teleost epiholy \\-err, in fact, a partial hasis for His’ theory [3] that morphogenelic movements are caused by tliffcrcntial grolvth. Various early workers subsequent to His concluded that epiholy anti the resulting increase in blastoderm area are accompanied by little or no increase in the mass of the blastoderm L-51. Richards and Porte1 [S] determined the mitotic index during gastrulation in eggs of Fnndu/rrs and found a rather uniform distribution of mitoses during both blastula anti gastrula stages, with a decrease in frequency during gastrulation. Therefore, tlin‘errntial growth, either by increase in mass or by cell division, has been rendered unlikely as an explanation of cpiboly. The importance of cells as such in epiboly has been further de-emphasized by the observations of Trifonowa [lo] that the blastoderm of certain parthenogenetic eggs may spread over the yolk in an epibolic movement lvithout cell or nuclear division. All of these studies lead to the same conclusion: epiholy is probably not to be thought of as a problem of growth, hut rather as a problem of mass movcmerits [ll]. But, damaging as these studies arc to the hypothesis of epiholy by differential growth, they do not exclude it as a possibility in normal gastrulation; anti the idea has persisted that in some forms differential cell division may play a causative role in gastrulation. For example, Agrell [ 1 ] studied the distribution of mitoses in the sea urchin embryo and came to the conclusion that increased cellular division at the vegetal pole was a supporting mechanism for endodermal invagination. This process aided in forcing cellular material, the cndodermal plate, into the interior of the hlastula. Like\\-ise, Spratt [9] Ezperirnentol
Cell Reseurch
20
R. G. Kessel reached the conclusion that cell division and gro\vth play a significant role in the inragination movements of the chick. ISvitience of a more critical nature is rrquirccl if the hgpothcsis is to he adequately tested. The present investigation xvas hegun with the hope that such evidence u~~~~ld hc provided 1-q inhibiting cell division during gastrdation. MATERIALS
AND
METHODS
In this study, the antimitotic alkaloid, colchicine, was used to block mitosis in the blastoderms of Fund&us heteroclitus. Preliminary experiments indicated that a 0.001 molar solution of colchicine completely blocked cell division in early cleavages and Oppenheimer [6] stage S-9 blastulae. A 0.025 molar solution of colchicine, stored in the dark in the refrigerator, was used as a stock from which the appropriate concentrations were prepared using unfiltered sea water. In each of the three experiments performed, approximately 50-100 eggs from a single female were stripped into a dish and artificially fertilized according to standard procedures. With the exception of experiment III, sperm from several males were used to fertilize the eggs. These eggs were then permitted to develop in sea water at 22%24°C to stage 11 (expanding blastula). At this time about one-half of the eggs were transferred to the colchicine and kept in this medium throughout the gastrulation process. The remainder in each experiment was left in sea water and served as controls. During gastrulation, at stages 12, 13, 14, and 15, several of both treated and control embryos were removed and fixed in Nouin’s. The eggs were dechorionated and much of the yolk removed to facilitate imbedding and sectioning. The eggs were sectioned at 6 microns and stained with Heidenhain’s iron hematoxylin. The mitotic index, representing the ratio of cells in mitosis to the total number of cells and expressed as per cent, was determined in each embryo on the basis of cell counts of every tenth section. Apparently the chorion offers little resistance to the penetration of colchicinc. Similarly Waterman [14] found that colchicine penetrated the chorion quickly in the fish, Oryzias lntipes. In many types of cells, the effect of colchicine is to prevent cytokinesis presumably through prevention of gelation of the spindle fibers. The cells may then go into interphase condition with larger, polyploid nuclei. This condition does not appear to occur in Fundulus. Mitotic figures were found rather than large polyploid nuclei. These figures were similar to those referred to by Eigsti and Dustin [2] as c-mitoses. OBSERVATIONS
Immersion in 0.001 molar colchicine had no apparent efkct on gastrulation which (with one exception to be mentioned presently) occurred at the same rate in the treated as in untreated eggs. Moreover, as far as could be detected, gastrulation was morphologically normal (i.e., similar to control eggs) in those eggs treated with colchicine. In colchicine-treated eggs characteristic c-mitoses hvere frequent. Mitotic index determinations for treated and control Experimental
Cell
Resccrch
20
Cell division
and gastrulatiorz
I \\-hich intticatc the numhrl eggs in t\\-0 experiments are given in l’ahlc of c-mitoscs in the colchicine-treatcti eggs increases during gastrulation. At the cntl of gastrulation, at least thirty per cent of the cells arc in mitosis. It is clear from this that mitosis \vas coml~letel~ I~lo~ltr~cl in eggs \\hicli tlrvel01~~1 in colrllicinc. In contrast, the mitotic indicts of control eggs arc relativeI\ lo\v throughout gastrulation. l~l~otoiiii~rogral~lis of a portion of lwtli trcatccl anti control eggs at stages 13 (atlraiwccl gastrula) and 15 (closuw of hlastopore) arc slio\\n in Figs. l-1. ‘l’.\nr.l-
1. Mitotic
indices
(Two experiments,
o/‘ trerrtd
trntl
one blastotlerrn
Fundulus
control
used for each dctelmination.)
hlitotic
index
(‘:A)
Control stage 12 1 :j l-1 15
1:spt. ‘2.9 2.9 3.3 3.5
1
cnlh~y0.s.
‘I‘lXltCtl lkpt.
II 2.1 ‘2.1 3.1 3.!)
1:spt. 3.0 19.0 X.8 Xl.0
1
1:rpt.
II
5.7 !).i 21.1 32.1)
In cslwrimcnt III, the milotic indicts for which are not lislctl in ‘I’ahlc I, a small lag \vas noted in the rate of morl)hogcnetic movements of Ireatcct as comparccl lo normal eggs to\varti the cncl of gastrulation. \\‘hcn control eggs w-w-e in stage 15, more than one-half of the ~olchi~inc-treated eggs \vcrc in stage 14. ‘I’hc mitotic intlcs in once of thcw trcatccl eggs \\-as founcl to he quite high (GO per cent). Ho\\-cavcr, it sliould tw c~nil~tiasizctl that gastrulation n-as morl~holog$callv normal and no la, tF \vas cletcctcd in treated hlastotlerms at stages 1% anal 13 of gastrulation. Thus it alqwars that when ccl1 division is quik rapid, as in this case, inhibition of division may cause some delay in gastrulation. It is also prohal~le that the grtbat rccluction in the numhcr of mitoscs from hlastula to gastrula [Xl does not occur in an instant. Thus, in some cases, \\-hat is morl~l~ologi~all~ a hcginning gastrula may still hare several final btastular ~~11 (Ii\-isions during the first moments of cpilwl~. If then treated with colchicinc and dcprivccl of a small portion of the rav material for morphogcnctic morcmcnts, a slight slo\\-ing of gastrulation might occur. In cilhcr case, gastrulation clots owur and in a normal fashion \vllrIl cell division is prevented. In vie\\- of these results, ccl1 division can be dismissed as an essential causative factor in early teleost morl,liogenesis. Cell tli\-ision apparcntl\provides
R. G. Kessel
Figs. l-4.-Photomicrographs of Fundulus blastoderms at stage 13 (Figs. 1 and 2) and stage 15 (Figs. 3 and 4). Control blastoderms are shown at Figs. 1 and 3; the colchicine treated blastoderms at Figs. 2 and 4. Heidenhain’s iron hematoxylin. 650 x. Experimental
Cell Research
20
Cell division
crnd gastrulation
only the ra\\- material for morphogcnetic morcmcnts [1]. ;\s ITogt [ 121 has cmphasizecl in Amphibians anti as csprcsscd in the spwulation of Rhumbler is not a problem of gro\\-th hut of mass mwcments. L-51, gastrulation These results with Frzndzzlzzs ~~ultl appear to reopen the prohlrm of the mechanism of gastrulation in the chick and Echinoderms as interpreted 1,~ Spratt [!I] and Agrell [l]. It wo~dd, of course, he of interest to appl\- the colchicinc method to gastrulation studies in these forms as \vcll. Experimental
Cell
Resecrrch
20
R. G. Kessel
282
SUMMARY
Llorphogenetic mo\-cmcnts in gastrulation of in a normal fashion in cases in which cytoltincsis the rate of occurrence of the procws of gastrulation of cell division. ‘I’hercforc, it is con~ludc~l that in little, if any, role in the morphogenetic movements I am indebted this investigation.
to Prof.
J. I’. Trinkaus,
k’undulus l~eteroc~litus occur is lx-e\-cntetl. In most caws is uninterruptrti hy aljscIlCC’
Frmdrdrrs ccl1 division l)lays of gastrulation.
Yale ITnivcrsity,
for his guidance
during
REFERENCES 1. AGRELL, I., drkiv Zool. 6, 213 (1954). 2. EIGSTI. 0. J. and J>USTIN, R. .JR., Colchicinr. Iowa State College Press, Ames, 1955. Ktirperfdrm und das physiologischc Problem-ihrer Entstehung. Ixipzig, 3. HIS, LG., Unserc 1874. .J., .J. Exptf. Zoof. 94, 2fil (1943). 4. HOLTFRETER, 5 MORGAN, T. H., J. dlorphof. 10, 419 (1895). ci: OPPENHEIMER, J. ;\I., Amt. Record 68, 1 (193i). 7. RHUYBER, L., Il’i/helm Koux’ Arch. Enfo,ick/ungsmec/,. 14, 401 (1902). 56. 365 (1935). 8. RICIIARDS, .\. and PORTER, R. P., Am. 97. And. 9 SPRATT, N. T., J. Exptl. Zoo/. 103, 259 (19%). ii TRIFONOWA, A., dctcf Zool. 15, 183 (1934). 11. TRINKAUS, J. I’.. J. Erpff. Zoof. 118, 269 (1951). Enfwickfunysmech. 120, 381 (1929). 12. VOGT, \V., T~~i/hcfrn Roux’ Arch. I<. I?., 17ntcrsuchunpen tihcr die Entwicltlurlgsgcschichte dcr E‘ischc, nebst ChClll 13. VON I~AER, Xnhangc iiber die Schwimmblase. Ikpzig, lS35. 14. \\~.~TERM.~s, L\. .J., [
Experimental
Cell
Research
20
THE
Cell
Research
ROLE
20, 277-2S2
OF CELL
(1960)
27’7
DIVISION
FUNDULUS
IN GASTRULATION
OF
HETEROCLITUS R. G. KESSEL
Department Woods
of Zoology, Ho/r, Muss.,
State University of Iowa, Iowa City, Marine and Department of Anatomy, Bowman Gray Winston-Salem, N.C., U.S.A. Received
July
Biological Laboratory. School q/’ Xfrdicinr.
15, 1959
T- E,T,T:OST _
gastrulation illustrates very well the process of epiboly; that process of morphogenctic movement \\-hich has been studied by embryologists in many difTerent organisms since it \vas first described in 1835 by van Raer [13]. The observations of van Raer on teleost epiholy \\-err, in fact, a partial hasis for His’ theory [3] that morphogenelic movements are caused by tliffcrcntial grolvth. Various early workers subsequent to His concluded that epiholy anti the resulting increase in blastoderm area are accompanied by little or no increase in the mass of the blastoderm L-51. Richards and Porte1 [S] determined the mitotic index during gastrulation in eggs of Fnndu/rrs and found a rather uniform distribution of mitoses during both blastula anti gastrula stages, with a decrease in frequency during gastrulation. Therefore, tlin‘errntial growth, either by increase in mass or by cell division, has been rendered unlikely as an explanation of cpiboly. The importance of cells as such in epiboly has been further de-emphasized by the observations of Trifonowa [lo] that the blastoderm of certain parthenogenetic eggs may spread over the yolk in an epibolic movement lvithout cell or nuclear division. All of these studies lead to the same conclusion: epiholy is probably not to be thought of as a problem of growth, hut rather as a problem of mass movcmerits [ll]. But, damaging as these studies arc to the hypothesis of epiholy by differential growth, they do not exclude it as a possibility in normal gastrulation; anti the idea has persisted that in some forms differential cell division may play a causative role in gastrulation. For example, Agrell [ 1 ] studied the distribution of mitoses in the sea urchin embryo and came to the conclusion that increased cellular division at the vegetal pole was a supporting mechanism for endodermal invagination. This process aided in forcing cellular material, the cndodermal plate, into the interior of the hlastula. Like\\-ise, Spratt [9] Ezperirnentol
Cell Reseurch
20
R. G. Kessel reached the conclusion that cell division and gro\vth play a significant role in the inragination movements of the chick. ISvitience of a more critical nature is rrquirccl if the hgpothcsis is to he adequately tested. The present investigation xvas hegun with the hope that such evidence u~~~~ld hc provided 1-q inhibiting cell division during gastrdation. MATERIALS
AND
METHODS
In this study, the antimitotic alkaloid, colchicine, was used to block mitosis in the blastoderms of Fund&us heteroclitus. Preliminary experiments indicated that a 0.001 molar solution of colchicine completely blocked cell division in early cleavages and Oppenheimer [6] stage S-9 blastulae. A 0.025 molar solution of colchicine, stored in the dark in the refrigerator, was used as a stock from which the appropriate concentrations were prepared using unfiltered sea water. In each of the three experiments performed, approximately 50-100 eggs from a single female were stripped into a dish and artificially fertilized according to standard procedures. With the exception of experiment III, sperm from several males were used to fertilize the eggs. These eggs were then permitted to develop in sea water at 22%24°C to stage 11 (expanding blastula). At this time about one-half of the eggs were transferred to the colchicine and kept in this medium throughout the gastrulation process. The remainder in each experiment was left in sea water and served as controls. During gastrulation, at stages 12, 13, 14, and 15, several of both treated and control embryos were removed and fixed in Nouin’s. The eggs were dechorionated and much of the yolk removed to facilitate imbedding and sectioning. The eggs were sectioned at 6 microns and stained with Heidenhain’s iron hematoxylin. The mitotic index, representing the ratio of cells in mitosis to the total number of cells and expressed as per cent, was determined in each embryo on the basis of cell counts of every tenth section. Apparently the chorion offers little resistance to the penetration of colchicinc. Similarly Waterman [14] found that colchicine penetrated the chorion quickly in the fish, Oryzias lntipes. In many types of cells, the effect of colchicine is to prevent cytokinesis presumably through prevention of gelation of the spindle fibers. The cells may then go into interphase condition with larger, polyploid nuclei. This condition does not appear to occur in Fundulus. Mitotic figures were found rather than large polyploid nuclei. These figures were similar to those referred to by Eigsti and Dustin [2] as c-mitoses. OBSERVATIONS
Immersion in 0.001 molar colchicine had no apparent efkct on gastrulation which (with one exception to be mentioned presently) occurred at the same rate in the treated as in untreated eggs. Moreover, as far as could be detected, gastrulation was morphologically normal (i.e., similar to control eggs) in those eggs treated with colchicine. In colchicine-treated eggs characteristic c-mitoses hvere frequent. Mitotic index determinations for treated and control Experimental
Cell
Resccrch
20
Cell division
and gastrulatiorz
I \\-hich intticatc the numhrl eggs in t\\-0 experiments are given in l’ahlc of c-mitoscs in the colchicine-treatcti eggs increases during gastrulation. At the cntl of gastrulation, at least thirty per cent of the cells arc in mitosis. It is clear from this that mitosis \vas coml~letel~ I~lo~ltr~cl in eggs \\hicli tlrvel01~~1 in colrllicinc. In contrast, the mitotic indicts of control eggs arc relativeI\ lo\v throughout gastrulation. l~l~otoiiii~rogral~lis of a portion of lwtli trcatccl anti control eggs at stages 13 (atlraiwccl gastrula) and 15 (closuw of hlastopore) arc slio\\n in Figs. l-1. ‘l’.\nr.l-
1. Mitotic
indices
(Two experiments,
o/‘ trerrtd
trntl
one blastotlerrn
Fundulus
control
used for each dctelmination.)
hlitotic
index
(‘:A)
Control stage 12 1 :j l-1 15
1:spt. ‘2.9 2.9 3.3 3.5
1
cnlh~y0.s.
‘I‘lXltCtl lkpt.
II 2.1 ‘2.1 3.1 3.!)
1:spt. 3.0 19.0 X.8 Xl.0
1
1:rpt.
II
5.7 !).i 21.1 32.1)
In cslwrimcnt III, the milotic indicts for which are not lislctl in ‘I’ahlc I, a small lag \vas noted in the rate of morl)hogcnetic movements of Ireatcct as comparccl lo normal eggs to\varti the cncl of gastrulation. \\‘hcn control eggs w-w-e in stage 15, more than one-half of the ~olchi~inc-treated eggs \vcrc in stage 14. ‘I’hc mitotic intlcs in once of thcw trcatccl eggs \\-as founcl to he quite high (GO per cent). Ho\\-cavcr, it sliould tw c~nil~tiasizctl that gastrulation n-as morl~holog$callv normal and no la, tF \vas cletcctcd in treated hlastotlerms at stages 1% anal 13 of gastrulation. Thus it alqwars that when ccl1 division is quik rapid, as in this case, inhibition of division may cause some delay in gastrulation. It is also prohal~le that the grtbat rccluction in the numhcr of mitoscs from hlastula to gastrula [Xl does not occur in an instant. Thus, in some cases, \\-hat is morl~l~ologi~all~ a hcginning gastrula may still hare several final btastular ~~11 (Ii\-isions during the first moments of cpilwl~. If then treated with colchicinc and dcprivccl of a small portion of the rav material for morphogcnctic morcmcnts, a slight slo\\-ing of gastrulation might occur. In cilhcr case, gastrulation clots owur and in a normal fashion \vllrIl cell division is prevented. In vie\\- of these results, ccl1 division can be dismissed as an essential causative factor in early teleost morl,liogenesis. Cell tli\-ision apparcntl\provides
R. G. Kessel
Figs. l-4.-Photomicrographs of Fundulus blastoderms at stage 13 (Figs. 1 and 2) and stage 15 (Figs. 3 and 4). Control blastoderms are shown at Figs. 1 and 3; the colchicine treated blastoderms at Figs. 2 and 4. Heidenhain’s iron hematoxylin. 650 x. Experimental
Cell Research
20
Cell division
crnd gastrulation
only the ra\\- material for morphogcnetic morcmcnts [1]. ;\s ITogt [ 121 has cmphasizecl in Amphibians anti as csprcsscd in the spwulation of Rhumbler is not a problem of gro\\-th hut of mass mwcments. L-51, gastrulation These results with Frzndzzlzzs ~~ultl appear to reopen the prohlrm of the mechanism of gastrulation in the chick and Echinoderms as interpreted 1,~ Spratt [!I] and Agrell [l]. It wo~dd, of course, he of interest to appl\- the colchicinc method to gastrulation studies in these forms as \vcll. Experimental
Cell
Resecrrch
20
R. G. Kessel
282
SUMMARY
Llorphogenetic mo\-cmcnts in gastrulation of in a normal fashion in cases in which cytoltincsis the rate of occurrence of the procws of gastrulation of cell division. ‘I’hercforc, it is con~ludc~l that in little, if any, role in the morphogenetic movements I am indebted this investigation.
to Prof.
J. I’. Trinkaus,
k’undulus l~eteroc~litus occur is lx-e\-cntetl. In most caws is uninterruptrti hy aljscIlCC’
Frmdrdrrs ccl1 division l)lays of gastrulation.
Yale ITnivcrsity,
for his guidance
during
REFERENCES 1. AGRELL, I., drkiv Zool. 6, 213 (1954). 2. EIGSTI. 0. J. and J>USTIN, R. .JR., Colchicinr. Iowa State College Press, Ames, 1955. Ktirperfdrm und das physiologischc Problem-ihrer Entstehung. Ixipzig, 3. HIS, LG., Unserc 1874. .J., .J. Exptf. Zoof. 94, 2fil (1943). 4. HOLTFRETER, 5 MORGAN, T. H., J. dlorphof. 10, 419 (1895). ci: OPPENHEIMER, J. ;\I., Amt. Record 68, 1 (193i). 7. RHUYBER, L., Il’i/helm Koux’ Arch. Enfo,ick/ungsmec/,. 14, 401 (1902). 56. 365 (1935). 8. RICIIARDS, .\. and PORTER, R. P., Am. 97. And. 9 SPRATT, N. T., J. Exptl. Zoo/. 103, 259 (19%). ii TRIFONOWA, A., dctcf Zool. 15, 183 (1934). 11. TRINKAUS, J. I’.. J. Erpff. Zoof. 118, 269 (1951). Enfwickfunysmech. 120, 381 (1929). 12. VOGT, \V., T~~i/hcfrn Roux’ Arch. I<. I?., 17ntcrsuchunpen tihcr die Entwicltlurlgsgcschichte dcr E‘ischc, nebst ChClll 13. VON I~AER, Xnhangc iiber die Schwimmblase. Ikpzig, lS35. 14. \\~.~TERM.~s, L\. .J., [
Experimental
Cell
Research
20