The stabilization of morphological field size during slime mold morphogenesis

Transcription

1 /. Embry!, exp. Mrph. Vl. 44, pp , Printed in Great Britain Cmpany f Bilgists Limited 1977 The stabilizatin f mrphlgical field size during slime mld mrphgenesis By MICHAEL PEACOCK 1 AND DAVID R. SOLL 1 Frm the Department f Zlgy, University f Iwa SUMMARY The relatinship between aggregate size and mrphlgical field size has been investigated in the cellular slime mld Dictystelium discideum. Evidence is presented that aggregate size and field size exhibit different temperature sensitivities and that an aggregate can be induced t separate int several mrphlgical fields by a decrease in temperature. In additin,, evidence is presented that field size is stabilized at a pint in time just prir t tip frmatin. INTRODUCTION When amebae f a lg-phase culture f Dictystelium discideum are dispersed as a multicellular carpet n a filter saturated with buffered salts slutin,, they separate int discrete aggregates. These aggregates then prgress thrugh a defined sequence f mrphlgical stages resulting in a fruiting bdy. Nrmally, each aggregate frms a single tip at the tp which appears t functin as an rganizer fr a single mrphlgical field (Raper, 1940; Farnswrth, 1973; Rubin & Rbertsn, 1975). Each field then gives rise t a single fruiting bdy. In this reprt we have investigated the relatinship between the size f the aggregate and the size f the mrphlgical field. Evidence will be presented that aggregate size and field size exhibit different temperature sensitivities and that an aggregate can be induced t separate int several mrphlgical fields by a decrease in temperature. These results indicate that different mechanisms cntrl aggregate size and field size, and that aggregate size des nt rigidly determine field size. In additin, it will be demnstrated that field size is stabilized at a pint in time just prir t tip frmatin. MATERIALS AND METHODS Grwth and maintenance f rganism Subclnes f the axenic strain f Dictystelium discideum, AX-3, clne RC-3, were maintained n lawns f Aerbacter aergenes n nutrient agar (Sussman, 1966). Axenic cultures were initiated by dispersing spres int 2 ml 1 Authrs' address: Department f Zlgy, University f Iwa, Iwa City, Iwa U.S.A. 4 EMB 44

2 46 M. PEACOCK AND D. R. SOLL f the axenic medium HL-5 (Ccucci & Sussman, 1970) cntaining 500 jug per ml f streptmycin sulfate in sterile test tubes. After several days these initial cultures were in turn inculated int 1000 ml Erlenmyer flasks cntaining 130 ml f liquid nutrient medium and rtated at 90 rev./min at 21 C. At this temperature amebae multiplied with a generatin time f apprximately 12 h and reached a final cell density f apprximately 2 x 10 7 per ml at statinary phase (Yarger, Stults & Sil, 1974; Sil, Yarger & Mirick, 1976). Cells were diluted int fresh medium when cell densities reached 5 x 10 6 per ml. Fr all experiments reprted in this cmmunicatin, cells were btained frm mid-lg-phase cultures at densities f 2 x 10 6 per ml. Initiating and mnitring mrphgenesis Lg-phase cells were washed twice in 10 ml f buffered salts slutin (0-02 M-KCI, 005 M-MgCl 2, 0-04 M phsphate buffer, ph 6-5, 35 mm streptmycin sulfate; Sussman, 1966); 5 x 10 7 washed amebae were then resuspended in ml f buffered salts slutin and dispersed n a black filter pad (4 cm diameter, Whatman n. 29; Sil & Waddell, 1975) verlaid n tw Millipre prefilters (number AP10037) saturated with buffered salts slutin. The develping cell culture and pads were centered in a Petri dish which in turn was placed in a humidity chamber at the desired temperature. T change the temperature during develpment, the black filter supprting the develping culture was transferred t saturated underpads preincubated at the desired temperature. Develpmental prgress as well as the number f fingers per aggregate were mnitred under a Nikn dissectin micrscpe with hrizntal lighting. Measuring aggregate diameters Aggregate diameters were measured under a Baush and Lmb dissectin micrscpe fitted with a calibrated micrmeter in ne eye-piece. Measurements were made at 60 x pwer. RESULTS When grwing Dictystelium amebae are washed free f nutrient medium and dispersed n a filter pad saturated with buffered salts slutin, they prgress thrugh an rdered sequence f stages t the final fruiting bdy. At 20 C the amebae begin aggregating after 6 h s that by 9 h they have separated int lse but discrete aggregates. In the next 2 h, each lse aggregate cnstricts at its perimeter s that by 11 h each aggregate appears as a near-perfect hemisphere referred t as a tight aggregate. After h, each tight aggregate frms a tip at its tp and appears slightly pyramidal, and then elngates rapidly int an inverted cne. By apprximately 13-5 h, the height f the cne is twice the diameter at the base. This mrphlgy is referred t as a finger. Each finger then gives rise t a fruiting bdy after 24 h ttal develpmental time.

3 Mrphlgical field size in slime mld mrphgenesis n I E rrrrn-n J i T-FR J Number f fingers per aggregate Fig. 1. The distributins f the number f fingers per aggregate at several different temperatures. Apprximately.100 aggregates were scred at each temperature. % CfJ 11 -, Cfl 1 Averat;c nuniber _ I 10 i I I 1 1 l l i Temperature ( C) Fig. 2. The relatinship between temperature and the average number ffingersper aggregate. The average number ffingersat each temperature represents the mean fr five separate experiments. The effect f lw temperatures upn the number f fingers per aggregate At 20 C the majrity f aggregates frm ne finger which in turn develps int ne fruiting bdy. In a standard experiment at 20 C apprximately 75 % f individual aggregates give rise t ne finger, 15 % t tw fingers, and 10 % t three fingers (Fig. 1); therefre, each aggregate n the average gives rise t 4-2

4 48 M. PEACOCK AND D. R. SOLL 4 -, I 2 I I T I I Temperature after transfer ( C) Fig. 3. The effect f a decrease in temperature at the tight aggregate stage n the average number f fingers per aggregate. Cultures were develped at 20 C t the tight aggregate stage and then shifted t the desired temperature and maintained at that temperature until fingers were frmed and culd be scred. 1-3 fingers. If amebae are allwed t develp at higher temperatures, the prprtin f aggregates giving rise t nly ne finger is even greater. Fr instance, at 24 C, 90 % f the initial aggregates give rise t ne finger and nly 10 % t tw fingers (Fig. 1); therefre, each aggregate n the average gives rise t 1-1 fingers. If amebae are allwed t develp at temperatures belw 20 C, the average number f fingers arising frm single aggregates increases dramatically. Fr instance, at 16 C each aggregate gives rise n the average t apprximately 2-8 fingers, the distributin ranging frm ne t nine fingers per aggregate (Fig. 1). At 10 C amebae aggregate int amrphus clusters which then separate int multiple fingers; each cluster gives rise n the average t ten fingers, the distributin ranging frm 1 t 22 fingers per cluster (Fig. 1). The relatinship f fingers per aggregate and develpmental temperature is pltted in Fig. 2. Althugh the average number f fingers per aggregate in a culture develping at 16 C is twice that in a culture develping at 20 C, the average tight aggregate diameters at the tw temperatures are nearly identical. At 20 C the average tight aggregate diameter is 316/mi (± 16S.D., 50 measurements) and at 16 C 296 jam (±77 s.d., 50 measurements). Since tight aggregates at bth temperatures appear t be near-perfect hemispheres, ne can cnvert average tight aggregate diameters int average vlumes using the frmula f a hemisphere. This cnversin results in average tight aggregate vlumes f 8-3 x 10 /*m 3 and 6-8 x 10 6 jltm 3 fr develpment at 20 and 16 C respectively. If ne then calculates

5 Mrphlgical field size in slime mld mrphgenesis i r 100 g 3-80 O - 60 f 2 ' - 40 a J LA I TA EC I I I I I 1 II ]5 16 Time f transfer (h) Fig. 4. The time f mrphlgical field size stabilizatin. Cultures develping at 20 C were shifted t 5 C beginning at the tight aggregate stage. Cultures were then maintained at 5 C until fingers were frmed and culd be scred. LA, TA, F, and EC represents the times at which the riginal ppulatin exhibited 50% lse aggregate, tight aggregate, finger, and early culminate mrphlgies respectively at 20 C. In additin, the percentage fingers in the riginal ppulatin at 20 C is pltted as a functin f time t demnstrate the pint that stabilizatin ccurs befre cmpletin f the finger mrphlgy. L 0 average field vlumes at 20 and 16 C by dividing the average tight aggregate vlumes by the average number f fingers, ne btains average mrphlgical field vlumes f apprximately 243 x 10 G /tm 3 and 106 x 10 6 /tm 3 respectively. Therefre, the increase in the number f fingers per tight aggregate resulting frm a decrease in temperature frm 20 t 16 C cannt be explained by an increase in aggregate vlume. Rather, the size f the mrphlgical field is sensitive t temperature and decreases with a decrease in temperature. In additin, it is clear that the size f the mrphlgical field is nt strictly dictated by the size f the aggregate. The effect f a temperature shift at the tight aggregate stage upn the number f mrphlgical fields per aggregate The sensitivity f field size t temperature can als be demnstrated by decreasing the temperature f a develping culture at the tight aggregate stage. When a culture which has develped t the tight aggregate stage at 20 C is shifted t 15 C r less, the average number f fingers per aggregate increases frm 1-3 t apprximately 3 (Fig. 3). When cultures which have frmed mre than ne finger per aggregate at temperatures belw 15 C are brught back t

6 50 M. PEACOCK AND D. R. SOLL 20 C, each finger gives rise t a single fruiting bdy. Therefre, the size f the mrphlgical field at the tight aggregate stage is nt rigidly determined and can still be decreased apprximately twfld by a decrease in temperature. Testing when field size is stabilized T test when field size stabilizes during mrphgenesis, develping cultures were shifted at varius times after the lse aggregate stage frm 20 t 5 C. In the particular experiment presented in Fig. 4, when the temperature was shifted between 10- and 12yh develpmental time, the average number f fingers per aggregate increased frm apprximately 1-25 t 30. Hwever, when shifted at 13 and 13^- h, at a time when cultures were still in the tight aggregate stage and exhibited virtually n fingers in the ppulatin (Fig. 4), the average number f fingers per aggregate remained unchanged at 1-3. When shifted after 13^ h, the average number f fingers per aggregate still remained unchanged at 1-3. Therefre, at a discrete pint midway in the interval between cmpletin f the tight aggregate and frmatin f the finger, an event is cmpleted which stabilizes the size f the mrphlgical field. DISCUSSION Raper (1940) demnstrated years ag that if a migrating slime mld pseudplasmdium is bissected int a frnt and rear prtin, each culd rerganize and frm a well prprtined fruiting bdy. Therefre, a single slime mld aggregate is nt restricted t a single field f rganizatin. In this reprt, we have demnstrated that decreasing develpmental temperature als causes an aggregate, which wuld nrmally have frmed ne fruiting bdy, t frm several fruiting bdies. Therefre, ne aggregate pssesses the ptential fr frming several fields f rganizatin and, cnversely, several fields f rganizatin can functin in a single aggregate. We have als presented evidence which indicated that the mechanism which dictates the size f the aggregate may be distinct frm the mechanism which dictates the size f the mrphlgical field. Aggregatin size appears t be insensitive t a change in temperature frm 20 t 16 C, but field size is sensitive, decreasing by mre than twfld. Hwever, it has been argued that bth the aggregatin prcess and field rganizatin rely upn the same signaling system, the release f camp (Durstn, 1974; Rubin & Rbertsn, 1975; Rubin, 1976). Therefre, it may be the respnse f cells t the signal rather than the signaling machinery which is different in the establishment f aggregatin and field size. Hhl & Raper (1964) presented evidence that the upper limit f mrphlgical field size is regulated by a critical mass value. By depsiting cell agglutinates which had frmed in suspensin n an agar surface, they fund that agglutinates f strain NC4(S2) with diameters less than 370 fim frmed ne pseudplasmdium and agglutinates with diameters greater frmed tw

7 Mrphlgical field size in slime mld mrphgenesis 51 pseudplasmdia. Therefre, it is quite likely that decreasing the temperature f mrphgenesis decreases the critical mass value apprximately tw- t threefld. By temperature shift experiments, we have als demnstrated that the size f the mrphlgical field is nt rigidly determined in a single aggregate until a pint apprximately ne hur prir t the frmatin f the finger mrphlgy. Therefre, a relatinship may exist between the frmatin f the tip f the finger and the stabilizatin f field size. Evidence has accumulated that the tip functins as an rganizer f the mrphlgical field in fruiting bdy cnstructin (Raper, 1940; Farnswrth, 1973; Rubin & Rbertsn, 1975). Since the stabilizatin event ccurs just prir t tip frmatin, the event may represent the cmpletin f prcesses invlved in tip frmatin. In this cntext, the temperature sensitivity f field size and therefre field number per aggregate indicates that the mechanism dictating size may als dictate the number f tips in a single aggregate. The relatinship between the mechanism dictating field size and the frmatin f the rganizing tip is nw under investigatin. This investigatin was supprted by grant PCM awarded by the Natinal Science Fundatin td.r.s. REFERENCES Ccucci, S. & SUSSMAN, M. (1970). RNA in cytplasmic and nuclear fractins f cellular slime mld amebae. /. Cell Bil. 45, DURSTON, A. J. (1976). Tip frmatin is regulated by an inhibitry gradient in the Dictystelium discideum slug. Nature, Lnd. 263, FARNSWORTH, P. (1973). Mrphgenesis in the cellular slime muld Dictystelium discideum; the frmatin and regulatin f aggregate tips and the specificatin f develpmental axes. /. Embryl. exp. Mrph. 29, HOHL, H. R. & RAPER, K. B. (1964). Cntrl f srcarp size in the cellular slime mld Dictystelium discideum. Devi Bil. 9, RAPER, K. (1940). Pseudplasmdium frmatin and rganizatin in Dictystelium discideum. J. Elisha Mitchel. Scient. Sc. 56, RUBIN, J. (1976). The signal frm fruiting bdy and cnus tips f Dictystelium discideum. J. Embryl. exp. Mrph. 36, RUBIN, J. & ROBERTSON, A. (1975). The tip f the Dictystelium discideum pseudplasmdium as an rganizer. /. Embryl. exp. Mrph. 33, SOLL, D. R. & WADDELL, D. (1975). The accumulatin and erasure f 'mrphgenetic infrmatin' in the cellular slime mld Dictystelium discideum. Devi Bil. 47, SOLL, D. R., YARGER, J. & MIRICK, M. (1976). Statinary phase and the cell cycle f Dictystelium discideum in liquid nutrient medium. /. Cell Sci. 20, SUSSMAN, M. (1966). Bichemical and genetic methds in the study f cellular slime mld develpment. In Methds in Cell Physilgy, vl. 2 (ed. D. M. Presctt), pp New Yrk: Academic Press. YARGER, J., STULTS, K. & SOLL, D. R. (1974). Observatins n the grwth f Dictystelium discideum in axenic medium: evidence fr an extracellular grwth inhibitr synthesized by statinary phase cells. /. Cell Sci. 14, {Received 21 June 1977, revised 11 August 1977)