Embryonic induction and cation concentrations in amphibian embryos

Cell Differentiation, 17 0 ~

~ 9 ~ ¼ f f Sdenfific Pubfi~ffs kdan~ Ltd.

209

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Embryonic induction and cation concentrations in amphibian embryos G g n ~ r N e g ~ ~, H o ~ t G r u n z ~, U l r i c h G r u n d m a n n ~, H N n z T i e d e m a n n 3 and Hildegard Tiedemann 3 1lnstitu~ ~ Physiolo~ ~ c ~ l Res~rch Gro~ The Free ~ e r s i ~ ~ ~rlin, D- 1000 ~rlin ~ ~D~artment ~ ~ h y s i o l o ~ ~e ~ ~ Ess~ ~ H ~ D- ~ Essen L ~ d 3Ins~ute ~ Modular ~olo~ ~ d ~ochem~t~, ~ e Free ~ ~ B~ D-I~O B ~ 3~ ~

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E x ~ a n t e d ectoderm I ~ m early g~trulae ~ Tdturus alpestds w ~ ~ e a ~ d ~ the N ~ K ~ n o p h o ~ ~ a m i c i ~ n (10 -9 to 1 0 - s M) and the Ca-ionophore A 23187 ( 1 0 - 7 ~ 1 0 - s M). The ~ t o d ~ m devdoped ~ m o ~ exclusively ~ a ~ c ~ e ~ d e r m ~ ~ ~ ~ e con~ol e x ~ a n ~ When the ~ d e r m was ~ e ~ e d ~ o u a h ~ n (10 -4 M), ~ d i ~ Na + ~ e ~ e d abom & ~ I d d and K + w ~ ~ d u c e d by h ~ M ~ e n c h y m e cells ~ sm~! number d i | f e ~ n t i a ~ d ~ a b o ~ 40% ~ the o u a b ~ e a ~ d e x ~ a m ~ The time course ~ ~ t ~ Na + and K + ~ n eoncen~ations w ~ m e ~ e d o v ~ a period ~ 72 h ~ ~ t o d e r m ~ ~ a ~ t r i s ~ r induction ~ ~ g e t ~ i z i n g factor and ~ ¢ o ~ d ex~ants. ~ the f i ~ t 15 h alter e x ~ a n ~ t i o n , no figni|icant ~ e r e n c e s between con~ol and ~dueed e x ~ a n ~ w e ~ f o u n ~ Tberea|ter, ~ e ~eady s t a ~ e o n c e n ~ m ~ n ~ K + d ~ r e a ~ d ~ the i o d u ~ d ex~ants, w h e ~ ~ e steady-state c o n ~ n ~ a f i o n ~ Na + ~ g h t ~ ~ e ~ e d . The membrane ~ s t i n g p o ~ n t i ~ ~ e o ~ e d ~ a e e l l u l a r l y ~ ~ t o d e r m ~ n d w ~ h e s I ~ m e ~ i y gastrula ~ a ~ s was ~ u n d ~ be - 4 1 3 mV ~ ~ o n ~ d and - 5 9 3 mV ~ induced e x ~ a n ~ F ~ m the specific conductances and permeahiliti~ ~ n o ~ d u c e d and ~duced cells R ~ concluded ~ ~ e ~duction p ~ ~ads ~ a ~enti~n ~ the cell m e m ~ a n ~ wh~h ~ q ~ s the c h a ~ c ~ f i ~ s ~ ~ c s d ~ t i f i ~ . Ectoderm I ~ m A m b y s t o m a m e x i c a n u m forms n e p a l or n e u r o ~ fissu~ m e ~ n c h y m e and melanophores ~ t e r e x ~ a n ~ f i o n ~ s ~ t sdufion ~ up ~ 50% of ~ e e x ~ a n ~ ~ o u t any additions. ~ d a ~ d A m b y s ~ m a ectoderm ~ ~ e ~ not s ~ b ~ for test e x p e ~ m e m ~ e m h ~ o n i e ~ d u e f i o ~ cation~ a m p ~ a

In~oduetion E c ~ d ~ m of ~ d y g ~ d a st~g~ of a m p ~ a is not yet irreversibly d ~ m ~ e d to its later fate in Abbmo~om: ~ t ,

[~t, ~ ~dld~ ~n e o n ~ n ~ n ; ion concen~afion,

0045~039/85/$03.30 © 1985 ~

~ff~; ~a+l~, [K+]~, ~ a + ]o, [K+]0, ~ ~

d ~ d o p m e m . In ~ e e m b ~ o the d ~ l ~d~m is induced by the u n d e r l i n g m ~ o d ~ m to the n e u r ~ pla~, the p f i m ~ u m of t~e n e u ~ l ~ e m , whereas the v e n ~ ~ d ~ m differentiates to epid ~ m ~ E ~ o d ~ m can, howeve~ be induced to tissues w ~ c h in n o r m ~ d e v d o p m e n t are derived ~ o m m ~ o d ~ m or e n d o d ~ m , when a p p ~ p r i ~ e inducer~ w ~ c h are p r o t o n in nature, are appl~d

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210 (Tiedemann, 1982). In some amphibian spede& e s p e d ~ A m b y ~ o m a , differentiation of the ectoderm into nerve and pigment cells can be easily caused by u n s p e d f i c means (Barth, 1941; Holffreter, 1944). The ectoderm of other spede& as Triturus torosus or Tr~urus a ~ e s ~ , can be neurahzed by more radic~ ~eatment, for in~ance with ~ k ~ i or a d d (Holffrete~ 1947). Treatment with Hthium chlo~de can b~ng about the differen6a6on of not only n e u r ~ but ~ s o m e s o d e r m ~ 6ssues (Grun~ 1968). The so-called ~auto-differe n 6 a t i o ~ probably depends on the ac6va6on of factors which are ~ r e a d y present in masked form in the ectoderm (Holffrete~ 1934; Tiedemann et ~.} 1961; John et ~.} 1984). Barth and Barth (1972) concluded from measuremen~ of ca6on concen~a6ons and fluxes in early embryos of Rana p ~ n s that embryonic induction ~ initiated by changes in in~acellular cations. On the other han& Slack et ~. (1973) concluded ~ o m t h d r measurement of Na + and K + acfi~ties in early embryos of Xenopus &ev~ that t o t ~ ionic concentrations are not a m ~ o r factor in tissue determination. To d u d d a t e fu~her the role of changes of the ionic concen~ation in cell determina~on we have inves6gated whether the apphca6on of ionophores or o u a b ~ n can induce 6ssue differentiafon in the ectoderm of ~ a~estris. We ha~e ~ s o determined the changes of N a + and K + ion concen~ations and of the membrane p o t e n 6 ~ a~er induction of gaswula ectoderm with vegetali~ng factor,

up (Becker et ~., 1959; Yamada and T a k a t ~ 1961). The explants were then cultured in Holffre~r solu6on (s~t concentration: 5.9 × 10 2 MNaC1,6.7×10-4MKC1,9.0X10-4MCaC12~ to which s ~ e p t o m y d n (100 rag/l), penidHin (100000 U / I ) and g e n t a m y d n (50 m g / l ) were added and the p H adjusted to about Z2-7.4~ for 9 - 1 4 days. For cu~ure at p H 6.8 ectoderm explants were r ~ s e d in 80% L d b o w i ~ medium (L-15) with N a H C O 3 (6 × 10 -3 M) added. The p H was kept at 6.8 in a CO2-incubator. A~er 24 h the explants were ~anfferred to Holffre~r solution and cultured for 10-14 days. The explan~ were fixed in Bouin solution and examined hi~ologically. The paraffin sec6ons were c o u n t e r m i n e d with aniline blue-orange G. The ionophores were di~olved in distil~d dimethylsulfoxide (DMSO) and then added to FI~kinger solu6on so that the fin~ concentration of D M S O did not exceed 0~2%. Con~ol e x p e ~ m e n ~ had shown that D M S O up to 0.1% is not toxic for the explants. All glass vessds and operation disks (without a g a 0 were ~nsed twice with the diluted ionophore or o u a b ~ n solu6ons to avoid a change of concentration due to adsorp6on to the glass surfaces. Ectoderm of early gas~ula stages of Ambystoma mex~anum was explan~d and cultured in Fl~kinger solution for up to 20 h and then in Holt~eter solution as described for Triturus ectoderm.

Measurement of Wn concentra#ons m exp&n~ M a t e f i ~ and Methods

Treatment of e~oderm with Wnophores or ouabain Ectoderm was isolated from eaflygastrula stages ( G 1 - G 2 ) of ~ a~estris (stage 10-11; Knight, 1938) and treated in Flickinger solution ( a ~ u ~ e d to p H 7.2-7.4; s~t concentrations: 5 . 8 × 10-~ M NaC1, 7 × 10 4 M KC1, 4 × 1 0 - 4 M CaCI~, 8 × 1 0 - 4 M MgSO 4, 7.8 × 10 4 M N a ~ H P O 4 , 1.5 × 10 - 4 M K H 2 P O 4, 2.4 × 10 -3 M NaHCO3) for the time indicated in the tables. For the incubation with ionophores the explan~ are covered with Mlk, filter paper or nylon sheets to prevent t h d r curling

Amphibian ectoderm was isolated from early gaswulae of ~ a~estris and treated with purified veget~izing factor for 2, 7, 24 and 72 h, respecfively, e m p l o y i n g the s a n d w ~ h - t e c h n i q u e ( H o l f f r e ~ 1933). The inducer was then removed. Ectoderm sandwiches of the control series (omi~ ting the Weatment with the induceO were incubated under the same conditions in sterile Holffre~r solution. Both, the sandwiches of the expefiment~ as well as the control series, were partially d i ~ e c ~ d so that they had the form of an open oyster. Histolo~cal examination of the explantK which were cultured for up to 12 days,

211 showed that the vegetaliNng Nctor used had s~ong

~o-

a yl ~a o~dhe m~°dermN/end°dermm gNrvegetNiZinc Ta pgh~keo h d nncarboxym~h~c~l~oe s e'am ct°ribndudnw r y °gafrSa~n~ee s f dxtracteC dapa~a "nb df~°mchr°el "ecl -1-

i'i~,~

trophor~N (Born et N., 1972) and diluted 1:1 with 7-~obuhn.

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Preparat~n of samples for ~ o m ~ absorp~on spectrophomm~ry

~ ~ ~

Four ~duced or c o n ~ sandwich~ per series wert ehree ve~ddi s~e~effontNn~a g n dquic~diYstil~t~ans~ew daterto thr°Ugre hmove sMts of the cMtu~ medium and the fl~d enclosed b~ween the two shee~ of ectoderm. Explan~ were finally placed ~to small qua~z v~sd~ The dry w~ght was determined and the mMeriM d i ~ v e d ash-~ee by adding 500 ~1 of 32% ~tfic acid (HNO 3 Suprapur, Merck). After evaporation ~ an cven (60°CL 1200 #1 of 32% HNO 3 were added to the var~sh-~ke refidu~ and atomic absorption measurements w~e made di~cfly ~om t~s fluid for

6

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~

~

~

~ [h~

~

~

~

~

du~d e ~ a1.e~od~m Na d + Fand~K+ ~nz~atioanS~estr~ y ~ 1 ~ ~f expNn~d m and~~ ° no~nrm ~du~d ~th ~getaliNngN~or as a functionof incubation time ~ H o l t ~ ~olutionfolding ~e ~Nam~n. The curves ~w~em the optimMexponentiM~fions ~und by c o m p m ~ f i ~ n g (cf. M ~ h o d ~

Na + ~ n ( ~

and K + ~ n

~c)on~mmtiOa ~ nKd+ ~ 0 2 ~ ~ )no~ndu~d ~ ~ ~omrin Oldu~d.~an~; ~#amNa+ ~n standard error of the mean

i~anthanuN ma
Sm= ± ~ Y'~(M (ni-n - 1~i)2

Mathem~ical evalua~on procedu~

and ~e numb~ of observations in bracket~ Stafi~ t~al Ngnificance was proven by uNng Studenfs ~test and pNred btest.

The graphs for mtM concentrations in F~. 1 were obtNned by fitting the function

(2)

Electrical recording C(t) = a +(b + a ) xe -d'

(1)

to the measu~ment pNnts by means of a ~NtM compumr (TR 440; Compu~r GmbH, Kon~an~ F.R.G.). The data were wNgh~d in correspondence to thNr scatter (Neg~ et N., 1974). For further ~ s c u ~ n the param~ers a, b, c and d we~ evNua~d i~r~N~y u~ng an Mgorithm for least squares estima~s of nonainear param~ers (Marquardt, 1963L T~s f u n ~ n has been appfied to describe faithful~ the concentration shif~ of mon~ and ~vNent c ~ n s in various mus~e ti~ sues after ~ N ~ n and fol~wing eq~l~rafion (Siegel et al., 197~. The resul~ of ion concentration measu~ments were expressed as mean vNues with the estimmed

In~ac~luNr recordings of the membrane po~ntiM of ectoderm sandwich~ from early gastrula stages (GI-G2) of Z a~estr# induced with veg~aliNng factor for 30-35 h were made with ~ass m~roe~mrod~ filled with 3 M KC1. The ~ s m n c ~ ranged ~om 30 to 100 M~ and the tip po~nfiNs ~om -40 to -80 inV. The ~e~ trodes were sN~ded to just b~ow ~e t~s; othe~ wise conventionN ~cording ~chniques were used (S~g~ and SchnNde~ 1981). Ectoderm sandw~hes with membrane po~ntiMs b~ween -35 and -55 mV in the case of noninduced explant~ and b~ween -50 and -80 mV ~ the case of induced exNant~ were s~e~ed for the finn averaNng.

212

Results and Discussion

~ e q u e n c y of mitoses was not ~ g n i f i c a n t l y different ~ o m the c o n w o l ~ At an ionophore concentration of 10 5 M the explants did, howeve~ lose cells and some explants d i h n t e g r a t e d after several days in culture. The C a - i o n o p h o r e activates sea urchin eggs ( C h a m b e r s et al., 1974; S t d n h a r d t a n d E p d , 1974L leads to cortical c o n ~ a c t i o n a n d a b n o r m a l deveb o p m e n t of Xenopus 16-cell embryos ( O s b o r n et al., 1979) a n d is mitogenic for lymphocytes (Luckasen et al., 1974; Hesketh et al., 1977) in the same range of c o n c e n t r a t i o n s as apphed to gastrula ectoderm. The activation of sea urchin eggs is i n d e p e n d e n t of an external source of Ca or even e n h a n c e d when the external c a l ~ u m is reduced ( C h a m b e r s et al., 1974). It has been supposed that the ionophore increases intraceHular ~ee c a M u m by r ~ e a ~ n g b o u n d calcium ( S t d n h a r d t and Epel, 1974; O s b o r n et al., 1979). The i n ~ a c d l u l a r c o n c e n ~ a t i o n of ~ e e Ca :+ is very low, and most calcium in the cells is complexed. N o i n d u c t i o n of the explants was observed when the c o n c e n t r a t i o n s of p r o t o n s a n d ~ee [Ca:+]~ were increased by a ~ d i f i c a t i o n of the culture m e d i u m to p H = 6.8. The explants also did not differentiate in a H o l t ~ e t e r solution, the Ca ~+ concenWation of which was decreased to 0.09 m M ( 1 / 1 0 n o r m a l conc.).

Treatment of gasWula ectoderm with the ionophore gramicidin D Ectoderm of early gaswula stages (G1 G2) of

T. alpesW# was explanted a n d treated with the K - N a ionophore gramicidin D in a c o n c e n t r a t i o n range of 10 -9 M to 10 -5 M (Table Ia). At a c o n c e n ~ a t i o n of 10 - s M one explant differenfiated partially to m e s e n c h y m ~ b u t differentiation to m e s e n c h y m e was also observed in one u n ~ e a t e d c o n ~ o l explant. A ~ e r w e a t m e n t with the highest concenWafion of gramicidin D (10 - s M for 3 h) some explants were somewhat retarded as cornpared to the conwol explants a n d a ~ e r 3 - 5 days of i n c u b a t i o n r d e a s e d dead cells. I n the other expefim e n t a l series the frequency of mitoses was not ~ g n i f i c a n f l y different ~ o m the controls,

Treatment of gas~ula ectoderm with the Ca-ionophore A23187 Explanted gasWula ectoderm was treated with C a - i o n o p h o r e in a c o n c e n t r a t i o n range of 10 -7 M to 10 ~ M. A small a m o u n t of neural tissue d i ~ ferentiated in only one explant of 94 (concentrafion of i o n o p h o r e 10 6 M, T a b l e Ib). The

TABLE I Gas~a ~d~m

exp~n~ of Z a~e~ns ~ea~d with ~ a m ~

Conc. and time of ~cubation

No. of exp~n~

Affp~ e p ~ m i s [%]

(~ G ~ m i ~ n D 10 9 ~ 10-~ M, 1-4 h Conw~

113 50

99 98

(b) C ~ n o p h o ~ A 23187 10-7 m10-5 Ma, 1 3h

94

(C) OuabNn 10 4 M, 3 h 10 -~ M, 3 h Con~

23 22 11

D, C ~ n o p h o ~ A23187 or ouabain M~enchyme [%]

Neur~ t~sue [%]

Blood c ~ [%]

1 2

0 0

0 0

99

0

1 b

0

74 91 1O0

22 ~ 9~ 0

0 0 0

4 0 0

~ At a concen~afion of 10 -5 M C>~nophore about 50% of the ex~an~ ~ n m ~ a m d . ~ Sm~l ~oup of neur~ cells in one exp~nt (~fim wi~ 10 -6 M C>~nophor~. ~ In most exp~n~ on~ a ~w m~enchyme calls were found.

213

Treatment of ga~ru& ectoderm wi~ ouabain OuabNn causes a blockade of the N a ÷ / K + p u m p (FrN 1974; D ~ k and Fry, 1975); it finally decreases [K+]i and increases [Na+]i, The pronounced changes of the t o r n concentration of N a ÷ and K ÷ ions under ouabNn u e a t m e n t are shown in TaMe II. The N a ÷ concen~ation is increased up to &4-fold, the K ÷ concentration reduced by h N [ The concen~ation of Ca ~+ did not change. The high t o r n Ca concen~ation of 45.4 m m o l / k g dry wt or 13.5 m m o l / k g wet wt of the explants is remarkab~. This ~ a d s to the cNculation that [C~i = 20.6 mM. In pregas~ular embryos of X. ~ev~, Slack et N. (1973) found internN Ca concentrations of up to 41.1 mM. Unt~ now nNther lhe c o m p a r t m e n t N location nor f u n ~ tion of this large amount of c N d u m is known, The ouabNn-~eated expNnts did not show the t y p ~ N appearance of ' undif%rentiateff emoderm, but o~en formed larger and smaller fluid-filed verities. At the higher ouabNn concen~ation the inner surface of the veMdes was fined with a few mesenchymN cells in about 40% of the cases, Some blood cells differentiated in one explant (Table Ic). Other tissues did not differentiat~ This is q u i ~ different ~ o m the high percentage of large inductions of mesoderm derived or neurN tissues which are obtNned when purified indudng facto~ are appfied to gas~ula ectoderm (Tiedemann, 1982, 1984). Whether the inMgnificant differentNtion of the

TABLE II Nm K and Ca con~m~tio~ d i~N~d ampNbNn ~md~m ~ early ga~m~e ~cub~ed ~r 7 h ~ H ~ f f ~ r s~m~n ~ ~ wi~out ouabMn ~ × 1 0 -4 M) ~n ~eci~

Na + K+ C~ +

~n ¢on~m~fion[mm~/kgd~ wq Conff~ + OuabMn (n = 5) (n = ~ 21.8±1~ 9&9± 2.5 P < ~005 147~±7.8 74.1±13.3 P < ~005 45A±7~ 43.7± 6.7

o u a b N n - ~ e a ~ d explants depends on the activation of masked factors ( H o l f f r e ~ 1934; T i e d ~ m a n n et N., 1961; John et N., 1984) which could be triggered off by conformationN changes of plasma membrane receptors on the ectoderm cells (Born et N., 1980) or perhaps on conformationN changes of nuclear proteins modifying thNr inte~ action with certNn D N A sequences in the drastic N ~ changed ionic e n ~ r o n m e n t is not yet known. It should be mentioned in th~ c o n ~ x t that ouabNn induces h e m o ~ o b i n syntheNs in Friend erythro~ u k e m ~ cells (Bernstein ~ N., 1976).

Autoneura~zatmn ofAmbys~ma ec~derm All experimen~ so far described were carried out with ectoderm explants ~ o m Z a ~ e s ~ . These explants showed very fitfle autoneuralization or autodifferentiation of mesenchyme and m d a n o phores when cultured in sNt solution under condifions where the curling up of the explants was prevented. E x p l a n ~ d gas~ula ectoderm of A. mexicanum ( T a b ~ III) show~ howevec conMde~ able a u t o d ~ f e r e n t i a t i o n of neural tissue, mesenchyme and m d a n o p h o r e s when cuRured under the same conditions in sNt solution without any addition to the culture medium. The p e ~ centage of autodif~rentiation is different in ectoderm ~ o m dif~rent batches of eggs ( T a b ~ III). The expefimenU show that under the same culture conditions as used for Triturus ectoderm, autodiG ferentiafion occurs e a ~ in explan~d Ambysmma ectoderm, espedMly under conditions where the inner surface of the ectoderm is exposed to the sNt s o l u t i o n as in small explan~ or in larger explants where the curling off N preven~d. Holtfreter (1944) observed that autoneurN~afion occurs in about 15% of the cases when ectoderm of young gasffulae of AmbyMoma punaatum was expMn~d in H o l t f f ~ e r solution under conditions where the c u f f i n g off was not prevented. E ~ o d e r m from older gas~ulae had a higher rate of autoneuralizafion (Barth, 1941; Holffre~L 1944). It has been r e p o s e d that the addition of the Ca-ionophore A 23187, of ouabNn or of severn other substances in very low concen~ation to small e x p l a n ~ d p~ces of A. mexkanum ectoderm leads to cell differentiation (the exact kind of calls was

214 TABLE 111 G ~ M a ~md~m cf A. mex~anum exNan~d wi~out speciN tre~ment N~ of exNanu

NeurN fi~ue [%] a 50-30%

46

2

30-10%

< 10%

28

20

Neurod b [%]

M~enchyme[%]

Mdanophor~ [%]

33

33

13

23 54

0 0

0 0

(

31 13

13 15

~ Percentageof the totN call mass of ~e exNam. b C l u s ~ of neurN cdN wi~out ~NcN o~an s~ucm~.

not ~ated; LCvtrup and Perfi~ 1983). Cyclic adeno~ne-3', 5'-monophosphate and Rs guano~ne analogue have been r e p o s e d to induce differentiation in small explan~ of Ambystoma (Wahn et al., 1975L but not in larger explants of P&urodeles wa##i (Wahn et al., 1976). Cyclic nucleotides (10 -8 to 10 -3 M) did not induce ectoderm of T. alpest~s and X. laev# dther (Grunz and Tiedemann, 1977, and unpub~shed experiment). The experiments on the inducing capabi~ty of the Ca-ionophore and of cyclic nucleotides are in disagreement with our resulu on ectoderm of Z alpestris. The d~crepancy ~ probably due to the fact that Ambystoma ectoderm has been used which spontaneously autodifferentiates, Cation concentra~ons in induced and uninduced explan~ The time course of the sodium and potasfium concen~ation of'noninduced amphibian ectoderm as well as ectoderm induced with vegetali~ng factor a~er explantation and following equihbration in Holffreter solution is shown in Fig. 1. Immediatdy a~er explantation the explan~ have a total Na concen~ation of 35.2 m m o l / k g dry wt and a total K concen~afion of 172.5 m m o l / k g dry wt. This corresponds to a [N~ t = 10.5 m m o l / k g wet wt and [K]t = 51.4 m m o l / k g wet wL respecfivdy (water content 70.2%). Assuming the ionic concentrations in the ex~acellular cle~ spaces (about 5% in noninduced and induced ectoderm as estimated by morphometric method~ to be equal with those in the Holt~eter solution, ~ can be

calculated that [Na+]i = 11.5 mM and [K÷]~ = 78.8 mM. The K + content of oocytes and early embryos of R. pipien~ X. laev# and A. mexicanum (Kostellow and Morrill, 1968; Dick and McLaughlin, 1969; Horowitz and Fenichel, 1970; Slack et al., 1973; Dick, 1978; Dick and Ibrahim, 1980) was found at concen~afions (49-100 mM) ~m~ar to those in various adult nerve (KrnjeviC 1955) and muscle tissues (Adrian, 1956; Page and Solomon, 1960; Siegd et al., 1974; Siegd et al., 1976; Siegel et al., 1980). The fa~ exchangeable Na + fraction in oocytes was determined to 8.5 mM (Horowitz and Fenichel, 1970) and the in~acellular Na ÷ activity in oocytes and early embryos to 14-15 mM (Slack et al., 1973; Dick, 1978). Thus our measurements are in good agreement with lhose in the fiterature. The Na + and K + concentrations of the explan~ change in dependence on the time of incubation in Holt~eter solution (Fi~ 1). A decrease of Na + concen~afion and a fimultaneous increase of K + concentration is observed within the first 5 h in the con~ol explants as wall as in the induced explants before the adju~ment of new, stationary concen~ations. These kinetics are a consequence of the concentration gradien~ between ex~aceHular and intracdlular Na + and K +, wh~h change when the explants are transferred into Holt~eter solution. The ex~acellular fluid in pregastrular embryos of X. laeo~ has Na + and K + concen~ations of 104 and 1.1 mM, respectively (Slack et al., 1973). The Na + concentration of the Holffreter solution is only 57.6%, the K + concentration only 60.9% of the concen~ation in the ex~acellular

215 fluid. The initi~ decrease of the cellular Na concen~ation is compensated by a ~multaneous increase of the K concentration. Thus, for osmotic reason~ the rule [Na+]0 + [K+]0 = [Na+]i + [K+]~ ~ready known from other cells seems to bE valid (Haas et ~., 1966; Siegd et ~., 1980). Cannon et ~. (1974) found the N a and K concentrations in Bufo bufo oocytes to be inversdy rdated: when the N a + concentration was high, the oocyte cont~ned tittle K + and v~e versa so that the sum of the intern~ N a + and K + concentrations was ~ m o ~ constan~ Within the first 10 h a~er explantation no differences were observed in the Na + and K + concen~ations between control explan~ and induced explants (Fig. 1). A~er 20-30 h, howeve~ differences in the steady-sta~ concen~ations appear which suggest permeability changes following induction. In the induced ectoderm the K + concentration decreases fignificanfl~ and the Na + concen~ation increases sfighfly. These observations are s u p p o s e d by computation of lhe parameters of function C(t) according to eqn. 1 which are comp~ed in Table IV for the control and ~st experiments. The two param~ers a and b descfibing the stationary end concen~ation and the di~ ference between inifiM and fin~ concentration vary fignificantly between con~ol and induced series. Howeve~ paramete~ c and d which, as flux and time constanL m ~ n l y determine the dynamic time course within the first 15 h are stafistic~ly equ~. This ind~ates that the presumed permeabifity change of the cell membranes ~ a conse-

quence of the induction process Therefore, differences of concen~ations are detectable only. after a longer latency. The in~aceHular N a ÷ and K ÷ concen~ations ([Na+]i and [K+]i; T a b ~ V) were calculated as 9.7 and 60.9 mM, respectivdG for the control and 10.9 and 46.9 mM, respectivdG for the induced ectoderm.

Effe~ of reduction on eaton permeabi~ A quantification of the membrane physiologic~ changes of induced explants requires the knowledge of the driving force V - E and the pasfive net current I of the ion spe~es in question. Table V summarizes extern~ and internal concen~ations of Na ÷ and K + ions, t h o r equilibrium potentials E and the membrane potenfi~ V. N a + and K + equilibrium po~ntials are in a range described for nerve and muscle cells (Krnjevi~ 1955; Adrian, 1956; Page and Solomon, 1960; Siegd et ~., 1974; Siegd et ~., 1980). The average membrane p o ~ n fial for the control explan~ was - 4 1 . 3 ± 1.0 mV (n = 18) whi~ that of induced explants was found to be fignificanfly more negative at - 5 9 . 3 ± 1.5 mV (n = 32). During neurulation of the amphibian embryo the resting potenti~ rises by 25 mV (Me~ senger and Warne~ 1976L and a resting potenti~ of - 6 4 mV was found for presumptive myotome cells ~nd somme muscle cells of X. ~ev~ (Blackshaw and WarneL 1976). Thus, our measuremen~ are in good agreement with the data ~ o m the hterature. Studies on unidirectional Na + and K + fluxes in

TABLE IV P ~ a m ~ s ~ ~und by compm~ fitting ~ ~e opfim~ c u ~ ~ e d P~ame~

Na + Co~r~ (n = 6)

~ Fig. 1 ~r a m p ~ a n e~od~m of early g a ~ l ~ K+

+ Inductor (n = 6)

Comr~ (n = 6)

+ Inductor (n = ~

(~ [mm~/kg d~ wt.]

31.3 ± 0.5 P < 0~25

3&0 ± 0.9

133.3 ± 3.9 P < 0.005

102.8 ± 2.7

~) [mm~/kg d~ wt.]

5.6 ± 1.8

2~ ± 1.7

39.0 ±4.0 P < 0~05

69.1 ±4.7

-4.4 ±0.8

-6.4 ±1.2

I6.7 ±1.5

18.2 ±2.8

(c)[mm~/kgd~wt~ (d) [h- ~]

~16 ± 0~2

~18 ± ~02

~18 ± ~01

~15 ± 0~1

216 TABLE V Io~c comen~afion~ eq~fibfium po~nfiMs (E), memb~ne po~nfi~s (VL u ~ d ~ e ~ n ~ flux~ (~), ~ c cur~nts (IL condu~anCea Smp~Nanfi~u~g ) and p~meab~fi~ (P) ~ embryo~c Contrd [Na+ ]0 [Na+ ]i E~ [K+ ]0 [K+ ]i EK ~Naa 1~ gNa py~ ~K ~

glKK p~

gK/gN~ P~/PYa

+ Inducer

59.88 59.88 9.7 10.9 +46.3 +43.3 0.67 0.67 6~9 46.9 -11~7 -108.0 --41"1.320 --59"1.320 -~112 -~114 1'28×10-6 1"11×10-6 0.99×10 -s 0.78×10 -s 1.33 1.33 1.65~ 121× 10 6 224 ~ ~ 1×0 10 9 5.46×10 -s 1:0.776 1:0.181

mM mM mV mM mM mV pmmN V cm_2s ~ ~Acm -2 n-~ cm-Z cm s -~ pm~ cm -z s -~ ~_~ 1ACm-c 2m 2

11.27×10 -s cms ~ 1:0.496 1:0~69

~ Dick and Lea ~96~; Nack et N. ~97~; Fry (197~. the steady state of amphibian gastrular ectoderm are not r e p o s e d in the hterature. Dick and Lea (1967) found ~ = 1.2 pmol cm -~ s -~ in the stationary equilibrium in ~olated toad oocytes, The ratio of unidirectional K + / N a + flux is 1.11 in single immature B. bufo oocytes (Fry, 1974), 1.13 in erythrocytes (Glynn, 1957L and 1.54 in single frog muscle fibres ( H o d g k i n and Horowicz, 1959 a, b). The data ~ o m Nolated toad oocytes were taken for the calculation of ~K = 1.33 pmol cm ~ s -~ in Table V. In view of the more negative m e m b r a n e potential in induced explants it is fik~y that the N a ÷ flux is decreased and the K ÷ flux is increased in comparison to noninduced cells. As no flux data are available for explanU, the following computations were started from ident~al unidirectional fluxes for noninduced and induced explants. If a change would have been taken into confideration, the values for the N a + conductance and permeability in Table V would be even lowe~ for the K + conductance and permeabifity even higher in the induced cells. Assuming an independence of both pasfive c o m p o n e n t s of the net currenk efflux ~0 and

influx ~i, the Ussing-Teorell flux relation (Teorell, 1949; U s f i n ~ 1950)

~tI)~- ~

e-vF/~r=e-~v-~)F/~r

(3)

leads to the c o m p u t a t i o n of pas~ve ionic net c u ~ rents IN~ and 1~ across the cell m e m b r a n e if the extra- and in~acellular ion concentrations as well as the m e m b r a n e potential V are known. Usually the c o n d u c t a n c e g or the permeability P are used to characterize the pas~ve m o v e m e n t of an ion species through c h a n n d s of the call m e m b r a n ~ According to H o d g k i n and Huxley (1952L the conductance is the quotient of the pas~ve net current I and its driving force ( V - E ) and means in general the reciprocal of the membrane resistance g~

- - , ~i = N a V- E i

+,K +...

(4)

In comparison to values from nerve or muscl~ the N a + and K + conductances in Table V are lower by a factor of 5 20 (Keynes, 1951; H o d g k i n and Keynes, 1955; H o d g k i n and Horowicz, 1959a, b; Haas, 1964). Interestingly, gya decreases in the i n d u c e d tissue while g~ increases c o m p a r e d to the c o n s o l s . The calculated ratios are gNa (conffol): gNa 0 n d u c t o O = 1:0.87, and gK (control): gK (inductoO = 1 : 1.36. F u ~ h e ~ it can be seen in T a b ~ V that the ratio K + / N a ÷ conductance is 1 : 0 . 8 in noninduced explant~ while in induced explants gNa is about half of g~. This 'differentiation' of the call m e m b r a n e with regard to its permeabihty for cations becomes even more evident by utiliNng the G o l d m a n equation (Goldman, 1943) for the calculation of specific ion permeabifities

RT

1 - e wT~r e(V-E~)F/Rr) '

Pi = ~ - - F 2V [Ci] 0 . ( 1 i = Na+, K + . . .

(5)

This equation can be appfied without restrictions as the comparison of N a + and K + p u m p rates in control and induced explants is not indicative of

217 ~gnificant electrogenic processes (cL Table V), so that an influence of the ion pumps on the calculation can be neglected. On an average, the PNa values in Table V are lower by a factor of fou~ the PK values by a factor of ~x, compared with corresponding values from nerve (Hodgkin and Katz, 1949L skdetal (Hodgkin and Horowicz, 1959a, b), cardiac (Haas, 1964) and smooth muscle (Siegel et al., 1980; Siegel and Schn~de~ 1981). The ratios PNa (c°n~ol)/PN~ OnductoO = 1 : 0.79 and PK ( c o n ~ o l ) / P n 0nductoO = 1 : 2.06 demonstrate again that following induction, the p e ~ meabihty of the cell membrane decreases for N a + ions and increases for K ÷ ions. It is obvious from the last hne of Table V that in con~ol explants the K ÷ permeabihty is five times higher than the N a ÷ permeability, while in induced explants P~ is about 14 times higher than PNa" The ratio PK/PNa = 1:0.069 for these explan~ is within the range which was measured for several differentiated tissues (Keynes, 1951 ; Hodgkin and Horowic~ 1959a, b; Haas et al., 1966; Siegel et al., 1980; Siegel and Schneide~ 1981). Our resul~ show that during the induction process a differentiation of the call membrane occur~ The membrane thereby acquires the characteristics of ion s p e ~ f i ~ t y and selectivity. The consequence of this permeability change is a variation in cation distribution across the call membrane as shown in Fig. 1 (cL Baud and Barish, 1984). If the induction process was supposed to be initiated by changes of ion~ concentration in a cell compartment, eqn. 5 could serve to calculate the Na + extra influx and K + extra efflux caused by an initial permeability change during induction using the initial N a + and K + concentrations (cL Table V). When data on cell geometry are taken into confideration, it follows from the calculation that under the condition of a constant ion p u m p rate, the internal N a + concen~ation should have increased by about 30 mM ( - 65 m m o l / k g dry wt) 7 h after explantafion, while the internal K + concentration should have fallen by about 30 m M already after 45 rain. Fig. 1, howeve~ shows no fignificant difference of ion concentrations between induced and noninduced explan~ within the first 15 h of the experiment. The experimen~ suggest that changes of the in~ac~lular ionic concentrations which occur di-

recfly after explantation are a consequence of variation of the ex~acellular environment (explantation to Holffreter solution) while the long time variations of the intracellular ionic concentrations are a consequence of cell differentiation following induction.

Acknowledgements The authors thank Mrs. Ch. Fuhrmann for her excellent technical asfistanc¢ We would also fike to thank the staff of the 'GroBrechenzentrum for die Wissenschaft in Berlin' for their cooperation and for generou~y making computer time available. This work was s u p p o s e d by the Deu~che Forschungsgemeinschaft (Si 182/1-4) within it~ overall program 'S~ucture and Function of Biological M e m b r a n e ~ and lhe Sonderforschungsberdch ' E m b r y o n a l e Entwicklung und Differen~erun~, the Stiftung Volkswagenwerk (Az. 111 327), and the Erwin Riesch-Stiftung (ERS 7/8).

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