acetylcholinesterase (ache an)d pseudocholinesterase ...the distributio onf acetylcholinesterase...

/. Embryol. exp. Morph. 86, 89-108 (1985) £ 9Printed in Great Britain © Company of Biologists Limited 1985

Acetylcholinesterase (AChE) andpseudocholinesterase (BuChE) activity distributionpattern in early developing chick limbs

CARLA FALUGI AND MARGHERITARAINERIIstituto di Anatomia Comparata delVUniversitd, Via Balbi5, 16126 Genova, Italy

SUMMARY

The distribution of acetylcholinesterase (AChE) and pseudocholinesterase (BuChE) activitieswas studied by histochemical, quantitative and electrophoretical methods during the earlydevelopment of chick limbs, from stage 16 to stage 32 H.H. (Hamburger & Hamilton, 1951).

By quantitative methods, true AChE activity was found, and increased about threefold duringthe developmental period, together with a smaller amount of BuChE which increased morerapidly in comparison with the AChE activity from stage 25 to 32 H.H.

Cholinesterase activity was histochemically localized mainly in interacting tissues, such as theectoderm (including the apical ectodermal ridge) and the underlying mesenchyme. True AChEwas histochemically localized around the nuclei and on the plasma membrane of ectodermal(including AER) and mesenchymal cells, and at the plasma membrane of mesenchymal cellprocesses reaching the basal lamina between the ectoderm and the mesenchyme. AChE togetherwith BuChE activity was found in the basal lamina between the ectoderm and the mesenchyme,in underlying mesenchymal cells and in deeper mesenchymal cells, especially during their trans-formation into unexpressed chondrocytes.

During limb morphogenesis, the cellular and regional localization of the enzyme activitiesshowed variations depending on the stage of development and on the occurrence of interactions.

The possibility of morphogenetic functions of the enzyme is discussed.

INTRODUCTION

Ectoderm-mesenchyme interactions are known to play an important role duringchick limb morphogenesis (Gumpel-Pinot, 1972,1973,1980,19Sla,b,c). An impor-tant part of these interactions is taken over by the apical ectodermal ridge andunderlying mesenchyme (Saunders & Gasseling, 1968; Zwilling, 1956a,b; Solurshetal. 1981; Murphy, Gasseling & Saunders, 1983). Apical ectodermal ridge (AER)is the name of a particular structure of ectodermal cells which increase in thicknessand become pseudostratified on the tip of the limb bud.

Generally, cell and tissue interactions may be mediated by chemical messengers(McMahon, 1974). Among the chemical transmitters acting during early morpho-genesis, neurotransmitters and correlated enzymes have been studied in particulardetail, and the hypothesis has been advanced that they may act as cell and tissueorganizers before taking on their mature neuromuscular function. The presence of

Key words: limb bud, acetylcholinesterase, pseudocholinesterase, cholinergic system.

90 C. FALUGI AND M. RAINERI

neurotransmitters and correlated enzymes during embryonic development wasstudied by the use of different methods in sea urchin (Buznikov, Chudakova,Berdischeva & Vyazmina, 1968; Gustafson & Toneby, 1970, 1971; Ozaki, 1974,1976; Buznikov & Shmukler, 1981), in Oryzias latipes (Ruck, 1978, 1982; Fluck,Wynshaw-Boris & Schneider, 1980; Fluck & Shih, 1981) and in chick (Drews,1975).

During chick limb morphogenesis, cholinesterase activity was detected by histo-chemical methods in the ectodermal hull, in the AER and also in the pre-cartilaginous mesenchymal cells during their aggregation (Drews, 1975). Thischolinesterase activity, independent of the neuromuscular system, was called 'em-bryonic' by the author, suggesting that it may be involved in the inductive processesand/or correlated with cellular movements of morphogenesis.

The name 'cholinesterase' includes two distinct enzyme activities: acetylcholin-esterase (AChE, E.C. 3.1.1.7.) andpseudocholinesterase (BuChE, E.C. 3.1.1.8.),whose presence and function has been studied mainly in the neuromuscular system.

Recently, a specific AChE activity was found in the plasma membrane of blas-tomeres during the embryonic development of an ascidian by the use of a modifiedhistochemical method (Minganti & Falugi, 1980). Subsequently, the method wasimproved and adapted to vertebrate embryo tissues, and the investigation wasextended to early chick embryos and developing chick limbs; preliminary reportsof these results were briefly communicated (Falugi, 1981; Minganti, Falugi, Raineri& Pestarino, 1981, 1982; Falugi, Guastalla & Faraldi, 1984).

In the present study we have extended the research into cholinesterase activityduring the early development of the chick limb by employing histochemical andelectrophoretic methods. Particular attention has been paid to the AER and to theother structures interacting with it during differentiation. The aim of this study wasto specify: a) the distribution respectively of AChE and BuChE in the differentcholinesterase-positive structures, in order to help us to understand their functions;b) the localization of AChE activity associated with cell membrane, because suchan AChE localization during cellular interactions may indicate that the enzyme isinvolved in the regulation of membrane functions correlated with these inter-actions.

MATERIALS AND METHODSChick embryos were obtained from fertilized eggs obtained from the Ladi's hatchery, Carasco

(Genova). Embryos were removed from the eggs, staged according to Hamburger & Hamilton(H.H., 1951) and the somite number determined.

Histochemical methodsFixation. Early embryos were fixed in toto prior to stage 19 H.H.

1) ethanol fixation: 10 drops of cold 80% ethanol (0-4 ml) were added directly onto theembryos, placed in 3-5 ml of cold PBS (phosphate-buffered saline, pH7-2, 7-4) and when theembryos came to the surface, they were washed with two changes of cold PBS.

AChE and BuChE distribution in developing chick limbs 912) two-step fixation: embryos were fixed in 0-1 % paraformaldehyde and 0-5% glutaral-

dehyde in PBS, for 10 min and then washed in 0-1 M cold maleate buffer pH 6, containing 0-168 %sucrose. Then they were incubated in the incubation medium at room temperature for 45 min,washed with cold PBS and fixed with ethanol as above, rinsed in maleate buffer-sucrose andagain immersed in the incubation medium.

After stage 19 H.H., the limb buds were removed from the embryos, and fixed using one ofthe above mentioned methods.

Enzyme reaction. The enzyme reaction was carried out at pH5-9-6 in the incubation mediumdescribed by Karnovsky & Roots (1964), with 0-312 % sucrose, added to make the mixture iso-osmotic to the chick embryo. The incubation medium was changed each 30min, to avoidprecipitation during the incubation period (from 3 to 8 h). Part of the material was incubated inthe medium described by Tsuji (1974). Both methods allow visualization of the enzyme reactionproduct; the first method is more sensitive, while the second allows longer incubation timeswithout non-specific precipitation.

Specificity of the enzyme reaction. To check the specificity of enzyme activity, incubation wascarried out using different substrates: acetylthiocholine iodide (AcTChI), which is hydrolysedmainly by AChE; butyrylthiocholine iodide (BuTChI), which is hydrolysed mainly by BuChE;acetyl-j3-methylthiocholine iodide (AcMTChI), which is specifically hydrolysed by AChE; all ofthese products were obtained from Sigma Chem. Co., U.S.A.

Determination of enzyme specificity by specific inhibition of the histochemical reaction. Sampleswere pre-incubated for 30 min in 10~3 to 10"6M specific inhibitors in Tyrode solution; then theywere incubated in the enzyme reaction mixture, containing the same inhibitors at the sameconcentration: eserine (physostigmine, BDH, England), specific for AChE and BuChE (usefulfor the evaluation of the presence of non-specific esterases); BW 284c51 (anticholinesterase,Burroughs-Wellcome Labs., U.S.A.), specific for AChE; iso-OMPA (tetraisopropylpyrophos-phoramide, ICN Pharm., Canada), specific for BuChE.

Preparation for light microscopy. After the enzyme reaction, samples were washed in PBS,dehydrated slowly in up to 95 % ethanol and then embedded in JB4 preparation (Polyscience,U.S.A.). 4jum sections were obtained with a glass knife, using a Pyramitome (LKB, Sweden).

Quantitative assayThe cholinesterase activity in developing limb buds was determined colorimetrically by the

method of Rappaport, Fischl & Pinto (1959), modified by the Sigma Chem. Co. (Techn. Bull.No. 420,1969). Crude homogenates or Triton X-100 extracts were incubated at 25 °C for 30 minin OlM-phosphate buffer, pH7-8, containing acetylcholine chloride as substrate and m-nitro-phenol as indicator. The absorption was measured by a Bausch and Lomb Spectronicspectrophotometer at 440 nm and compared to a standard acetic acid. Enzyme activity wasexpressed in Rappaport units (1RU = 1 jianole of AChE hydrolysed/30 min under the aboveconditions) per mg of protein N, as determined by the xanthoprotein method of Millon-Nasse(Oser, 1965).

Disc electrophoresisCholinesterase subunits were separated on 7-5 % polyacrylamide gel following the procedure

of Ornstein & Davis (1962). Crude homogenates, diluted from 1/1 to 1/10 (v/v) with distilledwater and Triton X-100 homogenates were centrifuged at 12000g; 150—200 jul of the super-natants, diluted 1/1 with 40 % sucrose were layered on the top of the gels, without a spacer.Current (2-5-4 mA) was applied to each tube for 2-3 h at 4°C, until the tracking dye(bromophenol blue) reached the end of the gels. 50jug of AChE purified from Electrophoruselectricus, type 6 (Sigma Chem. Co., U.S.A.), in 40 % sucrose was employed as the control.

After electrophoresis, gels were fixed (10 min in 80 % ethanol at 4 °C) and stained by the directcolouring thiocholine method of Karnovsky & Roots (1964). The gels were pre-incubated for0-5 h at 4°C in medium without substrate and incubated for 24 h at room temperature in thehistochemical mixture as above.


RESULTS

Histochemical localization of cholinesterase activity in developing limbFrom stage-16H.H. embryos, cholinesterase activity is localized in the whole

ectodermal layer, mainly in the dorsal side of the limb (Fig. 1). The positive re-action is found in most of the ectodermal cells, either in the outer flat cells or in theinner isodiametric ones, which are in contact to the basal lamina.

The enzyme staining of the ectodermal cells is localized around the nuclearenvelope, in the cytoplasm, and sometimes also in portions of the cell membrane(Fig. 1B,C,). A strong enzyme reaction is localized also in the cells of the AER (Fig.2, 3), in the basal lamina under the ectoderm and in mesenchymal cells near to thepositive ectoderm (Fig. 1, 2, 3). In the early limb, from stage 16 to 18H.H., thecholinesterase activity of ectoderm and AER cells is mainly localized on the nuclearenvelope (Fig. 1, 2). Then, from stage 20-21 H.H., the main localization is at theplasma membrane (Fig. 3). Positive mesenchymal cells are distributed in differentlocalizations; under the AER (Fig. 2A,B,C, 3D), under the dorsal ectoderm (Fig.1), at the posterior margin of the limb (Fig. 4), and, in the wing bud, also in a smallportion of the ventral side (Fig. IF, 4A, 10). In every one of these localizations,mesenchymal staining is present in well-defined areas. The exact distributiondepends on the section level of the limb, on the stage of development, and it isdifferent in leg and wing buds. However, in each of these cases, positive mesen-chymal cells have positive processes which reach the positive basal lamina (Fig. IBarrow, D,E arrows, F). In particular, the distribution of the enzyme activity in thebasal lamina and in mesenchymal cells situated under the AER is different atdifferent section levels and/or developmental stages: in the postaxial sections

Fig. 1. Histochemical localization of cholinesterase activity, obtained by the use ofAcTChI as substrate, in the dorsal and ventral side of limb buds. The enzyme activityis revealed by dark staining.

(A) General view of a stage-19H.H. leg bud. Bar equals 20jum(B),(C) Stage-19 limb bud dorsal margin: perinuclear and cytoplasmic staining is

present, the basal lamina is not continuous; enzyme activity localized around mesen-chymal cell processes reaching the basal lamina may be associated with plasma mem-brane or localized in nearby extracellular matrix. Bar equals 8 jitm

(D) Stage-21H.H. leg bud, general view; at this stage the positive mesenchymelocalized in the dorsal margin of the bud is deeper than in earlier stages. Bar equals8jum

(E) Stage-23 H.H. dorsal side of the leg bud: the enzymic staining is localized onectodermal cell membranes, in the basal lamina and in mesenchymal cells. Mesen-chymal cells have positive processes extending orthogonally to the basal lamina.Positive processes to the basal lamina also come from deeper mesenchymal cells(arrows). Bar equals 40/an

(F) Stage-21 H.H. wing bud, ventral side. In the wing bud a little portion of positivemesenchyme is also present in the ventral side and over it the ectoderm is thickened andstrongly positive to the enzymic reaction. Bar equals 40juma, AER; d, dorsal side; e, ectoderm; /, basal lamina; m, mesenchyme; o, outer layer ofectoderm; p, mesenchymal process; v, ventral side.

AChE and BuChE distribution in developing chick limbs 93

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Fig. 2. Localization of cholinesterase activity in the apical zone of the early limb bud, ob-tained by the use of AcTChI as substrate. The enzyme positivity is revealed by dark staining.

(A),(B),(C) Stage-18H.H., postaxial section. At this early stage the staining islocalized mainly at the nuclei and cytoplasm of AER cells, in the basal lamina and inunderlying mesenchymal cells. Bar equals 40jum

(D) Perinuclear localization of the enzyme. Bar equals 15 jtim(E) Stage-23 H.H. BuChE activity, obtained by the use of AcTChI as substrate and

BW 284c51 as inhibitor (the same result was obtained by the use of BuTChI as sub-strate). Bar equals 50 fima, AER; /, basal lamina; m mesenchyme.


Fig. 3. Localization of cholinesterase activity in the AER cells, obtained by usingAcTChI as substrate; the enzyme staining is dark.

(A),(B) Stage-21H.H. The AER is prominent, the staining is localized around thenuclei and on plasma membrane of AER cells. The staining of basal lamina is strong inthe dorsal side of the limb, fainter under the AER (pre-axial section); due to magnifica-tion of the picture, the dorsal mesenchyme positive to the histochemical reaction is cutoff. Bar equals 20jum (A); 8jum (B)

(C) Postaxial section of the same limb, showing cholinesterase activity under theAER. Bar equals 8jum

(D) Stage-24H.H., the staining is localized on the plasma membrane of AER cells.The AER is low, due to flattening of the ectodermal cells. Bar equals 20juma, AER; d, dorsal side; /, basal lamina; m, mesenchyme; o, outer layer of ectoderm.


and/or young stages the reaction is positive in these sites (Fig. 2, 3C); in pre-axialsections and/or later stages the reaction is less strong (Fig. 3A,B) and sometimesit is not apparent. In later stages (24H.H.) AER cells are still positive to thereaction, although they are flattened, and the AER appears less prominent (Fig.3D).

From stage 21H.H., cholinesterase activity is present also in deeper mesen-chymal cells (Fig. 1D,E,F, Fig. 4, Fig. 5A). This positive enzyme activity involvesalso chondrogenic mesenchymal cells (Fig. 5,6). The cholinesterase activity of thefirst chondrogenic cells is spatially connected with the ectodermal activity: theenzyme-positive chondrogenic cells are localized almost at the apex of a widetriangle of mesenchymal positive cells (defined anteriorly by a sharp boundary),whose base is contiguous with the basal lamina of the dorsal ectoderm (Fig. 5A,B).

When chondrogenic aggregation has begun (stage 23-25 H. H.), the enzyme activ-ity is present only in the chondrogenic blastema, surrounded by mesenchyme whichdoes not display 'embryonic' cholinesterase activity (Fig. 5C,D, 6A,B,C). Duringcartilage differentiation a little enzymic reaction is present only in cells, while it is notdetectable in the matrix by the histochemical methods employed (Fig. 6D).

4AFig. 4. Cholinesterase staining obtained by the use of AcTChI as substrate. The enzymepositivity is revealed by dark staining. Posterior margin of a stage-23 H.H. wing bud. Inthe middle of the positive area a blood vessel is visible. In the wing a little portion ofventral mesenchyme is also positive (arrow). (A) Bar equals 200/an. (B) Bar equals40/ime, ectoderm; d, dorsal side; /, basal lamina; m, mesenchyme.

ACHE and BuChE distribution in developing chick limbs 97

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Fig. 5. Cholinesterase staining obtained by the use of AcTChI as substrate. The enzymepositivity is revealed by dark staining.

(A) ,(B) Stage-24 H.H. wing bud: the triangle of positive mesenchyme is in contact withpositive ectoderm and with precartilaginous cells; a sharply defined boundary is presentbetween the positive and negative mesenchyme. Bar equals 200[im (A); 40jum (B)

(C) Stage-25, (D) stage-26 H.H. limb bud: the mesenchymal precartilaginous addens-ation appears stained, the mesenchyme surrounding the precartilaginous anlagen is notstained. Bar equals 40 /imc, precartilage mesenchymal cells.


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Fig. 6. Cholinesterase activity of precartilaginous addensation. The enzyme positivityis revealed by dark staining.

(A) Stage-25H.H. limb bud, section 5 jum thick. Bar equals 400 jum(B) Stage-25 leg bud, in toto. The staining was obtained by the use of BuTChI as

substrate. Bar equals 400 jum(C) Stage-26H.H. limb bud: the core of the precartilage anlagen does not display

enzyme activity, while the cells around it present nuclear and plasma membrane(arrows) staining. Incubation carried out with AcTChI as substrate. Bar equals 40/im

(D) Maturing cartilage: the enzyme staining is weak.cm, chondrogenic mesenchyme; c, determined cartilage.


Histochemical specification of enzyme activity (Table 1)By the use of specific substrates we had the possibility of distinguishing three

different distributions of cholinesterase activity: BuChE alone, AChE alone,AChE and BuChE together.

BuChE activity can be detected by histochemical methods using BuTChI assubstrate; AChE alone was detected by comparing the enzyme localizations ob-tained using respectively AcTChI (which hydrolyses AChE and a small percent ofBuChE) and AcMTChI (which hydrolyses merely AChE).

BuChE alone was never found in any of the structures examined; AChE, aloneor together with amounts of BuChE not detectable with the histochemical methodsemployed, was found only in the ectoderm and in the AER cells. AChE togetherwith BuChE was found: (1) in the basal lamina under the ectoderm and in thesubjacent mesenchymal cells, with major AChE activity mainly localized in thedorsal side of leg buds; (2) in clustered pre-cartilaginous mesenchymal cells (Fig.2E, 6).

Selective inhibition of histochemical reaction (Table 2)By the use of 10~4 to 10~5 M-eserine, the inhibition was complete in every one of

Table 1

Substrates Localization of enzyme activity

cell ectoderm AER basal mesench. pre-cart.membrane lamina mesench.

AcTChI ++ ++ ++ + + ++ + +AcMTChI ++ + + ++ + + +BuTChI + + + +

Revelation of cholinesterase activity in different localizations by specific substrates in earlydeveloping limb buds. ++ = strongly positive, + = positive, - = negative.

Table 2

Inhibitors Localization of enzyme activity staining inhibition

cell ectoderm AER basal mesench. pre-cart.membrane lamina mesench.

10"5 M-eserine ++ + + ++ + + ++ + +1(T4M-BW284C51 + + ++ ++ + + +10"3 M-iso-OMPA - - - + + +

Effect of cholinesterase activity specific inhibitors in different localizations in limb buds ofdifferent stages, incubated with AcTChI as substrate.

+ + = complete inhibition; + = partial inhibition; — = no inhibition


the studied structures. The same result was obtained by the use of 10~4 M-DFP. By

the use of 10~4 M-BW 284c51, only the reaction localized in the ectoderm and in theAER cells was strongly inhibited: a residual reaction was visible in the basal laminabetween ectoderm and mesenchyme, and in the cytoplasm of mesenchymal positivecells. Residual reaction was present throughout the clustered mesenchymal pre-cartilage cells (Fig. 6A,B)- The residual reaction was never associated with plasmamembranes or localized at the perinuclear envelope; it was often formed by tinygranules of precipitated salts. The histochemical reaction appeared unaffected bythe use of 10"3 to l(T6M-iso-C>MPA.

Quantitative analysis of cholinesterase activity during developmentWe have shown the results relating to wing and leg buds together, because the

differences between them revealed by this method are not significant. The meancholinesterase activity of the stage 17-18 H.H. limb is 0-15 RU/mgN; it rises to0-166RU/mgN at stage 24-25, and to 0-4 RU/mgN at stage 28-30-32. Overall,the cholinesterase activity increases about threefold from stage 17 to 32. Abdomi-nal skin of stage-32 H.H. chick embryos shows a mean cholinesterase activityamounting to 0-05 RU/mgN.

Since the cholinesterase activity is made up by AChE and BuChE activity, theuse of specific inhibitors has allowed to check the different specific activities. Theinhibition with 10~ 4 M-BW 284C51 causes a loss of nearly 60 % activity in the earlystages (16-18 H.H.), and of nearly 30 % in later development; iso-OMPA on thecontrary causes a 20% decrease in the early stages (17-18H.H.), and a furtherdecrease (about 50 %) from stage 25 H.H.

The inhibition with 10"4 M-eserine causes an activity that is different at differentstages: at stage 17-18H.H. residual activity is 0-03 RU/mgN, then it decreases tonearly zero at successive stages. The developmental changes in AChE and BuChEquantity are referred in the Fig. 7 histogram.

Electrophoretic analysis of the enzyme activity (Fig. 8)By electrophoresis, three forms of cholinesterase were separated in leg and wing

buds throughout development from stage 17 to 30H.H.At the earliest developmental stages examined (at the sample concentration)

band 1 is more heavily stained than the other bands with both AcTChI andAcMTChI as substrates. At later stages, from 20 to 30 H.H., the three bands areequally stained by the reagents. BuTChI gives a reaction only in band 1 at stage17 H.H. and in band 1 and 3 at later stages.

The stage 32 H.H. abdominal skin presents only the band 3 revealed withBuTChI. The bands are also sensitive to specific inhibitors (see Fig. 7). The AChEfrom Electrophorus electricus, used as control, gives five electrophoretically mobileforms, comprising bands more stained with AcTChI (1 and 2) and bands stainedequally by AcTChI and BuTChI.


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a b

Fig. 7. Quantitative changes of cholinesterase activity during the early development ofchick limb bud. 1RU = 1 /zmol of ACh hydrolysed/30 min in the assay conditions. (1)Stage-16 to 18H.H. limb buds; (2) Stage-23 to 25H.H. limb buds; (3) Stage-28 to32H.H. limb buds; (4) Stage-32H.H. abdominal skin, a, incubation with AcTChI assubstrate; b, is obtained from the elaboration of data obtained with either BuTChI assubstrate or AcTChI as substrate and specific AChE inhibitors. The difference betweena and b at each stage represents the amount of true AChE activity.

Association of enzyme activity to cell membranesThere was always specific AChE activity. It was found in the plasma membrane

of ectodermal cells (Fig. 1) and in the plasma membrane and around the nuclearenvelope of most of the AER cells (Fig. 2, 3). The plasma membrane activity wasfound on the whole plasma membrane or on portions of the plasma membrane, incontact with other cells or with the basal lamina (Fig. 1, 2, 3). In the mesenchymeof the early limb bud, such activity was present on the whole membrane, or on themembrane of the processes reaching the basal lamina between ectoderm andmesenchyme, mainly in the dorsal side of the leg, and also in the ventral side of thewing bud (Fig. IF). From stage 20 H.H., membrane AChE specific activity was alsofound in deeper mesenchymal cells (Fig. IE, 4). After stage 21 H.H. it was alsoassociated with the plasma membrane of some of the clustering precartilage mesen-chymal cells (Fig. 5B, arrow).

DISCUSSION

General localization of the cholinesterase activityAs expected, the general localization of the enzyme activity was similar to that

obtained by Drews (1975). Nevertheless some differences were observed, probablydue to the different methods employed. The first difference is the positive enzymereaction of the cell membrane; the second is the presence of enzymic activity in thebasal lamina under the ectoderm; the third is the detection of further enzyme

102

0C. FALUGI AND M. RAINERI

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iff 2 21 BW 211 i-OMFig. 8. Acrylamide gels stained for cholinesterase activity; method of Karnovsky &Roots (1964); a, substrate AcTChI; b, substrate BuTChI; I, stage-18H.H. limb buds;II, stage-25 H.H. limb buds; III, stage-32 H.H. limb buds; IV, stage-32 H.H. muscle; V,stage-32 H.H. brain; VI, stage-32 H.H. abdominal skin, VII, stage-32 H.H. limb budsensitivity to inhibitors: BW, 10~4M-BW 284C51, substrate AcTChI; i-OM, 10~4M-iso-OMPA, substrate BuTChI.

activity in superficial and deep mesenchymal cells and in processes of the formerreaching the basal lamina; the fourth is the presence of some staining in cartilagecells during their maturation. These findings may lead one to think that the enzymeactivity detected by us in such localizations could be represented by differentmolecular forms of the enzyme, less stable at embedding temperatures and/orusing some fixation techniques, as Whittaker (1982) points out referring to the

AChE and BuChE distribution in developing chick limbs 103membrane-localized enzyme found in ascidian embryos (Minganti & Falugi, 1980).Actually, it is known that both AChE and BuChE do exist in multiple molecularforms, with different functions (Witzeman & Bonstead, 1982). Some of thesemolecular forms are heavily inactivated at relatively low temperatures (Edwards &Brimijoin, 1983), such as the temperature required for embedding in polyethyleneglycol. 'Embryonic' AChE, both soluble and associated with cell membranes, wasfound to be present in sea urchin blastomeres in two of these less-stable forms(Ozaki, 1974,1976). The histochemical method used in the present work is basedon a mild fixation procedure, and small pieces were incubated in toto at roomtemperature in the reaction medium. The fixation method was carefully tested bythe detection of AChE activity associated with the human intact erythrocyte mem-brane (Minganti etal. 1981; Falugi, unpublished data), where the presence of theenzyme is well known, but it has never been detected by histochemical methods.

The two-step fixation which we have used with most of the samples is per se acontrol, because it also makes it possible to detect the cholinesterase activity foundwith usual fixation procedures. The localization of this cholinesterase activity isquite unaffected by the first step of the method.

Developmental changes of cholinesterase activityQuantitative and electrophoretic analyses of cholinesterase activity show the

presence of a true AChE, increasing threefold from early to later stages. Togetherwith AChE there is also BuChE activity. The AChE activity is pre-eminent in earlystages; from stage 24 BuChE activity increases more rapidly than the AChE activityand at stage 28H.H. its amount is equal to that of AChE. The rapid increase inBuChE activity and its appearance in two of the three electrophoretically mobilebands, corresponds to the beginning of cartilage condensation and differentiation.

The specification of histochemically detected cholinesterase activity, obtainedeither with different incubation substrates, or with specific inhibitors of differentcholinesterases, showed the presence of a true AChE activity alone in the ectodermand AER, but AChE activity accompanied by BuChE activity in the basal lamina,in mesenchymal cells and their processes reaching the basal lamina, and in precar-tilage mesenchymal cells. These findings are consistent with the results of Schroder(1980). This author, using both colorimetry and disc electrophoresis techniques,found that cholinesterase activity of the limb chondrogenic blastema is made up ofAChE and BuChE activities, and concluded that the cholinergic system revealedby the presence of these enzymes may be involved in the regulation of the em-bryonic development of the limb.

AChE activity associated with cell membranesIn this study, we have paid particular attention to the localization of enzyme activ-

ity at the plasma membrane of ectodermal and mesenchymal cells and of the mesen-chymal cell processes which reach the basal lamina. This was shown to be a specificAChE activity (see Table 1,2). AChE activity associated with cell membranes is


recognized in the neuromuscular system of vertebrates (Tennyson, Brzyn & Slot-winer, 1971; Tennyson, Brzyn & Kremzner, 1973; Miki & Mizoguti, 1982) andinvertebrates (Spielholtz & Van der Kloot, 1973; Booth, Statton & Larsen, 1975;Raineri & Falugi, 1983). AChE activity associated with the plasma membrane ofnon-neuromuscular cells has been detected by histochemical methods in culturedhuman tumour cells in contact with each other or confluent (Falugi, Balza & Zardi,1983a; Falugi, Castellani & Zardi, 19836), in early chick embryo structures, suchas 'area opaca' endoderm (Falugi et al. 1984), in blastomeres of ascidian earlyembryos (Minganti & Falugi, 1980), of a teleost (Fluck, 1982) and of sea urchins(Ozaki, 1974,1976). In erythrocyte membranes it has been thoroughly studied byquantitative methods and recently also by histochemical methods (Geyer & Linss,1980; Halbhiiber etal. 1982). In all of the cases mentioned, the enzyme was thoughtto be involved in the regulation of membrane functions. For erythrocytes, suchinvolvement was shown by recording the variation of transmembrane ion flux afterthe addition of specific AChE inhibitors (Finin, Volotovskii & Konev, 1979). So,we may suppose that AChE localized on plasma membrane may also be involvedin the regulation of membrane functions in limb buds, where it may trigger morpho-genetic events.

The hypothesis that neurotransmitters and their correlated enzymes may beinvolved in induction processes during morphogenesis has been advanced by anumber of authors (Buznikov et al. 1968; Gustafson & Toneby, 1970, 1971;McMahon, 1974; Drews, 1975; Satoh, 1979; Le Douarin, 1980; Misawa, Doull,Kitos & Uyeki, 1981; Minganti etal. 1981, 1982; Fluck, 1982; Layer, 1983). Thiskind of intracellular signalling is thought to be a link in the evolutionary progressionfrom the prenervous function of neurotransmitters and correlated enzymes as tissueorganizers to their participation in cell-cell interactions occurring during synaptictransmission (Buznikov & Shmukler, 1981; Minganti etal. 1981). Electron micro-scopy studies are needed to provide evidence of the exact localization of thisenzyme activity.

ConclusionsAChE and BuChE activities in the different cholinesterase-positive structures of

developing chick limb are localized. The presence of AChE activity associated withcell membrane is evident. This activity is found in structures such as the ectoderm,the AER and the underlying mesenchymal cells. These structures have beendemonstrated as interacting with each other during limb differentiation (GumpelPinot, 1972,1973,1980,1981aAc; Solursh etal. 1981; Murphy etal. 1983). Mesen-chymal cell processes that reach the basal lamina have been shown to be necessaryfor the normal development of the limb (Sawyer, 1982) and for the control ofcartilage development. The postaxial margin of the limb, where we have foundenzyme activity in the basal lamina and in mesenchymal cells more than in the pre-axial zone, is known to play an important role in the guidance of limb development(Rowe & Fallon, 1981). Furthermore, the localization of AChE activity at the

ACHE and BuChE distribution in developing chick limbs 105

C D

Fig. 9. A possible model for the control exerted by ectoderm and AER on a target tissue(T).

The figures (A),(B) represent leg bud postaxial sections (where the mesenchyme isalso positive under the AER).

(A) Stage-19H.H.; (B) stage-21H.H.; (C) stage-21H.H. wing bud, postaxial sec-tion; (D) stage-21H.H. leg bud, pre-axial section. The dotted areas represent themesenchyme positive to the cholinesterase reaction; the arrows represent the possibledirections of control exerted on T cells. The double arrow under the AER of (A)represents the reciprocal interaction between AER and mesenchyme.

plasma membrane of precartilage mesenchymal cells has a spatiotemporal corres-pondance in cell interactions. In these cells, two processes have been implicated;the first involves the conversion of mesenchyme to unexpressed chondrocytes andthe second involves mesenchyme-dependent expression of chondrogenic dif-ferentiation (Solursh et al. 1982). At the first step we have detected strong cholin-esterase positivity, at the second less enzyme activity.

In early stages (18 to 21H.H.) the enzymatic reaction of the mesenchyme isstrictly defined in small areas, under the AER (in postaxial sections) and in thedorsal side of the leg bud. A possible explanation of this particular feature may bethe control exerted by the ectoderm (including AER) on the developmental eventsinside the mesenchyme (Solursh etal. 1981). Such control may be directed towardsparticular target cells or territories, e.g. the differentiating chondrocytes. One ofthe possible models of the action of this control may be that represented in Fig. 9.The regulation mechanism has to be complex, and most probably the cholinergicsystems represent only a part of it. Hopefully some in vitro experiments will provideinformation on the function of the cholinergic system in ectoderm-mesenchymeinteractions during limb bud differentiation. In the wing bud, there is also a ventral


site of mesenchymal cholinesterase activity. The ectoderm is higher and morestained with AChE reaction than in the remaining ventral surface (Fig. IF, 4Aarrow, 5A, 9C). This difference may be due to the different symmetry of the futureskeletal pattern of the wing.

These findings may give further support to the hypothesis that plasma membraneassociated AChE may be part of a cholinergic system involved in the regulation ofmembrane functions during morphogenesis. BuChE activity, which we have foundtogether with AChE activity in the basal lamina of the ectoderm, the cytoplasm ofmesenchymal cells, chondrogenic blastema cells and maturing cartilage, has beenstudied in the central nervous system (Graybiel & Ragsdale, 1982), but its role inthese non-neuromuscular structures is not known, although the possibility ofmorphogenetic functions of this enzyme has been discussed (Layer, 1983).

The authors wish to thank Dr Madeleine Gumpel for her helpful advice and critical review ofthe manuscript and Dr P. Cochard for suggestions and for having supplied some references. Thework was supported by a grant from the Italian Ministry of Education (MPI).

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{Accepted 2 November 1984)

acetylcholinesterase (ache an)d pseudocholinesterase ...the distributio onf acetylcholinesterase...

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