J. Cell Sci. i, 85-108 (1966J 85

Printed in Great Britain

CHROMOSOMES AND NUCLEOLI OF THE

AXOLOTL, AMBYSTOMA MEXICANUM

H. G. CALLANDepartment of Zoology, The University, St Andrews, Fife

SUMMARY

Amongst the axolotl's haploid complement of fourteen mitotic chromosomes, one of the fourlargest, with a greater arm asymmetry than the other three, shows a nucleolar constrictionsubterminally in its shorter arm. Low-temperature treatment causes further secondary con-strictions to appear; these constrictions enable most of the mitotic chromosomes to be identified;the constrictions occur at similar sites in the chromosomes of tail-fin epithelial cells, hepatocytes,and brain cells.

Homology between the mitotic and oocyte (lampbrush) nucleolar organizers has beenestablished, and thus the several hundred free nucleoli in oocytes are genetically related to thetwo nucleoli of diploid somatic interphases. During oocyte development the free nucleolitransform from solid structures to rings and back to solid structures again without detectableincrease in number. During the contraction and aggregation of the lampbrush chromosomeswithin the oocyte nucleus as maturity approaches, in most axolotls the free ring-shaped nucleolibecome stretched between the nuclear periphery and central chromosome group, and take ona characteristic beaded appearance. These transformations of the free nucleoli are largelyparalleled by forms which nucleoli attached subterminally to the shorter arm of lampbrushchromosome III concurrently assume.

The question as to whether fully developed nucleoli detach from the organizer loci and addto the population of free nucleoli in oocytes remains undecided. It may well be that virtuallyall the DNA-generators of free nucleoli detach from the organizer loci before starting to carryout nucleolar functions, and before there is any significant accumulation of protein andRNA around them. If so, the variability in quantity of attached nucleolar material may notreflect different states in a nucleolar synthesis and detachment cycle, but rather variation in thenumber of nucleolar DNA Anlagen which happen to remain attached to the organizer lociafter the synthesis and detachment of the great majority of the Anlagen has ceased.

In occasional oocytes the only chromosomal continuity maintained across the organizer locusconsists of a nucleolar' double bridge'; this indicates that the genetically persistent (i.e. chromo-somal) organizer DNA bears the same structural relationship to neighbouring parts of a lamp-brush chromosome as any other chromomere with its attendant pair of lateral loops.

The lampbrush chromosomes of the axolotl have been provisionally mapped. The centro-meres are represented by short portions of chromosome axis without lateral loops, and thereare two spheres close to the centromeres of both chromosome VI and chromosome XIII.Other recognition characters are inconspicuous or not very reliable, and features of the lamp-brush chromosomes related to the low-temperature induced secondary constrictions of mitoticchromosomes have not been identified.

INTRODUCTION

There are two particular reasons why axolotl chromosomes warrant detailed study.First, this is one of the very few urodeles about which there is any genetic information(see Signoret, Briggs & Humphrey, 1962; Humphrey, 1962, 1964). If one hopes to beable to correlate some particular genetic trait with a lateral loop characteristic on the

86 H. G. Callan

oocyte lampbrush chromosomes, both the genetical and cytological attributes of aspecies, for preference a urodele, need to be known. Secondly, the axolotl, like its nearrelative Ambystoma tigrinum, is unusual among urodeles in having a simple somaticnucleolar condition. There is one nucleolar organizer per haploid chromosome set.Having failed to determine whether or not there is a genetic relationship betweensomatic nucleolar organizers and free oocyte nucleoli in Triturus cristatus, where thesomatic nucleolar condition is complex, there seemed better hope of deciding thisquestion by working with the axolotl. Moreover, there is an established nucleolarmutant in the axolotl (Humphrey, 1961) and hence the possibility that this might berecognized in the lampbrush chromosome complement.

These two considerations were put to me by Dr J. G. Gall of Yale University, earlyin 1964. An opportunity to take up the study was presented soon afterwards whenI was invited by Dr T. M. Sonneborn to spend a year at the Zoology Department ofIndiana University, where Dr R. R. Humphrey maintains an axolotl colony.

In order to gain some general idea of the relative lengths of the various chromosomesmaking up the axolotl complement, the positions of their centromeres and of thenucleolar organizer constriction, mitotic preparations were first studied. Thereafter,in the hope that the mitotic chromosomes might be further characterized, prepara-tions from cold-treated animals were examined in order to learn the distribution ofseveral more secondary constrictions which become apparent after appropriate ex-posure to low temperature.

With this information as a guide, the lampbrush chromosomes were examined. Inthe course of this study I was struck by the remarkable variability in form of the freenucleoli, and this led me to investigate the developmental history of the free nucleoliduring the growth of axolotl oocytes.

The various topics are discussed section by section in this paper.

THE NORMAL MITOTIC CHROMOSOMES

The axolotl's diploid chromosome number of 28 was established by Fankhauser &Humphrey in 1942. In order to study details of the normal karyotype, axolotl larvaeabout 15 mm long, raised at room temperature (21 °C), and which had just started tofeed, were placed for 12 h in 0-5 % colchicine in pond water, and then fixed entire for12 h in Clarke's (1851) 3:1 ethyl alcohol/acetic acid fixative. From these fixed larvaethe tail fins and livers were excised and placed on separate slides. To each preparationa drop or two of 0-5 % aceto-orcein (G. T. Gurr, synthetic) was added, a small watchglass was set over each preparation to reduce evaporation, and 30 min of stainingfollowed. Each preparation was then tapped out to dissociate the cells from one another,and covered with a long, siliconed cover glass. The preparations were squashed byfinger-pressure between folded filter paper, the cover glasses removed by the ' dri-ice'method, and the slides dehydrated and mounted in' Permount' (Fisher Scientific Co.).

Better chromosome spreads were obtained from hepatocytes than from tail-finepithelial cells; an example is shown in Figs. 1 and 7. The range in chromosome sizefrom largest to smallest is without well-marked discontinuities, and I have found

Chromosomes and nucleoli of the axolotl 87

individual identification difficult. In practice one can readily subdivide the haploidcomplement into eight larger chromosomes with median or submedian centromeres,in no case with arm ratios approaching 2:1, and six smaller chromosomes withmarked arm asymmetry, all with ratios greater than 2:1.

Fig. 1. Camera lucida drawing of axolotl hepatocyte mitotic chromosomes from anormal larva. A photograph of this same complement is shown in Fig. 7; in the drawingthe chromosomes have been spaced out to avoid overlaps, n, nucleolar organizerconstriction.

Of the eight larger chromosomes, two with virtually median centromeres (VII andVIII) are substantially smaller than the rest; they are indistinguishable from oneanother. Among the remaining six larger chromosomes there is one with a greaterarm asymmetry than the others (it has a ratio of about 5:3), and subterminally in itsshort arm there is a secondary constriction. This is the only axolotl chromosome whichnormally shows such a constriction; the constriction is more evident in hepatocytethan in tail-fin epithelial chromosomes. In the light of Dearing's (1934) classic studyon the related A. tigrinum, where a subterminal constriction in the short arm of alarge chromosome of similar arm ratio was shown to be the site of production of thenucleolus, this secondary constriction in the axolotl can be assumed to mark thenucleolar organizer. The nucleolar chromosome of the axolotl can be further rankedas one of the four largest chromosomes in the complement, but I have not found itpossible, from a study of mitotic plates alone, to rank it more precisely than this.

The three smallest chromosomes XII, XIII and XIV can be recognized with easeand arranged in length order without ambiguity. However, I have been unable todifferentiate with certainty between chromosomes IX, X and XL

H. G. Callan

THE MITOTIC CHROMOSOMES IN LARVAE EXPOSED TO LOW TEMPERATURE

When larvae of three species of Triturus were subjected to low temperature for afew days prior to fixation, their mitotic chromosomes showed many secondary con-strictions which are not apparent in normal mitoses (Callan, 1942). The same is trueof the axolotl.

Fig. 2. Camera lucida drawing of axolotl hepatocyte mitotic chromosomes from a low-temperature treated larva. A photograph of this same complement is shown in Fig. 8 ;in the drawing the chromosomes have been spaced out to avoid overlaps. Identifiedchromosomes (all but I, II and IV) are marked by Roman numerals, n, nucleolarorganizer constriction.

Axolotl larvae which had just started to feed were placed in a refrigerator adjustedso that the water temperature fell to 2-5 °C. A 0-5% solution of colchicine in pondwater was also brought to this temperature, and after 3 days in plain cold water thelarvae were transferred to the cold colchicine solution, and left in the cold for a further12 h. They were then fixed and preparations made as already described. From a fewof the larvae samples of brain tissue as well as liver and tail-fin epithelium wereexcised to make preparations.

The mitotic chromosomes in these preparations show many secondary constrictions.The constrictions are rather more sharply defined in tail-fin epithelial cells (Figs. 3, 9)

Chromosomes and nucleoli of the axolotl 89

than in hepatocytes, but the latter spread much better, and provided more fullyanalysable complements (Figs. 2, 8).

No attempt was made to determine the minimum duration of cold treatmentnecessary to make these constrictions evident, but a point concerning temperaturedeserves mention. Larvae treated at o to 0-5 °C show constricted chromosomes, butin tail-fin preparations in particular the chromosomes are poorly spread and have fuzzyoutlines, which I think is connected with a failure of the nuclear membrane to breakdown. Larvae treated at 3-5-4-0 °C have chromosomes with less-pronounced con-strictions, inadequate for consistent identification.

Fig. 3. Camera lucida drawing of axolotl tail-fin epithelial cell chromosomes froma low-temperature treated larva. A photograph of this same complement is shown inFig. 9; in the drawing the chromosomes have been spaced out to avoid overlaps.Identified chromosomes (all but I, II and IV) are marked by Roman numerals.n, nucleolar organizer constriction.

The cold-induced secondary constrictions in axolotl chromosomes occupy well-defined and constant positions, and these positions are the same in hepatocytes as intail-fin epithelial cells. I have classified the constrictions into two groups, A and B.Group A consists of those which form more readily, in the sense that if a particularchromosome set shows any cold-induced constrictions, one may expect to find all ofthose in group A. The group B constrictions are apparent in fewer cells, they are moreevident in tail fin than in liver, and when they are present one can be sure to find allthe group A constrictions too.

As I had established from measurements on the lampbrush chromosomes a reason-ably accurate rank order for the complement, with centromere positions, I did not

90 H. G. Callan

attempt'to make systematic length measurements of the mitotic chromosomes. Forthis reason in Fig. 4 the positions of the cold-induced secondary constrictions havebeen superimposed on a lampbrush karyotype. It will be apparent that the cold-induced secondary constrictions aid considerably in the identification of the axolotl's

r-hB I A

" 1 1B ^ B

IVtB

tA

ttBA

t\ B

tn

VI

VII

VIII

IX

X

XI

XII

XIII

ffBA

Fig. 4. Axolotl karyotype based on relative lengths of the lampbrush chromosomes.The positions of centromeres are indicated by arrows pointing down. The arrowspointing up mark the sites of mitotic secondary constrictions, n, nucleolar organizer;A and B, first- and second-order low-temperature induced constrictions.

mitotic chromosomes, and the only troublesome ambiguity which remains concernsthree of the four largest members of the complement.

I was unable to make any well-spread preparations from brain tissue. However, thechromosomes show constrictions much as in hepatocytes, and the ability to recognizeseveral individual chromosomes by the familiar distribution of their constrictions,

tA

tA

tA

I

i

I

I

\

\

1

ttAB

t 'A

tA

tA

t tA B

ttAB

tA

Chromosomes and nucleoli of the axolotl 91

notably III, XII, XIII and XIV, leads me to suppose that the constriction pattern isthe same in brain cells as in liver or tail fin.

The origin and significance of these cold-induced constrictions is obscure. In 1942,when Darlington's nucleic acid 'starvation' theory was being energetically propounded,

I ft

IV

V

VI

VII

VIII

IX

XI

XII

XIII

XIV

Fig. 5. Working map of the axolotl's lampbrush chromosomes. The positions ofcentromeres are indicated by arrows pointing down, n, nucleolar organizer.

I accepted the possibility that the cold-induced constrictions in Triturus mightrepresent 'under-charged' regions, possibly even regions devoid of nucleic acid. Thiswhole concept has now rightly fallen into disrepute, and another explanation is calledfor. By analogy with McClintock's (1934) description of the origin of the nucleolarconstriction in metaphase chromosomes of maize, where the nucleolus at prophaseremains for a time attached to the nucleolar chromosome and interferes with its

92 H. G. Callan

condensation, one might suppose that the action of an abnormally low temperatureis to cause certain other gene products to remain for an abnormally long time associ-ated with their sites of production on the chromosomes, and likewise interfere withcondensation during prophase.

Whether or not this explanation is valid, it is clear that the cold-induced constric-tions are not related to tissue-specific gene products, for otherwise one wouldexpect quite different constriction patterns in tissues as unlike as liver and ectodermalepithelium.

THE LAMPBRUSH CHROMOSOMES

Axolotl lampbrush chromosomes were studied by the techniques devised forTriturus viridescens (Gall, 1954) and T. cristatus (Callan & Lloyd, i960). Threeparticular technical problems presented by axolotl oocytes were overcome, and thesedeserve attention.

The nuclear sap within axolotl germinal vesicles is a stiff jelly, stiffer than I haveever encountered in T. cristatus, and very much stiffer than the nuclear sap of T. viri-descens. This sap must be made to disperse if one wishes to study the lampbrushchromosomes in detail. The standard saline in which germinal vesicle nuclei areisolated, o-1M KC1 or NaCl or a mixture of the two, can be modified in two ways toachieve sap dispersal within the observation chamber. It may be diluted, though onlyto a limited extent, for if the saline is too dilute the lateral loop ribonucleoproteins(matrix) dissolve. CaCl2 may be added to the saline, though again only to a limitedextent, for at concentrations in excess of IO~*M the chromosomes contract and stiffenas though fixed.

A saline suitable for dispersing the axolotl oocyte's nuclear sap, and which leavesthe chromosomes in good condition, consists of o-i M K/NaCl containing 0-5 x I O ^ MCaCl diluted to three-fifths of its original concentration with distilled water. A four-fifth dilution of this saline fails to disperse the sap completely, and a two-fifth dilutioncauses appreciable loss of matrix from the loops. Furthermore, a three-fifth dilutionof saline containing 0-25 x I O ^ M CaCl2 fails to disperse the sap completely.

If one wishes to make long-lasting preparations of lampbrush chromosomes it iscommon practice to expose the nuclear contents, in dispersing saline, to the fumes ofconcentrated, neutralized formalin for a minute or two before the preparation iscovered. This treatment kills some bacteria which might otherwise contaminate thepreparation, and apparently stabilizes the lateral loop ribonucleoproteins against •autolysis. With oocytes of several species of urodeles this short exposure to formalinfumes has no detectable influence on nuclear sap dispersal, i.e. dispersal in an appro-priately chosen saline occurs whether or not the formalin treatment has been given. Inaxolotl oocytes, on the contrary, a short exposure to formalin vapour is necessary; with-out such treatment the nuclear sap forms a coarse meshwork of fibres as it dilutes withdispersing saline, and these fibres anchor the lampbrush chromosomes to one another.

The membrane surrounding the axolotl's germinal vesicle nucleus adheres to anyclean glass surface on which it comes to rest. If a considerable area of adhesion occurs

Chromosomes and nucleoli of the axolotl 93

it is difficult to remove the membrane without damage to the chromosomes, for whenthe membrane is seized by forceps and the nucleus lifted, a hole is torn around thearea of adhesion and some of the nuclear contents ooze out through this hole beforea tungsten needle can be manipulated to tear the membrane clear away. ' Strangled'preparations result. This trouble can be avoided by keeping the nucleus continuallyon the move while the cytoplasm is pumped away with a pipette, and by manipulatingforceps and needle without delay once the cleaned nucleus has been placed in itsobservation chamber.

Until the developmental stage is reached when the chromosomes start to aggregatein the middle of the germinal vesicle nucleus (oocyte diameter of about 1-5 mm)axolotl chromosomes are long objects, the longest being 1-5 mm or thereabouts;furthermore, the lateral loops which they bear are especially numerous, and also verylong. Thus there is a great congestion of chromosomal material within the nuclei ofsmaller oocytes, and I have been unable to obtain fully analysable preparations fromoocytes below 1-4 mm diameter; even after the membrane has been removed withoutdamage to the nuclear contents, the chromosomes become entangled with one another,stretched and broken, while the sap disperses.

The oocyte size-range from within which analysable preparations can be obtainedis therefore limited, much more restricted than is the case with T. viridescens orT. cristatus. The lower limit is 1-4 mm, the upper 1-7 mm. In oocytes larger than1-7 mm in diameter retraction of the lateral loops is well under way, and most of thelandmark structures, including the centromeres, are difficult or impossible to identify.In the ovaries of mature axolotls, oocytes within the range 1-4-1 -7 mm are notnumerous; it is rare for such oocytes to exceed 5 % of the pigmented oocytes present.

A provisional working map of the axolotl's lampbrush chromosomes has been con-structed (Fig. 5) based on detailed drawings of five complete undamaged complements,

Table 1. Relative lengths of axolotl lampbrush chromosomes

Chromosome

III

III (nucleolar)IVV

VI (bearingspheres)

VIIVIII

IXX

XIXII

XIII (bearingspheres)

XIV

Longer arm

64-460-365-3S4-o50-2

5O-S

33-33i-642-041-0

39-o32-321-9

17-8

Shorter arm

52-655-736746-037-836-S

30-73°-418-016-013-0

9 78-i

6 2

Total

Overall

117116

1 0 2

1 0 0

8887

646260

57524 23 0

2 4

1001

94 H. G. Callan

with subsidiary data taken from over ioo other preparations. By convention, the longerchromosome arms are called ' left' arms. Relative lengths of the fourteen chromosomes(Table i), and the positions of landmarks, were worked out in the manner describedby Callan & Lloyd (i960). In order that the data should be roughly comparable to thecorresponding data for T. cristatus, an overall map length of 1000 units was allocatedto the haploid complement.

Centromeres

The positions of the centromeres in the mitotic chromosomes were already known,and the corresponding loci in the lampbrush chromosomes were identified withoutdifficulty. The centric regions are short lengths (10/iora little less) of chromosomeaxis devoid of lateral loops. These stretches of axis are of irregular width, in generalsomewhat wider than chromomeres bearing loops but nowhere thicker than 1 fi(Figs. 10, 13 and 14). Unlike the centric regions of T. cristatus karelinii, there is nosign of a discrete granule flanked by axial bars in the centric regions of axolotl lamp-brush chromosomes.

Other landmarks

In contradistinction to the lampbrush chromosomes of T. cristatus, axolotl lamp-brush chromosomes do not possess many strikingly distinctive landmarks apart fromthe centromeres. Nevertheless, there are objects which permit the identification ofvarious chromosomes, and these will be described.

Spheres. There are two spheres just to the left of the centromeres of chromosomes VIand XIII (Figs. 10, 14 and 16). These objects closely resemble the structures alsocalled spheres on chromosomes V and VIII of T. cristatus. They are directly attachedto the chromosome axis. Homologous sphere-generating loci are often jointly involvedin the production of single spheres, i.e. sphere fusions are common. Attached spheresvary in size up to a maximum diameter of about 10 /*, and free spheres of similardimensions and refractility are frequently encountered in oocytes within the sizerange which I have studied. Just as in T. cristatus it is probable that detachment of thespheres from their generating loci occurs, and is followed by the production of morespheres; a further comment on this point is made in the section concerned with thenucleolar organizer.

The disposition of the spheres with respect to one another, and to the centromeres,is so similar in chromosomes VI and XIII that these two chromosome regions maywell be duplicate.

Suspended granules. This term has been introduced to define spherical objectswhich hang on short stalks from the chromosome axes (Fig. 11). Except for one sus-pended granule on chromosome III, the maximum size reached by these objects(about 5 /i) is decidedly smaller than the maximum size of spheres.

I am not sure whether all the terminal regions of axolotl lampbrush chromosomescan carry suspended granules (e.g. Figs. 15, 34 and 35), but this is certainly true ofmost. When a suspended granule is present it is often possible to see that its attach-ment is not strictly to the telomere (which in axolotl chromosomes are small structures

Chromosomes and nucleoli of the axolotl 95

no larger than nearby chromomeres) but to an immediately adjacent chromomere.These near-terminal suspended granules are of no value as landmarks, for they arequite variable in their occurrence. They are not indicated on the working map.

On the other hand, the distribution of interstitial suspended granules, even thoughindividually these too may be present or absent in a given preparation, are of greathelp in identifying particular chromosomes and parts of chromosomes. Thus, forexample, chromosomes I and II are much of a muchness for length and centromereposition, but the places where suspended granules are likely to be found are distinctivefor the two chromosomes, and the right arm of chromosome I bears none. The posi-tions occupied by suspended granules also help to discriminate between chromo-somes IV and V, VII and VIII, and they are the only sure means I have found todiscriminate between chromosomes IX, X and XL

Suspended granules are composed of somewhat more refractile material than spheresof similar size. As with spheres, though less frequently, suspended-granule fusionsoccur. Moreover, the variable presence or absence of granules at particular loci, andthe presence of granules of comparable size and refractility free in the nuclear sap(especially abundant in larger oocytes) suggest that cycles of formation and detach-ment exist.

In the right arm of chromosome III, about one-third the way out from the centro-mere, there is a suspended granule which is often as large (up to IO/J diameter) asthe spheres (Fig. 13).

Axial granules. These are defined as dense, swollen regions of the chromosome axis,about 3 /i wide, and they are regularly present at two sites. One lies about one-eighththe way in from the telomere in the left arm of chromosome VI (Fig. 12), while theother lies very close to the telomere of the right arm of chromosome VII. Neither ofthese axial granules bears lateral loops; either may fuse with its homologue.

Fluffy loops. These are more bulky structures than the generally long and slenderlateral loops of axolotl lampbrush chromosomes, and their matrix has a fluffy texture.They are often so contorted and compacted around the chromosome axes that theirsites of attachment appear as fuzzy, ill-defined regions. They are of more diagnosticvalue in oocytes at the lower end of the size range which I have studied, for in largeroocytes, where some degree of loop retraction has set in, they may no longer showtheir distinctive character.

It is only in the case of chromosome III that I have made much use of fluffy loopsfor chromosome identification. The nucleolar organizer locus lies on the right arm ofchromosome III, but in some oocytes there is little or no nucleolar material present atthe organizer locus (Fig. 38), and when the long left arm of this chromosome happensto be broken in a preparation the position of the centromere no longer serves indiagnosis. Provided, however, that the chromosome can be followed through from itsright end to just beyond the centromere, the distribution of suspended granules, andstill more of the fluffy loops relative to the centromere (Fig. 13), allow of certainidentification.

Stiff loops on chromosome XIII. Chromosome XIII can be readily identified onaccount of its small size and two spheres located near its centromere. In addition it

96 H. G. Callan

bears two adjacent pairs of loops subterminally in the left arm which, unusually forthe axolotl, are of a character found nowhere else in the complement (si in Fig. 16).These loops are strikingly asymmetric (in itself unusual for axolotl lateral loops) andtheir matrix is stiff and dense.

THE FREE NUCLEOLI IN OOCYTES

In the smallest oocytes which I have examined, of diameter about 0-25 mm, thefree nucleoli are spherical objects of uniform refractility and they all lie immediatelyadjacent to the smoothly rounded nuclear membrane. Oocytes of this size lack yolk,and the nucleus can be observed directly within the oocyte. The several hundrednucleoli are not flattened in the plane of the nuclear membrane, so the area of contactbetween each nucleolus and the membrane is slight. Nevertheless, when nuclei fromthese small oocytes are isolated and ruptured, all the nucleoli remain attached to themembrane. The nucleoli range from minute dots near the limit of resolution to amaximum diameter of about 6 [i.

Axolotl oocytes become fully opaque and white at a diameter of about o-6 mm.By the time this stage is reached the free nucleoli are noticeably larger, are of eventexture and refractility, and are much more uniform in size (Fig. 17). Most nucleoliremain round objects ranging in diameter from about 7 to 10 fi, but a small proportionare of irregular shape. In some oocytes there are occasional nucleoli 2 or more times aslarge as the general run (upper right in Fig. 18); these in all probability arise by fusionbetween normal-sized nucleoli.

The largest all-white oocytes are of about I-I mm diameter. By this stage all thefree nucleoli are of irregular shape (Figs. 18 and 26). Slightly larger oocytes developa uniform pigmentation over the surface, and oocytes of 1-25 mm diameter are intensedark brown, almost black, all over. In oocytes of this size the nucleoli of most irregularshape are clearly in the process of transforming into rings. The nucleolar substancefirst appears as though it were perforated by an eccentric hole. Later it becomes con-stricted into a few lumps, usually between 5 and 10, which remain strung togetherin a ring (Figs. 19, 27). Increase in volume of nucleolar substance accompanies thistransformation.

Oocytes of diameter 1 -\ mm are dark brown all over except for a small whitish area;as growth continues the white area extends until in the mature oocyte there are roughlyequal hemispheres of brown and white. In young axolotls about a year old the oocytesmature at i-8 mm diameter, but in older axolotls they reach a larger diameter, about2 mm.

The transformation of free nucleoli into lumpy rings is complete in an oocyte of1-5 mm diameter. In some axolotls, but not all, this transformation is accompaniedby release of granular material from the nucleoli, and in such animals the edges of thenucleoli appear very ragged at this and the immediately subsequent stage.

In oocytes up to a diameter of 1-5 mm the lampbrush chromosomes extend through-out the nuclear volume and bear long lateral loops. Further increase in oocyte size isaccompanied by extensive movements of materials within the nuclei. In the axolotl

Chromosomes and nucleoli of the axolotl 97

oocyte as it approaches maturity, and as in other urodeles, the lampbrush chromosomesstart withdrawing their lateral loops, shorten, and retire towards the middle of thenucleus. This contraction and aggregation of the chromosomes is a well-known thoughby no means well-understood phenomenon. In many axolotls (23 out of the 27 whichI have examined) pointers to the mechanism involved are provided by the free nucleoli,whose appearance during the aggregation process is bizarre in the extreme. I will firstdescribe the more common situation, and will leave consideration of the exceptionaluntil later.

Each nucleolus, which starts out as a ring of interconnected lumps, extends inwardsat one or a few places and each extension takes the form of a loop (Figs. 20, 21). Alongthese loops, which all point towards the middle of the nucleus, the nucleolar substanceis distributed as a series of beads of rather uniform size (Figs. 23-25), giving very muchthe appearance of droplets of dew on a spider's web. I deliberately draw the comparisonto a spider's web, for it is conceivable that the texture of the nucleolar substance andthe boundary it makes with the nuclear sap are such that surface tension determines theform assumed. Be this as it may, the nucleoli are evidently responding to strains set upin the nuclear sap, which in the axolotl, just as in T. cristatus and Xenopus laevisoocytes, consists of two colloid phases, one rigid and one fluid, co-extensive within thenuclear volume (Callan, 1952). The initially variable number of stretched loops pernucleolus (as stretching proceeds these merge into a single, radially extended ring)suggests that each nucleolus is responding passively to strains in its general neigh-bourhood rather than that it is being pulled towards the middle of the nucleus at onespecial point, like a chromosome at anaphase.

The rigid colloid of the nuclear sap impedes free movement of the ring nucleoli,and for a time at least tethers some of the lumpy components of individual rings atthe nuclear periphery. With continuing aggregation of the chromosomes in the middleof the nucleus, more and more nucleolar material is drawn inwards and, in the caseof most of the nucleoli, the peripheral residues ultimately break loose from theiranchorage. Those nucleoli which succeed in passing across the nuclear sap come toform a layer investing the chromosome group. Here they are no longer subject to theradially arranged stresses of the contraction phase, and they assume the form of shortrings with rough contours (Fig. 29). However, some nucleoli (and it may be up to ahundred or so; the number varies from oocyte to oocyte) fail to make the passage; atthe end of the contraction phase these remain peripheral, and continue so untilimmediately prior to ovulation.

Each ring nucleolus, whether it be centrally or peripherally located, finally amalga-mates its substance to form a solid, roughly spherical object of somewhere between10 and 20 ju, diameter (Figs. 22 and 30). In oocytes of i-8 mm and above all the nucleoliare round and solid.

The nucleolar transformations just described are illustrated diagrammatically inFig. 6. In order to take the series of photographs of whole isolated nuclei shown inFigs. 17-22, nuclei were isolated and cleaned of cytoplasm in o-i M K/NaCl, and thentransferred to an observation chamber containing the same saline. Optical sections ofthe nuclei were photographed using an ordinary (not phase) objective, with the con-

7 Cell Sci. 1

98 H. G. Callan

denser iris stopped down sufficiently to enhance contrast. Ino-iM K/NaCl the rigidcolloid of the nuclear sap maintains its form for several minutes but the fluid colloidexerts osmotic pressure and rapidly distends the membrane. This accounts for thegap between the membrane and the peripheral nucleoli in Figs. 18-22; it is an artifact.There is no such gap in Fig. 17; in oocytes of this stage (and younger ones) the nucleoliare attached to the membrane, and follow the membrane as it distends.

Nuclear membrane

Fig. 6. A sketch to show the transformations of axolotl nucleoli in oocytes developingfrom about 1 mm (extreme left) to 1-9 mm (extreme right) diameter.

During the contraction phase some nucleoli move towards the middle of the nucleusearly, others late or not at all. Those which move early suffer less deformation at thehands of the contraction mechanism than those which move late. Altogether there isa substantial lack of synchrony in the migration of the nucleoli during the contractionphase, and this accounts for the at first sight perplexing range of nucleolar form inpreparations of dispersed nuclear contents from oocytes of between i-6 and i-8 mmdiameter. To take an extreme example, a single preparation may contain short lumpyrings (nucleoli which have not yet started their migration inward, or which never will(Fig. 27)), multiple beaded loops arising from partly unstretched lumpy rings (nucleoliat the beginning of migration (Fig. 28)), single beaded rings ranging up to 300 /i long(nucleoli at full stretch between the nuclear periphery and central chromosome group(Fig. 25)), short rings of irregular outline, often markedly distorted at one point(nucleoli which have been subjected to stretching, but which have now contracted onreaching the chromosome group (Fig. 29)), and various condensed forms representing

Chromosomes and nucleoli of the axolotl 99

the final stages in the amalgamation of nucleolar substance to form solid roundedobjects (Fig. 30).

The appearance of those nucleoli which consist of several beaded loops attachedtogether (Fig. 28) suggests at first sight that a process of nucleolar self-replicationoccurs in axolotl oocytes, and this is made the more plausible because Dr J. Kezer(personal communication) and Miller (1964) have recently established that a threadof DNA forms an axis within ring-shaped nucleoli in Plethodon cinereus and Trituruspyrrhogaster. However counts of total nucleoli in single oocytes do not support suchan interpretation.

Accurate counts of nucleoli cannot be obtained from oocytes containing ringnucleoli, because in the early stages of transformation it is often impossible to decidewhether two or three neighbouring objects are separate, individual nucleoli; further-more, during the contraction phase it is often impossible to decide whether severalbeaded loops lying jumbled together are parts of one nucleolus, or of more than one.Though laborious, it is possible to make accurate counts of nucleoli from oocytes atthe stages just before transformation to rings (diameter 0-9 mm) and just after ringnucleoli have reverted to solid round objects (diameter i-8 mm).

From five oocytes of each of these two sizes preparations were made in the usualway in dispersing saline, except that to guard against the possibility of nucleolibecoming trapped in the nuclear membrane at the time of its removal, and not counted,the membrane was laid down elsewhere on the floor of the observation chamberinstead of being discarded. This precaution proved necessary for the smaller oocytes,but not for the large ones. The preparations were then photographed, and the nucleoliin each preparation were counted directly at the microscope, marking off areas on thephotographic prints as these were covered. Such a process is necessary if accuratecounts are to be made, for the nucleoli are scattered over an area of about one squaremillimetre and there is insufficient resolution in single photographs of such a largearea to permit direct counting from the print.

The nucleolar counts are given in Table 2. There is a wide variation in nucleolarnumber from oocyte to oocyte, but no evidence that large oocytes, after the ring

Table 2. Counts of total nucleoli in preparations from axolotl 1671-2

Preparationnumber

1

349

1 0

2

5678

Oocytediameter

(mm)

0-90-90-90 90 9

i-8i-8i-8i-8i - 8

Number offree nuclei

415523S°i667493

417484724

S°7607

7-2

ioo H. G. Callan

transformations have occurred, have more nucleoli than smaller ones. It is arguable,though improbable, that an increase in nucleolar number does occur, but that it iscompensated by nucleolar disintegration. This would need to be on a substantial scale,for during the early part of the contraction phase fully 50% of the nucleoli, indeedoften more, take the form of multiple loops. There is no evidence for any such dis-integration except in over-ripe oocytes,, and in normal oocytes at the time of ovulation;consequently, in my opinion, the counts establish that the multiple loop forms arenucleoli in the early stages of their stretching and migration across the nuclear sap,not nucleoli in the process of replication.

The mechanism responsible during the contraction phase for the aggregation of thechromosomes and nucleoli at the middle of the oocyte nucleus is obscure. Like theanaphase movement of chromosomes on a cell division spindle, it operates within anoverall volume which does not alter substantially. There is, of course, a progressiveincrease in nuclear volume which keeps pace with increasing oocyte size, but nountoward disturbance of this progression occurs before, during or after the contractionphase. One is tempted to envisage a series of radially arranged canals within thenuclear colloid, with fluid streams moving outward in the canals compensated by areturn flow inward, between canals, which would exert a generally distributed centri-petal pressure on chromosomes and nucleoli. However, this is merely one of severalpossible explanations, and further study will be necessary to resolve the problem.

The degree to which the free nucleoli are stretched and deformed during the aggrega-tion process varies between one axolotl and another, and in Table 3 I have indicatedthis variation by a scale ranging from o to + + + . The variation may be connectedwith nuclear sap consistency; certainly those axolotls which showed greatest nucleolarstretch had particularly stiff sap.

Four relatively young axolotls were exceptional in showing no stretching of freenucleoli. In these animals, in oocytes of between 1-2 and 1-4 mm diameter, the trans-formation to rough rings took place as usual, but re-amalgamation of the nucleolarsubstance to form smooth spheres had already occurred in oocytes of 1 -5 mm diameter,i.e. before the beginning of the movement of chromosomes and nucleoli towards themiddle of the germinal vesicle nucleus.

THE NUCLEOLAR ORGANIZER IN OOCYTES

The locus responsible for the production of free nucleoli in axolotl oocytes has beenidentified. It lies subterminally in the shorter (right) arm of chromosome III. Thischromosome has a much greater arm asymmetry (roughly 5:3) than any other of thesix largest chromosomes of the axolotl. The ratio of lengths: centromere to nucleolarorganizer, and nucleolar organizer to telomere, in the right arm of chromosome III,is nearly 9:1. These characteristics are in good accord with those which define thelocus of the nucleolar constriction in mitotic chromosomes, so with a fair measure ofassurance, just as Gall (1954) has maintained for A. tigrinum, homology between thenucleolar organizer in mitotic and lampbrush chromosomes can be assumed.

The objects attached laterally to the nucleolar organizing locus of lampbrush

Chromosomes and nucleoli of the axolotl 101

chromosome III exceed all other chromosomal materials in their refractility. Even ifsmall, they appear brightly illuminated when viewed with Zeiss Neofluar phaseobjectives, whereas all other chromosomal components appear darker than background.

The nucleolar organizing region is immensely variable in form in oocytes within thesize range on which I have concentrated, and this variability was at first bewildering.However it turned out to provide particularly strong evidence for homology betweenthe free nucleoli and objects attached at the organizer locus.

In preparations from the smallest oocytes in which I have been able to identify thenucleolar organizer (oocyte diameter of about i mm) the one or a few (up to six)objects attached to the chromosome axis are irregular and jagged in outline, and ofany size up to, but not greater than, that of the free nucleoli. The free nucleoli in thesesmaller oocytes are likewise irregular rough objects and they are of nearly uniformsize. This description holds good up to an oocyte diameter of about 1-25 mm, and inoccasional oocytes of slightly larger size (Fig. 31).

In oocytes within the size range 1-4-1-7 mm the nucleolar organizer locus may beoccupied (and the chromosome axis obscured) by a cluster of rounded objects (Figs. 32,and 39-43) or by an array of long drawn-out beaded loops (Figs. 33-36). Exceptionallythere are no, or quite minute, objects attached at the organizer locus, which is thenvisible as a cylindrical, not very refractile chromomere about 4 fi long by 2 fi wide(Figs. 38, 39). The cluster of round objects at the organizer locus is precisely com-parable in morphology to free nucleoli which have transformed into lumpy rings, andthe arrays of beaded loops similarly correspond to free nucleoli which have beenstretched and deformed during the contraction phase.

There is a considerable range in the number of round objects or beaded loopsattached to the organizer locus, and this is apparently due to variation in the originalnumber of attached nucleoli. If several nucleoli happen to be attached, each transformsinto a lumpy ring, with the rings stacked in a row along the chromosome. This causesthe chromosome axis to stretch, and in extreme cases nucleolar material may occupya length of 50/t or so (Figs. 41, 43).

There is great variation between axolotls, and between oocytes from a single axolotl,in the degree to which the attached lumpy-ring nucleoli become drawn out intobeaded loops during the contraction phase. This variation has been scored on a o to+ + + scale and is indicated in Table 3. There is a rough correspondence between thedegree of deformation of free and attached nucleoli, but even in those axolotls inwhich the stretching of the free nucleoli is extreme, there are always some oocytesin the appropriate size range whose attached nucleoli are not deformed. It seemsprobable that in such oocytes the nucleolar organizing loci happen to lie centrallywithin the germinal vesicle; the attached nucleoli would then be less exposed to thestresses of the contraction phase than they are when the organizers lie nearer thenuclear periphery.

The morphological similarity between attached and free nucleoli, and the verycomparable transformations which they undergo, is evidence that the free and attachednucleoli are homologous to one another in the sense that they share a common geneticorigin. The variable number (o to about 6) of attached nucleoli, be they solid or rings,

102 H. G. Callan

may further suggest that nucleoli periodically detach from the organizer locus of theaxolotl and add to the population of free nucleoli. Such an assertion could be madewith some assurance regarding the generation and release of spheres from the lamp-brush chromosomes of T. cristatus (Callan & Lloyd, i960) for in small oocytes ofT. cristatus there are no free spheres, and it is only in oocytes above a certain size,and where large attached spheres occur on the chromosomes, that free spheres makean appearance. In that instance the evidence based on sequence was compelling. Butregarding the free nucleoli of T. cristatus the situation is quite otherwise, as Mac-gregor (1965) has pointed out. Similar considerations apply to the axolotl.

The overwhelming majority of nucleolar 'Anlagen' in axolotl oocytes have alreadytaken up position at the nuclear membrane in oocytes of 0-25 mm diameter. The originof these nucleolar Anlagen by shedding from the organizer locus can only be inferred;it has not been demonstrated. The crux of the problem of deciding whether in olderoocytes well-developed nucleoli are shed lies in the fact that if such a process occurs,as well it may, free nucleoli are being added to a number that is already in the hundreds.That any such addition must be marginal only can be inferred from the total countsof nucleoli in five smaller (0-9 mm) and five larger (i-8 mm) oocytes given in Table 2.

Therefore there are two alternative ways to explain the observed variation in bulkof nucleolar material attached to the organizer loci. Either there is a continuing lowrate of production at, and detachment from, the organizer locus, or for some unknownreason a variable number of the last nucleolar DNA Anlagen synthesized at theorganizer do not detach, but grow, transform and develop, and carry out the charac-teristic nucleolar functions (such as RNA-synthesis in connexion with ribosome forma-tion (Brown & Gurdon, 1964)) in much the same fashion as their earlier synthesizedand detached sisters.

In the axolotl, Humphrey (1961) has described a chromosomal variant n whichdetermines the production of a smaller nucleolus than that determined by its normalallelic alternative JV. Somatic interphase nuclei of Nn heterozygotes, provided thenucleoli are not fused, generally contain two nucleoli of markedly different size, andthe heterozygotes can be readily distinguished from either homozygote by thischaracter. Fankhauser & Humphrey (1959) had earlier shown that the white mutant d,whose dominant normal allele D determines dark skin colour, lies on the nucleolar-organizing chromosome, and they gave evidence supporting the view that the locusfor D (or d) lies close to the nucleolar organizer.

By studying the lampbrush chromosomes of axolotls of various genetic constitu-tions from Dr Humphrey's colony I hoped to obtain critical evidence for the geneticidentity of oocyte and somatic nucleolar organizers. I also hoped that I might be ableto locate the gene d on the lampbrush chromosomes.

Twenty-six axolotls of the constitutions shown in Table 3 were studied. The rightarm of bivalent III was identified in a convenient number of preparations (or as manyas it proved possible to obtain from a fragment of ovary about 0-5 to 1 ml in volume)and sketches were made of the region extending from the nearest chiasma to the leftof the nucleolar organizer out to the end of the right arm. The preparations werescored for symmetry or otherwise of the nucleolar material attached at the two homo-

Tab

le 3

. Gen

etic

con

stit

utio

n an

d nu

cleo

lar

cond

itio

n in

axo

lotl

s

Axo

lotl

num

ber

2171-3

2145

-41793-1

11690-5

1647

-21518-2

2165-1

42111-1

30

1988

-319

68-1

1968

-317

37-2

1660

-41230-1

1919-1

19

19

-21671-2

1

1898

-117

08-8

1708

-37

1540-2

4

1498

-4H

98-5

1671-7

1671-1

61625-2

Age

r

Yea

rs

1 1 3 3 3 4 1 1 2 2 2 3 3 5 2 2 3 2 3 3 4 4 4 3 3 3

-*

v

Mon

ths

0 1 0 5 9 3 1 2 1 2 2 2 9 6 ^ 1

4 6 J

6 4 4 2 4 1

4 J

6 1

6

Gen

etic

cons

titut

ion

DN

/DN

• dN

/dN

• dn

/dn

DN

/dN

DN

/dn

- dN

/dn

• f ( I ( I

Stat

us o

f nu

cleo

liat

tach

ea a

t the

orga

nize

r lo

cus

A.

(

Lar

ger Sym

-m

etri

cal

0 0 0

IS 10 7 0 2 0 8 4 4 1 3

10 138

[ 9 7 6

1 0 4 0 2 3 6

AS

Asy

m-

met

rica

l

0 0 0 0 0 0 0 0 0 0 6 0 1 6 2 1 2 5 8 8 5

12

14 5 0 3

Smal

lor

abse

nt

6 71

0 0 0 0 7 5 6 0 11

0

10 1 0 0 0 0 0 1 1 2 0 3 6 6

Deg

ree

ofst

retc

hing

of

atta

ched

nuc

leol

i

0 0 0

Var

iabl

e to

+ +

+V

aria

ble

to +

+V

aria

ble

to +

+0 0 0

Var

iabl

e to

+ +

+V

aria

ble

to +

+0 0 0

Var

iabl

e to

+ +

Var

iabl

e to

+V

aria

ble

to +

+V

aria

ble

to +

+V

aria

ble

to +

Var

iabl

e to

+V

aria

ble

to +

Var

iabl

e to

+V

aria

ble

to +

+

0 0 0

Deg

ree

ofst

retc

hing

of

free

nuc

leol

i

+ + 0 +

+ +

++

+ +

+ +

+0 0 0

+ +

+ |

+ +

+ 1

+ +

I+

+

)+

+ +

++

++

+ +

+ +

++

+ + +

+ +

++

+ +

+ +

++

++

++

+

Rem

arks

Rec

ent

impo

rtat

ion

from

Mex

ico

Pure

Wis

tar

whi

teR

emot

e da

rk a

nces

tor

Pure

Wis

tar

whi

teFr

om m

atin

g D

d x

Dd

Pure

Wis

tar

whi

te

Rem

ote

dark

anc

esto

rSe

vera

l da

rk a

nces

tors

s "** s 3 icleoh r S" w 0

104 H. G. Callan

logous organizer loci, an assessed volume ratio of 2: i or more being arbitrarilydesignated asymmetric, of less than 2:1 symmetric. When both organizer loci hadattached nucleolar material as small or smaller in volume than is to be seen in Figs. 31and 39 the nucleolar status was classed separately as 'small or absent', without regardto symmetry. I came to adopt this method of classification because of the great vari-ability in size of attached nucleolar material that is encountered in most axolotis, andbecause it seemed to me that more significance should be attributed to symmetry orasymmetry of large masses of nucleolar material than to small. Whether this methodof scoring is justifiable or not (and it was adopted before I had properly appreciatedthe metamorphoses characteristic of free nucleoli) it has had the effect of discriminatingbetween bivalents where both nucleolar organizers carry a single nucleolus (or itssingle derivative ring), and bivalents where one or both nucleolar organizers carryseveral nucleoli (or their derivative rings).

The data are set out in Table 3. The observations are not conclusive. There areindications that nucleolar organizers in the oocytes of young axolotis tend to carrysmall (i.e. single) nucleoli, and that in regard to this character the ' pure Wistar white'strain may remain juvenile longer than dark axolotis. As regards the symmetry orasymmetry of larger nucleolar masses, the uniform symmetry of three older DN/DNanimals 1690-5, 1947-2, and 1518-2 (Figs. 32, 34 and 35), the preponderant symmetryof the three dnjdn animals (Fig. 33) and the preponderant asymmetry of the twoDN/dn animals (Figs. 41, 42) certainly suggest that the different potentialities ofN and n are expressed in oocytes. Further, Dr Humphrey has told me that, based onthe relative sizes of somatic interphase nucleoli, he suspects there may be a differencein nucleolar organizing capacity between N in the pure Wistar white strain and N indark axolotis; if so this might at least in part account for the extensive occurrence ofnucleolar asymmetry in the four DN/dN animals (Fig. 36). However, it must beclearly stated that animals which are genuinely homozygous AW may show in Someoocytes quite as striking asymmetry of attached nucleoli as heterozygotes, and viceversa animals which are known to be Nn heterozygotes may in some oocytes showsymmetry (Fig. 43).

The n variant was first detected by Humphrey in heterozygous combination in anexceptional diploid white axolotl among offspring from a mating DD x dd. Dr Humphreyhas told me that another exceptional diploid white offspring from an earlier mating ofthe same kind was also heterozygous for somatic nucleolar determinants, though hedid not detect it as such until later. This led Humphrey to propose (1961) that theseexceptional diploid white animals owe their origin to a deletion including the locus Dand part of N. I have examined with particular care the regions immediately to leftand right of the nucleolar organizers in the lampbrush chromosomes of two animalsof the constitution DN/dn, but have not found evidence of heterozygosity for a dele-tion. Often enough there are chiasmata close to the organizers, and any gross asym-metry in the chromosome axes between the two organizers and a nearby chiasma oughtto be apparent; but such asymmetry has not been observed.

At nucleolar organizing loci where the attached ring nucleoli were drawn out intobeaded loops I have occasionally noticed a pair of beaded strands extended in the long

Chromosomes and nucleoli of the axolotl 105

axis of the chromosome and forming the only connexion between the chromosomeregions to left and right of the organizer (Figs. 34, 35). Now this is exactly comparableto the characteristic 'double bridges' formed by the lateral loops of lampbrushchromosomes when their parent chromomere has cleaved transverse to the chromo-some axis. The observation is significant because it indicates that the nucleolarorganizer locus bears exactly the same structural relationship to the rest of the chromo-some as any other chromomere with its attendant pair of lateral loops. Moreover, itmust also mean that those DNA strands of the nucleolar organizer which remain aspersisting components of the chromosome are capable, once the cycles of replicationof DNA for the free nucleoli are over, of themselves becoming involved in the syn-thesis of nucleolar RNA and protein. This being so, the length relationship betweenthe persisting (chromosomal) nucleolar DNA and the detached nucleolar DNA is ofinterest. Is the detached DNA of the same length as the attached? If it is, and if N islonger than n, one would expect to find nucleoli with two different DNA lengths inNn heterozygotes. This question deserves further investigation.

At the end of the contraction phase there can be no doubt that attached nucleoli areshed from the organizers. However, if at the stage when the free nucleoli have roundedup, there remain any attached nucleoli, these too are no longer rings but instead solidround objects (see Fig. 37).

MITOTIC SECONDARY CONSTRICTIONS VIS-A-VIS LAMPBRUSH CHROMOSOME

LANDMARKS

The evidence for a straightforward genetic relationship between the nucleolarorganizer constriction in mitotic chromosomes and the nucleolar locus in lampbrushchromosomes has already been presented. However I have been unable to detect anyoutstanding landmarks on the lampbrush chromosomes corresponding in distributionto those secondary constrictions in mitotic chromosomes which appear when the somatictissues are exposed to low temperature (compare Figs. 4 and 5). As earlier mentioned,one might suppose that these constrictions owe their origin to a mechanism analogousto that responsible for the nucleolar constriction, i.e. exceptional accumulation andtardy detachment of certain gene products such as to interfere with prophase spiraliza-tion. These secondary constrictions are constant and identical in their distributionover the chromosome complements of three different tissues; moreover, similarconstrictions appear in the male meiotic metaphase chromosomes of Triturus helveticus,T. vulgaris and T. cristatus when the diplotene stage (corresponding to the lampbrushphase of female meiosis) is subjected to low temperature (Callan, 1942).

The short arm of the XlVth mitotic chromosome of the axolotl is divided by twocold-induced secondary constrictions into three regions of roughly equal length, butthere are no landmarks along this arm of the lampbrush bivalent (Fig. 15). The shortarm of the Xlllth mitotic chromosome has a nearly median secondary constriction,but again there is no landmark along this arm of the lampbrush bivalent (Fig. 16);contrariwise in the left arm of the lampbrush bivalent there are two characteristicspheres near the centromere (Figs. 14, 16), yet there are no cold-induced secondary

106 H. G. Callan

constrictions anywhere in the left arm of mitotic chromosome XIII. A similar state-ment holds for all the other chromosomes, and a general negative conclusion isunavoidable. This is to say that loci which one may presume to carry out some specialfunction in somatic interphase nuclei are represented by quite unremarkable lateralloops in oocytes.

CHIASMATA IN OOCYTES

Chiasmata are formed at a very high frequency in axolotl oocytes, and as few otherfusions occur between the lampbrush chromosomes, and as most of these can bediscriminated from chiasmata, accurate determination of chiasma frequency is possible.Centromere fusions such as occur abundantly in T. cristatus karelinii (Callan & Lloyd,i960; Watson & Callan, 1963) and occasionally in T. viridescens (Gall, 1954) have notbeen observed in the axolotl, and scarcely any terminal fusions have been noticed.

Total chiasmata per nucleus in the five lampbrush chromosome complements whichwere fully analysed are: 114, 126, 101, 101, 123, the mean of these figures being 113.The distribution of chiasmata looks to be substantially at random, unlike the situation inT. cristatus lampbrush chromosomes. If it were truly random, taking the mean chiasmafrequency at 113 per nucleus and the total haploid chromosome length at 1000 arbi-trary units, this would amount on the average to 0-34 chiasmata per 3 units of length.The one chromosome region where I have systematically scored chiasmata in manypreparations is the 3 units between the nucleolar organizer and the end of the rightarm of chromosome III. The recorded chiasma frequency in this region (based oneighty-one observations) is o-88, so in detail the distribution is probably not random.

Female heterogamety in the axolotl has been firmly established (Humphrey, 1945).I have found no cytological evidence for an extensive sex-differential chromosomesegment, devoid of crossing-over, in this animal, and this is in accord with Humphrey's(1948) conclusion, based on extensive breeding experiments with sex-reversedaxolotls, that: ' the W chromosome probably lacks nothing found in the Z except thegene or genes of the differential segment responsible for stimulating gonad develop-ment in the male direction'.

Brunst & Hauschka (1963) and Hauschka & Brunst (1965), from studies of axolotlmitotic chromosomes, have claimed that in the female complement the XHIth pairof chromosomes are heteromorphic, the longer arm of one being substantially longerthan that of the other. My studies of the lampbrush chromosomes indicate that nosuch differences exist between the XHIth or any other chromosome pair. An exampleof bivalent XIII is shown in Fig. 16.

I am greatly indebted to Dr R. R. Humphrey for providing me with axolotls from his colony,to Dr R. Briggs for supplying several laboratory materials and facilities, and to Mrs L. Lloydfor help with the illustrations accompanying this paper.

Chromosomes and nucleoli of the axolotl 107

REFERENCES

BROWN, D. D. & GURDON, J. B. (1964). Absence of ribosomal RNA synthesis in the anucleolatemutant of Xenopus laevis. Proc. natn. Acad. Sci. U.S.A. 51, 139-146.

BRUNST, V. V. & HAUSCHKA, T . S. (1963). Length measurements of the diploid karyotype of theMexican axolotl (Siredon mexicanum) with reference to a possible sex difference. Proc. XVIthint. Congr. Zool., Wash. 2, 274.

CALLAN, H. G. (1942). Heterochromatin in Triton. Proc. R. Soc. B 130, 324-335.CALLAN, H. G. (1952). Experimental work on amphibian oocyte nuclei. Symp. Soc. exp. Biol.

6, 243-255-CALLAN, H. G. & LLOYD, L. (i960). Lampbrush chromosomes of crested newts Triturus

cristatus (Laurenti). Phil. Trans. R. Soc. B 243, 135—219.CLARKE, J. L. (1851). Researches into the structure of the spinal chord. Phil. Trans. R. Soc.

141, 607-621.DEARING, W. H. (1934). The material continuity and individuality of the somatic chromosomes

of Ambystoma tigrinum, with special reference to the nucleolus as a chromosomal component.J. Morph. 56, 157-179-

FANKHAUSER, G. & HUMPHREY, R. R. (1942). Induction of triploidy and haploidy in axolotleggs by cold treatment. Biol. Bull. mar. biol. Lab., Woods Hole 83, 367-374.

FANKHAUSER, G. & HUMPHREY, R. R. (1959). The origin of spontaneous heteroploids in theprogeny of diploid, triploid and tetraploid axolotl females. J. exp. Zool. 142, 379-417.

GALL, J. G. (1954). Lampbrush chromosomes from oocyte nuclei of the newt. J. Morph. 94,283-351.

HAUSCHKA, T . S. & BRUNST, V. V. (1965). Sexual dimorphism in the nucleolar autosome of theaxolotl {Siredon mexicanum). Hereditas 52, 345-356.

HUMPHREY, R. R. (1945). Sex determination in ambystomid salamanders: a study of theprogeny of females experimentally converted into males. Am. J. Anat. 76, 33-66.

HUMPHREY, R. R. (1948). Reversal of sex in females of genotype WW in the axolotl (Siredon orAmbystoma mexicanum) and its bearing upon the role of the Z chromosomes in the develop-ment of the testis. J. exp. Zool. 109, 171-185.

HUMPHREY, R. R. (1961). A chromosomal deletion in the Mexican axolotl (Siredon mexicanum)involving the nucleolar organizer and the gene for dark colour. Am. Zool. 1, 361 (Abstr.).

HUMPHREY, R. R. (1962). A semilethal factor (v) in the Mexican axolotl (Siredon mexicanum)and its maternal effect. Devi Biol. 4, 423-451.

HUMPHREY, R. R. (1964). Genetic and experimental studies on a lethal factor in the axolotlwhich induces abnormalities in the renal system and other organs. J. exp. Zool. 155, 139-150.

MCCLINTOCK, B. (1934). The relation of a particular chromosomal element to the developmentof the nucleoli in Zea mays. Z. Zellforsch. mikrosk. Anat. 21, 294-328.

MACGREGOR, H. C. (1965). The role of lampbrush chromosomes in the formation of nucleoliin amphibian oocytes. Q. Jl microsc. Sci. 106, 215—228.

MILLER, O. L. (1964). Extrachromosomal nucleolar DNA in amphibian oocytes. J. Cell Biol.23, 60A.

SIGNORET, J., BRIGGS, R. & HUMPHREY, R. R. (1962). Nuclear transplantation in the axolotl.Devi Biol. 4, 134-164.

WATSON, I. D. & CALLAN, H. G. (1963). The form of bivalent chromosomes in newt oocytesat first metaphase of meiosis. Q.jfl microsc. Sci. 104, 281-295.

(Received 26 August 1965)

io8 H. G. Callan

Fig. 7. Normal complement of mitotic chromosomes from the liver of an axolotl larva.n, nucleolar organizer constriction.Fig. 8. Mitotic chromosomes from the liver of a cold-treated axolotl larva, showingthe secondary constrictions induced by low temperature, n, nucleolar organizerconstriction.

Journal of Cell Science, Vol. i, No. i

8J ' * »i.

II. G. CALLAN {Facing p. 108)

Fig. 9. Mitotic chromosomes from a tail-fin epidermal cell of a cold-treated axolotllarva, showing the secondary constrictions induced by low temperature.Figs. 10-13. Parts of axolotl lampbrush bivalents, all to the scale drawn on Fig. 13.Phase contrast. Fig. 10. Part of bivalent VI. c, centromere; r, towards right end;s, sphere. Fig. 11. Part of right arm of bivalent II. r, towards right end; sg, sus-pended granule. Fig. 12. Part of left arm of bivalent VI. ag, axial granule; le, left end.Fig. 13. Part of bivalent III. c, centromere;/, fluffy loops; Isg, large suspended granule;r, towards right end.

Journal of Cell Science, Vol. i, No. 1

H. G. CALLAN

Figs. 14-16. All phase contrast. Fig. 14. Part of axolotl bivalent XIII. c, centromere;r, towards right end; s, sphere. Fig. 15. Axolotl bivalent XIV entire, c, centromere;sg, suspended granule (at right end). Fig. 16. Axolotl bivalent XIII entire, c, centro-mere ; re, right end; s, sphere; si, stiff loops.

Journal of Cell Science, Vol. i , No. i

H. G. CALLAN

Journal of Cell Science, Vol. i, No. i

Figs. 17-19. Isolated axolotl oocyte nuclei photographed with ordinary optics and con-tracted condenser iris. Fig. 17. From oocyte of diameter o-6 mm. Fig. 18. Fromoocyte of diameter i-o mm. Fig. 19. From oocyte of diameter 1-4 mm.

H. G. CALLAN

Journal of Cell Science, Vol. i, No. i

20

200//

21.

Figs. 20, 21. Isolated axolotl oocyte nuclei photographed with ordinary optics andcontracted condenser iris. Fig. 20. From oocyte of diameter i-6 mm. Fig. 21. Fromoocyte of diameter i '8 mm.

H. G. CALLAN

Fig. 22. Isolated axolotl oocyte nucleus, from oocyte of diameter i 9 mm, photographedwith ordinary optics and contracted condenser iris.Figs. 23-25. Phase-contrast photographs of sectors of isolated axolotl oocyte nuclei,from oocytes of diameter i-6-r8 mm.

Journal of Cell Science, Vol. i, No. i

200/<

H. G. CALLAN

Figs. 26-30. Phase-contrast photographs of free nucleoli from preparations of thedispersed contents of axolotl oocyte nuclei. Fig. 26. From oocyte of diameter 10 mm.Fig. 27. From oocyte of diameter 1-5 mm. Fig. 28. From oocyte of diameter i-6 mm.Figs. 29, 30. From oocyte of diameter 17 mm.

Fig. 31. Phase-contrast photograph of left end of axolotl (dN/dN) bivalent III, in-cluding the nucleolar organizers, from an oocyte of diameter 1-4 mm.

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H. G. CALLAN

Figs. 32-37. Phase-contrast photographs of left ends of axolotl bivalents III, in-cluding nucleolar organizers (marked by arrows in Fig. 37). Fig. 32. From oocyte ofdiameter i-6mm, DN/DN. Fig. 33. From oocyte of diameter 155 mm, dn/dn.Fig. 34. From oocyte of diameter 1-55 mm, DN/DN. Fig. 35. From oocyte of diameter1-5 mm, DN/DN. Fig. 36. From oocyte of diameter 1-75 mm, DN/dN. Fig. 37. Fromoocyte of diameter 1-85 mm, dN/dN.

Journal of Cell Science, Vol. i, No. i

H. G. CALLAN

Figs. 38-43. Phase-contrast photographs of left ends of axolotl bivalents III, in-cluding nucleolar organizers (marked by arrows in Figs. 38 and 39). Fig. 38. Fromoocyte of diameter 145 mm, dN/dn. Fig. 39. From oocyte of diameter 15 mm, dN/dn.Fig. 40. From oocyte of diameter 155 mm, dN/dn. Fig. 41. From oocyte of diameter1-55 mm, DNIdn. Fig. 42. From oocyte of diameter i-8 mm, DN/dn. Fig. 43. Fromoocyte of diameter 1-7 mm, dN/dn.

Journal of Cell Science, Vol. i, No. i

H. G. CALLAN