CHROMOSOMES AND NUCLEOLI OF THE AXOLOTL, AMBYSTOMA MEXICANUM
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J. Cell Sci. i, 85-108 (1966J 85
Printed in Great Britain
CHROMOSOMES AND NUCLEOLI OF THE
AXOLOTL, AMBYSTOMA MEXICANUM
H. G. CALLAN
Department of Zoology, The University, St Andrews, Fife
SUMMARY
Amongst the axolotl's haploid complement of fourteen mitotic chromosomes, one of the four
largest, with a greater arm asymmetry than the other three, shows a nucleolar constriction
subterminally 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 been
established, and thus the several hundred free nucleoli in oocytes are genetically related to the
two nucleoli of diploid somatic interphases. During oocyte development the free nucleoli
transform from solid structures to rings and back to solid structures again without detectable
increase in number. During the contraction and aggregation of the lampbrush chromosomes
within the oocyte nucleus as maturity approaches, in most axolotls the free ring-shaped nucleoli
become stretched between the nuclear periphery and central chromosome group, and take on
a characteristic beaded appearance. These transformations of the free nucleoli are largely
paralleled by forms which nucleoli attached subterminally to the shorter arm of lampbrush
chromosome III concurrently assume.
The question as to whether fully developed nucleoli detach from the organizer loci and add
to the population of free nucleoli in oocytes remains undecided. It may well be that virtually
all the DNA-generators of free nucleoli detach from the organizer loci before starting to carry
out nucleolar functions, and before there is any significant accumulation of protein and
RNA around them. If so, the variability in quantity of attached nucleolar material may not
reflect different states in a nucleolar synthesis and detachment cycle, but rather variation in the
number of nucleolar DNA Anlagen which happen to remain attached to the organizer loci
after the synthesis and detachment of the great majority of the Anlagen has ceased.
In occasional oocytes the only chromosomal continuity maintained across the organizer locus
consists 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 there
are 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 mitotic
chromosomes 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 be
able to correlate some particular genetic trait with a lateral loop characteristic on the86 H. G. Callan oocyte lampbrush chromosomes, both the genetical and cytological attributes of a species, for preference a urodele, need to be known. Secondly, the axolotl, like its near relative Ambystoma tigrinum, is unusual among urodeles in having a simple somatic nucleolar condition. There is one nucleolar organizer per haploid chromosome set. Having failed to determine whether or not there is a genetic relationship between somatic nucleolar organizers and free oocyte nucleoli in Triturus cristatus, where the somatic nucleolar condition is complex, there seemed better hope of deciding this question by working with the axolotl. Moreover, there is an established nucleolar mutant in the axolotl (Humphrey, 1961) and hence the possibility that this might be recognized in the lampbrush chromosome complement. These two considerations were put to me by Dr J. G. Gall of Yale University, early in 1964. An opportunity to take up the study was presented soon afterwards when I was invited by Dr T. M. Sonneborn to spend a year at the Zoology Department of Indiana University, where Dr R. R. Humphrey maintains an axolotl colony. In order to gain some general idea of the relative lengths of the various chromosomes making up the axolotl complement, the positions of their centromeres and of the nucleolar 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 of several more secondary constrictions which become apparent after appropriate ex- posure to low temperature. With this information as a guide, the lampbrush chromosomes were examined. In the course of this study I was struck by the remarkable variability in form of the free nucleoli, and this led me to investigate the developmental history of the free nucleoli during 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 larvae about 15 mm long, raised at room temperature (21 °C), and which had just started to feed, were placed for 12 h in 0-5 % colchicine in pond water, and then fixed entire for 12 h in Clarke's (1851) 3:1 ethyl alcohol/acetic acid fixative. From these fixed larvae the tail fins and livers were excised and placed on separate slides. To each preparation a drop or two of 0-5 % aceto-orcein (G. T. Gurr, synthetic) was added, a small watch glass was set over each preparation to reduce evaporation, and 30 min of staining followed. 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 by finger-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-fin epithelial cells; an example is shown in Figs. 1 and 7. The range in chromosome size from 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 haploid
complement into eight larger chromosomes with median or submedian centromeres,
in no case with arm ratios approaching 2:1, and six smaller chromosomes with
marked arm asymmetry, all with ratios greater than 2:1.
Fig. 1. Camera lucida drawing of axolotl hepatocyte mitotic chromosomes from a
normal larva. A photograph of this same complement is shown in Fig. 7; in the drawing
the chromosomes have been spaced out to avoid overlaps, n, nucleolar organizer
constriction.
Of the eight larger chromosomes, two with virtually median centromeres (VII and
VIII) are substantially smaller than the rest; they are indistinguishable from one
another. Among the remaining six larger chromosomes there is one with a greater
arm asymmetry than the others (it has a ratio of about 5:3), and subterminally in its
short arm there is a secondary constriction. This is the only axolotl chromosome which
normally shows such a constriction; the constriction is more evident in hepatocyte
than in tail-fin epithelial chromosomes. In the light of Dearing's (1934) classic study
on the related A. tigrinum, where a subterminal constriction in the short arm of a
large chromosome of similar arm ratio was shown to be the site of production of the
nucleolus, this secondary constriction in the axolotl can be assumed to mark the
nucleolar organizer. The nucleolar chromosome of the axolotl can be further ranked
as one of the four largest chromosomes in the complement, but I have not found it
possible, 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 ease
and arranged in length order without ambiguity. However, I have been unable to
differentiate with certainty between chromosomes IX, X and XLH. G. Callan
THE MITOTIC CHROMOSOMES IN LARVAE EXPOSED TO L O W TEMPERATURE
When larvae of three species of Triturus were subjected to low temperature for a
few days prior to fixation, their mitotic chromosomes showed many secondary con-
strictions which are not apparent in normal mitoses (Callan, 1942). The same is true
of 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. Identified
chromosomes (all but I, II and IV) are marked by Roman numerals, n, nucleolar
organizer constriction.
Axolotl larvae which had just started to feed were placed in a refrigerator adjusted
so that the water temperature fell to 2-5 °C. A 0-5% solution of colchicine in pond
water was also brought to this temperature, and after 3 days in plain cold water the
larvae were transferred to the cold colchicine solution, and left in the cold for a further
12 h. They were then fixed and preparations made as already described. From a few
of the larvae samples of brain tissue as well as liver and tail-fin epithelium were
excised 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 fully
analysable complements (Figs. 2, 8).
No attempt was made to determine the minimum duration of cold treatment
necessary to make these constrictions evident, but a point concerning temperature
deserves mention. Larvae treated at o to 0-5 °C show constricted chromosomes, but
in tail-fin preparations in particular the chromosomes are poorly spread and have fuzzy
outlines, which I think is connected with a failure of the nuclear membrane to break
down. 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 from
a low-temperature treated larva. A photograph of this same complement is shown in
Fig. 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 in
tail-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 particular
chromosome set shows any cold-induced constrictions, one may expect to find all of
those in group A. The group B constrictions are apparent in fewer cells, they are more
evident in tail fin than in liver, and when they are present one can be sure to find all
the 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 not90 H. G. Callan
attempt'to make systematic length measurements of the mitotic chromosomes. For
this reason in Fig. 4 the positions of the cold-induced secondary constrictions have
been 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-h
B A
I
" 1 1
B ^ B
tt tn
BA
IV
t t
B B
\
t
A
VI
VII
t tt
A AB
I
VIII
t t '
A A
IX
i
t
A
I
X
t
A
XI
I
t t
A B
\
XII
t tt
A AB
XIII
\
t
1A
ff
BA
Fig. 4. Axolotl karyotype based on relative lengths of the lampbrush chromosomes.
The positions of centromeres are indicated by arrows pointing down. The arrows
pointing 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 concerns
three of the four largest members of the complement.
I was unable to make any well-spread preparations from brain tissue. However, the
chromosomes show constrictions much as in hepatocytes, and the ability to recognize
several individual chromosomes by the familiar distribution of their constrictions,Chromosomes and nucleoli of the axolotl 91
notably III, XII, XIII and XIV, leads me to suppose that the constriction pattern is
the 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 of
centromeres are indicated by arrows pointing down, n, nucleolar organizer.
I accepted the possibility that the cold-induced constrictions in Triturus might
represent 'under-charged' regions, possibly even regions devoid of nucleic acid. This
whole concept has now rightly fallen into disrepute, and another explanation is called
for. By analogy with McClintock's (1934) description of the origin of the nucleolar
constriction in metaphase chromosomes of maize, where the nucleolus at prophase
remains for a time attached to the nucleolar chromosome and interferes with its92 H. G. Callan condensation, one might suppose that the action of an abnormally low temperature is 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 with condensation 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 would expect quite different constriction patterns in tissues as unlike as liver and ectodermal epithelium. THE LAMPBRUSH CHROMOSOMES Axolotl lampbrush chromosomes were studied by the techniques devised for Triturus viridescens (Gall, 1954) and T. cristatus (Callan & Lloyd, i960). Three particular technical problems presented by axolotl oocytes were overcome, and these deserve attention. The nuclear sap within axolotl germinal vesicles is a stiff jelly, stiffer than I have ever 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 lampbrush chromosomes in detail. The standard saline in which germinal vesicle nuclei are isolated, o-1M KC1 or NaCl or a mixture of the two, can be modified in two ways to achieve sap dispersal within the observation chamber. It may be diluted, though only to 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 limited extent, for at concentrations in excess of IO~*M the chromosomes contract and stiffen as though fixed. A saline suitable for dispersing the axolotl oocyte's nuclear sap, and which leaves the chromosomes in good condition, consists of o-i M K/NaCl containing 0-5 x I O ^ M CaCl 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 dilution causes appreciable loss of matrix from the loops. Furthermore, a three-fifth dilution of 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 is common practice to expose the nuclear contents, in dispersing saline, to the fumes of concentrated, neutralized formalin for a minute or two before the preparation is covered. This treatment kills some bacteria which might otherwise contaminate the preparation, and apparently stabilizes the lateral loop ribonucleoproteins against • autolysis. With oocytes of several species of urodeles this short exposure to formalin fumes 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. In axolotl 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 with dispersing saline, and these fibres anchor the lampbrush chromosomes to one another. The membrane surrounding the axolotl's germinal vesicle nucleus adheres to any clean 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 when
the membrane is seized by forceps and the nucleus lifted, a hole is torn around the
area of adhesion and some of the nuclear contents ooze out through this hole before
a tungsten needle can be manipulated to tear the membrane clear away. ' Strangled'
preparations result. This trouble can be avoided by keeping the nucleus continually
on the move while the cytoplasm is pumped away with a pipette, and by manipulating
forceps and needle without delay once the cleaned nucleus has been placed in its
observation chamber.
Until the developmental stage is reached when the chromosomes start to aggregate
in 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 very
long. Thus there is a great congestion of chromosomal material within the nuclei of
smaller oocytes, and I have been unable to obtain fully analysable preparations from
oocytes below 1-4 mm diameter; even after the membrane has been removed without
damage 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 obtained
is therefore limited, much more restricted than is the case with T. viridescens or
T. cristatus. The lower limit is 1-4 mm, the upper 1-7 mm. In oocytes larger than
1-7 mm in diameter retraction of the lateral loops is well under way, and most of the
landmark 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 not
numerous; 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 offivecomplete undamaged complements,
Table 1. Relative lengths of axolotl lampbrush chromosomes
Chromosome Longer arm Shorter arm Overall
I 64-4 52-6 117
II 60-3 55-7 116
III (nucleolar) 65-3 367 102
IV S4-o 46-0 100
V 50-2 37-8 88
VI (bearing 5O-S 36-S 87
spheres)
VII 33-3 30-7 64
VIII 3i-6 3°-4 62
IX 42-0 18-0 60
X 41-0 16-0 57
XI 39-o 13-0 52
XII 32-3 97 42
XIII (bearing 21-9 8-i 30
spheres)
XIV 17-8 62 24
Total 100194 H. G. Callan with subsidiary data taken from over ioo other preparations. By convention, the longer chromosome arms are called ' left' arms. Relative lengths of the fourteen chromosomes (Table i), and the positions of landmarks, were worked out in the manner described by Callan & Lloyd (i960). In order that the data should be roughly comparable to the corresponding data for T. cristatus, an overall map length of 1000 units was allocated to 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 without difficulty. The centric regions are short lengths (10/iora little less) of chromosome axis devoid of lateral loops. These stretches of axis are of irregular width, in general somewhat 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 no sign 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 from the centromeres. Nevertheless, there are objects which permit the identification of various chromosomes, and these will be described. Spheres. There are two spheres just to the left of the centromeres of chromosomes VI and XIII (Figs. 10, 14 and 16). These objects closely resemble the structures also called spheres on chromosomes V and VIII of T. cristatus. They are directly attached to the chromosome axis. Homologous sphere-generating loci are often jointly involved in the production of single spheres, i.e. sphere fusions are common. Attached spheres vary in size up to a maximum diameter of about 10 /*, and free spheres of similar dimensions and refractility are frequently encountered in oocytes within the size range which I have studied. Just as in T. cristatus it is probable that detachment of the spheres from their generating loci occurs, and is followed by the production of more spheres; a further comment on this point is made in the section concerned with the nucleolar 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 may well be duplicate. Suspended granules. This term has been introduced to define spherical objects which 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 chromosomes can carry suspended granules (e.g. Figs. 15, 34 and 35), but this is certainly true of most. 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 are
quite variable in their occurrence. They are not indicated on the working map.
On the other hand, the distribution of interstitial suspended granules, even though
individually these too may be present or absent in a given preparation, are of great
help in identifying particular chromosomes and parts of chromosomes. Thus, for
example, chromosomes I and II are much of a muchness for length and centromere
position, but the places where suspended granules are likely to be found are distinctive
for 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 to
discriminate between chromosomes IX, X and XL
Suspended granules are composed of somewhat more refractile material than spheres
of similar size. As with spheres, though less frequently, suspended-granule fusions
occur. Moreover, the variable presence or absence of granules at particular loci, and
the 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) as
the 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-eighth
the way in from the telomere in the left arm of chromosome VI (Fig. 12), while the
other lies very close to the telomere of the right arm of chromosome VII. Neither of
these axial granules bears lateral loops; either may fuse with its homologue.
Fluffy loops. These are more bulky structures than the generally long and slender
lateral loops of axolotl lampbrush chromosomes, and their matrix has afluffytexture.
They are often so contorted and compacted around the chromosome axes that their
sites of attachment appear as fuzzy, ill-defined regions. They are of more diagnostic
value in oocytes at the lower end of the size range which I have studied, for in larger
oocytes, where some degree of loop retraction has set in, they may no longer show
their distinctive character.
It is only in the case of chromosome III that I have made much use of fluffy loops
for chromosome identification. The nucleolar organizer locus lies on the right arm of
chromosome III, but in some oocytes there is little or no nucleolar material present at
the organizer locus (Fig. 38), and when the long left arm of this chromosome happens
to be broken in a preparation the position of the centromere no longer serves in
diagnosis. Provided, however, that the chromosome can be followed through from its
right end to just beyond the centromere, the distribution of suspended granules, and
still more of the fluffy loops relative to the centromere (Fig. 13), allow of certain
identification.
Stiff loops on chromosome XIII. Chromosome XIII can be readily identified on
account of its small size and two spheres located near its centromere. In addition it96 H. G. Callan
bears two adjacent pairs of loops subterminally in the left arm which, unusually for
the 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) and
their matrix is stiff and dense.
THE FREE NUCLEOLI IN OOCYTES
In the smallest oocytes which I have examined, of diameter about 0-25 mm, the
free nucleoli are spherical objects of uniform refractility and they all lie immediately
adjacent to the smoothly rounded nuclear membrane. Oocytes of this size lack yolk,
and the nucleus can be observed directly within the oocyte. The several hundred
nucleoli are not flattened in the plane of the nuclear membrane, so the area of contact
between each nucleolus and the membrane is slight. Nevertheless, when nuclei from
these small oocytes are isolated and ruptured, all the nucleoli remain attached to the
membrane. The nucleoli range from minute dots near the limit of resolution to a
maximum 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 even
texture and refractility, and are much more uniform in size (Fig. 17). Most nucleoli
remain round objects ranging in diameter from about 7 to 10 fi, but a small proportion
are of irregular shape. In some oocytes there are occasional nucleoli 2 or more times as
large as the general run (upper right in Fig. 18); these in all probability arise by fusion
between normal-sized nucleoli.
The largest all-white oocytes are of about I-I mm diameter. By this stage all the
free nucleoli are of irregular shape (Figs. 18 and 26). Slightly larger oocytes develop
a uniform pigmentation over the surface, and oocytes of 1-25 mm diameter are intense
dark brown, almost black, all over. In oocytes of this size the nucleoli of most irregular
shape are clearly in the process of transforming into rings. The nucleolar substance
first 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 together
in a ring (Figs. 19, 27). Increase in volume of nucleolar substance accompanies this
transformation.
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 roughly
equal hemispheres of brown and white. In young axolotls about a year old the oocytes
mature at i-8 mm diameter, but in older axolotls they reach a larger diameter, about
2 mm.
The transformation of free nucleoli into lumpy rings is complete in an oocyte of
1-5 mm diameter. In some axolotls, but not all, this transformation is accompanied
by release of granular material from the nucleoli, and in such animals the edges of the
nucleoli 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 is
accompanied by extensive movements of materials within the nuclei. In the axolotlChromosomes and nucleoli of the axolotl 97
oocyte as it approaches maturity, and as in other urodeles, the lampbrush chromosomes
start withdrawing their lateral loops, shorten, and retire towards the middle of the
nucleus. This contraction and aggregation of the chromosomes is a well-known though
by no means well-understood phenomenon. In many axolotls (23 out of the 27 which
I 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 first
describe the more common situation, and will leave consideration of the exceptional
until later.
Each nucleolus, which starts out as a ring of interconnected lumps, extends inwards
at one or a few places and each extension takes the form of a loop (Figs. 20, 21). Along
these loops, which all point towards the middle of the nucleus, the nucleolar substance
is distributed as a series of beads of rather uniform size (Figs. 23-25), giving very much
the appearance of droplets of dew on a spider's web. I deliberately draw the comparison
to a spider's web, for it is conceivable that the texture of the nucleolar substance and
the boundary it makes with the nuclear sap are such that surface tension determines the
form assumed. Be this as it may, the nucleoli are evidently responding to strains set up
in the nuclear sap, which in the axolotl, just as in T. cristatus and Xenopus laevis
oocytes, consists of two colloid phases, one rigid and one fluid, co-extensive within the
nuclear volume (Callan, 1952). The initially variable number of stretched loops per
nucleolus (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 one
special 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 at
the nuclear periphery. With continuing aggregation of the chromosomes in the middle
of the nucleus, more and more nucleolar material is drawn inwards and, in the case
of most of the nucleoli, the peripheral residues ultimately break loose from their
anchorage. Those nucleoli which succeed in passing across the nuclear sap come to
form a layer investing the chromosome group. Here they are no longer subject to the
radially arranged stresses of the contraction phase, and they assume the form of short
rings with rough contours (Fig. 29). However, some nucleoli (and it may be up to a
hundred or so; the number varies from oocyte to oocyte) fail to make the passage; at
the end of the contraction phase these remain peripheral, and continue so until
immediately 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 between
10 and 20 ju, diameter (Figs. 22 and 30). In oocytes of i-8 mm and above all the nucleoli
are round and solid.
The nucleolar transformations just described are illustrated diagrammatically in
Fig. 6. In order to take the series of photographs of whole isolated nuclei shown in
Figs. 17-22, nuclei were isolated and cleaned of cytoplasm in o-i M K/NaCl, and then
transferred to an observation chamber containing the same saline. Optical sections of
the nuclei were photographed using an ordinary (not phase) objective, with the con-
7 Cell Sci. 198 H. G. Callan
denser iris stopped down sufficiently to enhance contrast. Ino-iM K/NaCl the rigid
colloid of the nuclear sap maintains its form for several minutes but the fluid colloid
exerts osmotic pressure and rapidly distends the membrane. This accounts for the
gap 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 nucleoli
are 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 developing
from about 1 mm (extreme left) to 1-9 mm (extreme right) diameter.
During the contraction phase some nucleoli move towards the middle of the nucleus
early, others late or not at all. Those which move early suffer less deformation at the
hands of the contraction mechanism than those which move late. Altogether there is
a substantial lack of synchrony in the migration of the nucleoli during the contraction
phase, and this accounts for the at first sight perplexing range of nucleolar form in
preparations of dispersed nuclear contents from oocytes of between i-6 and i-8 mm
diameter. To take an extreme example, a single preparation may contain short lumpy
rings (nucleoli which have not yet started their migration inward, or which never will
(Fig. 27)), multiple beaded loops arising from partly unstretched lumpy rings (nucleoli
at 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 on
reaching the chromosome group (Fig. 29)), and various condensed forms representingChromosomes and nucleoli of the axolotl 99
the final stages in the amalgamation of nucleolar substance to form solid rounded
objects (Fig. 30).
The appearance of those nucleoli which consist of several beaded loops attached
together (Fig. 28) suggests at first sight that a process of nucleolar self-replication
occurs in axolotl oocytes, and this is made the more plausible because Dr J. Kezer
(personal communication) and Miller (1964) have recently established that a thread
of DNA forms an axis within ring-shaped nucleoli in Plethodon cinereus and Triturus
pyrrhogaster. However counts of total nucleoli in single oocytes do not support such
an interpretation.
Accurate counts of nucleoli cannot be obtained from oocytes containing ring
nucleoli, because in the early stages of transformation it is often impossible to decide
whether two or three neighbouring objects are separate, individual nucleoli; further-
more, during the contraction phase it is often impossible to decide whether several
beaded 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 at
the stages just before transformation to rings (diameter 0-9 mm) and just after ring
nucleoli have reverted to solid round objects (diameter i-8 mm).
From five oocytes of each of these two sizes preparations were made in the usual
way in dispersing saline, except that to guard against the possibility of nucleoli
becoming 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 chamber
instead of being discarded. This precaution proved necessary for the smaller oocytes,
but not for the large ones. The preparations were then photographed, and the nucleoli
in each preparation were counted directly at the microscope, marking off areas on the
photographic prints as these were covered. Such a process is necessary if accurate
counts are to be made, for the nucleoli are scattered over an area of about one square
millimetre and there is insufficient resolution in single photographs of such a large
area to permit direct counting from the print.
The nucleolar counts are given in Table 2. There is a wide variation in nucleolar
number 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
Oocyte
Preparation diameter Number of
number (mm) free nuclei
1 0-9 415
3 0-9 523
4 0-9 S°i
9 09 667
10 09 493
2 i-8 417
5 i-8 484
6 i-8 724
7 i-8 S°7
8 i-8 607
7-2ioo 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 is compensated 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, indeed often 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 are nucleoli 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 the chromosomes and nucleoli at the middle of the oocyte nucleus is obscure. Like the anaphase movement of chromosomes on a cell division spindle, it operates within an overall volume which does not alter substantially. There is, of course, a progressive increase in nuclear volume which keeps pace with increasing oocyte size, but no untoward disturbance of this progression occurs before, during or after the contraction phase. One is tempted to envisage a series of radially arranged canals within the nuclear colloid, with fluid streams moving outward in the canals compensated by a return flow inward, between canals, which would exert a generally distributed centri- petal pressure on chromosomes and nucleoli. However, this is merely one of several possible 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 indicated this variation by a scale ranging from o to + + + . The variation may be connected with nuclear sap consistency; certainly those axolotls which showed greatest nucleolar stretch had particularly stiff sap. Four relatively young axolotls were exceptional in showing no stretching of free nucleoli. 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 nucleolar substance 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 the middle of the germinal vesicle nucleus. THE NUCLEOLAR ORGANIZER IN OOCYTES The locus responsible for the production of free nucleoli in axolotl oocytes has been identified. It lies subterminally in the shorter (right) arm of chromosome III. This chromosome has a much greater arm asymmetry (roughly 5:3) than any other of the six largest chromosomes of the axolotl. The ratio of lengths: centromere to nucleolar organizer, 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 the locus of the nucleolar constriction in mitotic chromosomes, so with a fair measure of assurance, just as Gall (1954) has maintained for A. tigrinum, homology between the nucleolar 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 if small, they appear brightly illuminated when viewed with Zeiss Neofluar phase objectives, whereas all other chromosomal components appear darker than background. The nucleolar organizing region is immensely variable in form in oocytes within the size range on which I have concentrated, and this variability was at first bewildering. However it turned out to provide particularly strong evidence for homology between the free nucleoli and objects attached at the organizer locus. In preparations from the smallest oocytes in which I have been able to identify the nucleolar 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 of any size up to, but not greater than, that of the free nucleoli. The free nucleoli in these smaller oocytes are likewise irregular rough objects and they are of nearly uniform size. This description holds good up to an oocyte diameter of about 1-25 mm, and in occasional oocytes of slightly larger size (Fig. 31). In oocytes within the size range 1-4-1-7 mm the nucleolar organizer locus may be occupied (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). Exceptionally there are no, or quite minute, objects attached at the organizer locus, which is then visible 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, and the arrays of beaded loops similarly correspond to free nucleoli which have been stretched and deformed during the contraction phase. There is a considerable range in the number of round objects or beaded loops attached to the organizer locus, and this is apparently due to variation in the original number of attached nucleoli. If several nucleoli happen to be attached, each transforms into a lumpy ring, with the rings stacked in a row along the chromosome. This causes the chromosome axis to stretch, and in extreme cases nucleolar material may occupy a 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 into beaded 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 the degree of deformation of free and attached nucleoli, but even in those axolotls in which the stretching of the free nucleoli is extreme, there are always some oocytes in the appropriate size range whose attached nucleoli are not deformed. It seems probable that in such oocytes the nucleolar organizing loci happen to lie centrally within the germinal vesicle; the attached nucleoli would then be less exposed to the stresses of the contraction phase than they are when the organizers lie nearer the nuclear periphery. The morphological similarity between attached and free nucleoli, and the very comparable transformations which they undergo, is evidence that the free and attached nucleoli are homologous to one another in the sense that they share a common genetic origin. 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 the axolotl and add to the population of free nucleoli. Such an assertion could be made with some assurance regarding the generation and release of spheres from the lamp- brush chromosomes of T. cristatus (Callan & Lloyd, i960) for in small oocytes of T. 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 make an appearance. In that instance the evidence based on sequence was compelling. But regarding 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 already taken up position at the nuclear membrane in oocytes of 0-25 mm diameter. The origin of 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 older oocytes 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 counts of 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 bulk of nucleolar material attached to the organizer loci. Either there is a continuing low rate of production at, and detachment from, the organizer locus, or for some unknown reason a variable number of the last nucleolar DNA Anlagen synthesized at the organizer 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 synthesized and detached sisters. In the axolotl, Humphrey (1961) has described a chromosomal variant n which determines the production of a smaller nucleolus than that determined by its normal allelic alternative JV. Somatic interphase nuclei of Nn heterozygotes, provided the nucleoli are not fused, generally contain two nucleoli of markedly different size, and the heterozygotes can be readily distinguished from either homozygote by this character. 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 locus for 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 genetic identity of oocyte and somatic nucleolar organizers. I also hoped that I might be able to locate the gene d on the lampbrush chromosomes. Twenty-six axolotls of the constitutions shown in Table 3 were studied. The right arm of bivalent III was identified in a convenient number of preparations (or as many as 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 left of the nucleolar organizer out to the end of the right arm. The preparations were scored for symmetry or otherwise of the nucleolar material attached at the two homo-
Table 3. Genetic constitution and nucleolar condition in axolotls
Status of nucleoli
attachea at t he
organizer locus
A.
(
Large
A
Age SmallS Degree of Degree of
r
Axolotl r -* v Genetic Sym- Asym- or stretching of stretching of
number Years Months constitution metrical metrical absent attached nucleoli free nucleoli Remarks
2171-3 1 0 0 0 6 0 ++
2145-4 1 1 0 0 7 0 0
1793-11 0 0 0 10 0
3 + Recent importation from Mexico
s"*
1690-5
DN/DN 0 0
3 5 IS Variable to + + + +++
1647-2 3 9 10 0 0 Variable to + + +++
1518-2 0 0
4 3 7 Variable to + + + ++
s3
2165-14 1 1 0 0 7 0 0 Pure Wistar white
2111-130 1 2 2 0 5 0 0 Remote dark ancestor
1988-3 2 1 0 0 6 0 0 Pure Wistar white
2 2 0 0
i
1968-1 8 Variable to + + + +++ |
• dN/dN • From mating Dd x Dd
1968-3 2 2 6 1 Variable to + + + + +1
cleoh
4
1737-2 3 2 4 0 10 0
++ I Pure Wistar white
3 9 1 1 10 0
1660-4 ++ )
1230-1 5 6 3 6 1 0 + Remote dark ancestor
1919-1 2
^ 10 2 0 Variable to + + +++ Several dark ancestors r
2 • f 1 0
1919-2 4
1 dn/dn 13 Variable to + ++ S"
1671-21 3 8 2 0 Variable to + + + ++
6 J
1898-1 2 6 9 5 0 Variable to + + +++
1708-8 3 4
[ 7 8 0 Variable to + ++
DN/dN 8 1
1708-37 3 4 6 Variable to + ++
1540-24 4 2 1 0 5 1 Variable to + +++
1498-4 4 4 4 12 2 Variable to + +++
H98-5 4 4 J 1DN/dn (I 0 14 0 Variable to + + +++
1671-7 3
6 2 5 3 0 ++ w
0
(I
1671-16 3 6 3 0 6 0
1- dN/dn ++
1625-2 3 6 3 6 0 ++104 H. G. Callan logous organizer loci, an assessed volume ratio of 2: i or more being arbitrarily designated asymmetric, of less than 2:1 symmetric. When both organizer loci had attached nucleolar material as small or smaller in volume than is to be seen in Figs. 31 and 39 the nucleolar status was classed separately as 'small or absent', without regard to 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, and because it seemed to me that more significance should be attributed to symmetry or asymmetry of large masses of nucleolar material than to small. Whether this method of scoring is justifiable or not (and it was adopted before I had properly appreciated the metamorphoses characteristic of free nucleoli) it has had the effect of discriminating between bivalents where both nucleolar organizers carry a single nucleolus (or its single derivative ring), and bivalents where one or both nucleolar organizers carry several nucleoli (or their derivative rings). The data are set out in Table 3. The observations are not conclusive. There are indications that nucleolar organizers in the oocytes of young axolotis tend to carry small (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 or asymmetry of larger nucleolar masses, the uniform symmetry of three older DN/DN animals 1690-5, 1947-2, and 1518-2 (Figs. 32, 34 and 35), the preponderant symmetry of the three dnjdn animals (Fig. 33) and the preponderant asymmetry of the two DN/dn animals (Figs. 41, 42) certainly suggest that the different potentialities of N and n are expressed in oocytes. Further, Dr Humphrey has told me that, based on the relative sizes of somatic interphase nucleoli, he suspects there may be a difference in nucleolar organizing capacity between N in the pure Wistar white strain and N in dark axolotis; if so this might at least in part account for the extensive occurrence of nucleolar asymmetry in the four DN/dN animals (Fig. 36). However, it must be clearly stated that animals which are genuinely homozygous AW may show in Some oocytes quite as striking asymmetry of attached nucleoli as heterozygotes, and vice versa animals which are known to be Nn heterozygotes may in some oocytes show symmetry (Fig. 43). The n variant was first detected by Humphrey in heterozygous combination in an exceptional diploid white axolotl among offspring from a mating DD x dd. Dr Humphrey has told me that another exceptional diploid white offspring from an earlier mating of the same kind was also heterozygous for somatic nucleolar determinants, though he did not detect it as such until later. This led Humphrey to propose (1961) that these exceptional diploid white animals owe their origin to a deletion including the locus D and part of N. I have examined with particular care the regions immediately to left and right of the nucleolar organizers in the lampbrush chromosomes of two animals of 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 ought to be apparent; but such asymmetry has not been observed. At nucleolar organizing loci where the attached ring nucleoli were drawn out into beaded 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 chromosome regions to left and right of the organizer (Figs. 34, 35). Now this is exactly comparable to the characteristic 'double bridges' formed by the lateral loops of lampbrush chromosomes when their parent chromomere has cleaved transverse to the chromo- some axis. The observation is significant because it indicates that the nucleolar organizer 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, it must also mean that those DNA strands of the nucleolar organizer which remain as persisting components of the chromosome are capable, once the cycles of replication of 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 between the persisting (chromosomal) nucleolar DNA and the detached nucleolar DNA is of interest. Is the detached DNA of the same length as the attached? If it is, and if N is longer than n, one would expect to find nucleoli with two different DNA lengths in Nn heterozygotes. This question deserves further investigation. At the end of the contraction phase there can be no doubt that attached nucleoli are shed from the organizers. However, if at the stage when the free nucleoli have rounded up, there remain any attached nucleoli, these too are no longer rings but instead solid round objects (see Fig. 37). MITOTIC SECONDARY CONSTRICTIONS VIS-A-VIS LAMPBRUSH CHROMOSOME LANDMARKS The evidence for a straightforward genetic relationship between the nucleolar organizer constriction in mitotic chromosomes and the nucleolar locus in lampbrush chromosomes has already been presented. However I have been unable to detect any outstanding landmarks on the lampbrush chromosomes corresponding in distribution to those secondary constrictions in mitotic chromosomes which appear when the somatic tissues 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 analogous to that responsible for the nucleolar constriction, i.e. exceptional accumulation and tardy detachment of certain gene products such as to interfere with prophase spiraliza- tion. These secondary constrictions are constant and identical in their distribution over the chromosome complements of three different tissues; moreover, similar constrictions appear in the male meiotic metaphase chromosomes of Triturus helveticus, T. vulgaris and T. cristatus when the diplotene stage (corresponding to the lampbrush phase of female meiosis) is subjected to low temperature (Callan, 1942). The short arm of the XlVth mitotic chromosome of the axolotl is divided by two cold-induced secondary constrictions into three regions of roughly equal length, but there are no landmarks along this arm of the lampbrush bivalent (Fig. 15). The short arm 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 characteristic spheres 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 is unavoidable. This is to say that loci which one may presume to carry out some special function in somatic interphase nuclei are represented by quite unremarkable lateral loops in oocytes. CHIASMATA IN OOCYTES Chiasmata are formed at a very high frequency in axolotl oocytes, and as few other fusions occur between the lampbrush chromosomes, and as most of these can be discriminated 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 not been observed in the axolotl, and scarcely any terminal fusions have been noticed. Total chiasmata per nucleus in the five lampbrush chromosome complements which were 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 in T. cristatus lampbrush chromosomes. If it were truly random, taking the mean chiasma frequency 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 many preparations is the 3 units between the nucleolar organizer and the end of the right arm of chromosome III. The recorded chiasma frequency in this region (based on eighty-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 chromosome segment, devoid of crossing-over, in this animal, and this is in accord with Humphrey's (1948) conclusion, based on extensive breeding experiments with sex-reversed axolotls, that: ' the W chromosome probably lacks nothing found in the Z except the gene 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 axolotl mitotic chromosomes, have claimed that in the female complement the XHIth pair of chromosomes are heteromorphic, the longer arm of one being substantially longer than that of the other. My studies of the lampbrush chromosomes indicate that no such differences exist between the XHIth or any other chromosome pair. An example of 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. Lloyd for help with the illustrations accompanying this paper.
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