/ . Embryol. exp. Morph. Vol. 39, pp. 97-113, 1977
Printed in Great Britain
97
Magnesium deficiency in embryos of Xenopus laevis
By JOHN C. MILLER 1 AND RICHARD LANDESMAN 1
From the Department of Zoology, The University of Vermont
SUMMARY
Embryos of Xenopus laevis reared in media with various low (^ 10~5 M) magnesium ion
concentrations will exhibit differing degrees of a potentially lethal magnesium starvation
syndrome depending on the ion concentration and rearing temperature. The higher the
rearing temperature or the lower the magnesium ion content of the medium the more severely
the syndrome will be expressed. (Normal development can be expected at temperatures
13-30 °C and magnesium ion concentrations > 10~5 M to 10~2 M.)
The magnesium deficiency syndrome in Xenopus embryos is described in detail and compared with the normal and anucleolate conditions. The deficiency condition becomes manifest
after hatching as retarded growth and differentiation with progressive paralysis and edema.
At the same time alterations are observed in the pattern of soluble proteins. The use of magnesium ions as a probe for investigating developing systems is discussed.
INTRODUCTION
In conjunction with studies on the biochemistry of early amphibian development it was observed that 10 % Holtfreter's solution used as an artificial pond
water was inadequate for rearing Rana pipiens embryos (Brown, 1961; Brown
& Caston, 1962). From other studies it also became apparent that embryos of
Xenopus laevis were similarly affected (Brown, 1964). When either Rana or
Xenopus embryos are reared in a medium with low magnesium ion levels
( ^ 10~ 6 M) they will be arrested in their growth and exhibit edema, paralysis
and death at a time their normally reared siblings are beginning to feed. Since
embryos of both species accumulate magnesium ions from their environment
by an unknown mechanism beginning at hatching and continuing at least until
the onset of feeding, the cause of the syndrome is believed to be magnesium
starvation (Brown, 1964).
The externally visible effects of magnesium deficiency in Xenopus are sufficiently similar in appearance to the anucleolate mutant to be considered to some
extent its morphological phenocopy (Brown & Gurdon, 1964). However,
Wallace, who has described the anucleolate condition (1960, \962a, b) points
out that although these syndromes are quite similar, they can be readily
distinguished from each other (see discussion following Brown, 1966). Since
the anucleolate and magnesium-deficient syndromes are in principle quite
1
Authors' address: Department of Zoology, The University of Vermont, Burlington,
Vermont 05401, U.S.A.
98
J. C. MILLER AND R. LANDESMAN
different, it is the purpose of this investigation to describe the magnesium-deficient
condition in greater detail and to indicate some of those factors which modify
its expression and distinguish it from the anucleolate.
MATERIALS AND METHODS
Embryos of Xenopus laevis were obtained from spawnings induced by injections of chorionic gonadotrophin into adults, 500 i.u. per female and 250 i.u.
per male. The developing embryos were chemically dejellied (Dawid, 1965)
prior to staging (Nieukoop & Faber, 1956). Although the dejellied embryos
emerge from their vitelline membrane at stages 31-32 instead of the normal
hatching at stages 35-36, neither the presence nor absence of the jelly coats
alters either normal or magnesium-deficient development. The advantage gained
by this procedure is cleaner culture conditions and greater ease in handling and
observation.
The embryos were reared in glass finger bowls generally at either 15-18 °C
or 27-30 °C in 10% Holtfreter's solution modified for Mg 2+ concentrations of
0-1 M to 10~7 M or in various percent concentrations of Holtfreter's solution.
Cultures were limited in size to small cultures of 50 embryos or large cultures
of 500 embryos and contained from 2 to 4 ml of culture medium per embryo.
The medium was changed daily until the embryos reached stage 35.
The magnesium content of embryos at various developmental stages was
calculated from hydrochloric acid digests of groups of 100 embryos using a
Perkin Elmer atomic absorption spectrophotometer. The embryos were counted,
washed three times with distilled water, homogenized in a small volume of
distilled water, and then digested by adding 2 ml of concentrated hydrochloric
acid and heating to boiling. The cooled solution was then filtered to remove
any residue, brought to a standard volume and assayed, giving results similar
to the more traditional wet ashing method (Dean, 1960).
Determinations of ribonucleic acid synthesis rates were made by dissociating
groups of 200-300 normal and magnesium-deficient embryos from the same
spawning in calcium and magnesium free Steam's solution containing 0-0020-005 M EDTA (Landesman & Gross, 1968). The dissociation medium was then
replaced by 2 ml of the rearing medium containing 100-200 JLLC'I of L(methyl-[3H])
methionine (specific activity 5000 mCi/mmole) for 3 h. Following incubation,
half the embryos in each group were processed to yield a ribosome preparation
using the methods of Landesman & Gross (1968, 1969) and the remainder used
whole for RNA extraction. The RNA was extracted from whole embryos and
ribosome pellets at 4 °C using the SDS phenol technique of Penman (1966) and
then precipitated at - 20 °C with ethanol. Sedimentation of the isolated RNA
was carried out in 13 ml linear sucrose gradients (15-30% sucrose, 0-01 M Tris,
pH7-4, 10" 3 M EDTA, 0-1 M-NaCl, 0-5% SDS) at 22000 rpm, IEC SB283
rotor, for 17-5 h at 22 °C. These gradients were eluted through a 2 mm flow
Magnesium deficiency in embryos 0/Xenopus
99
200
Control
150
/o
100
50
Magnesium
deficient
26
37
41
44
46
Stage
Fig. 1. Comparison of the magnesium content of post-hatching normal and
magnesium-deficient Xenopus embryos. (Calculated as percent of the stage 6-10
magnesium content.)
cell of a Gilford Spectrophotometer and collected in fractions. The RNA was
then precipitated with an equal volume of 15% TCA, collected on Millipore
filters, and counted in a Packard Tri-Carb liquid scintillation counter.
Embryos chosen for histological examination were fixed in Clark's fixative
(Culling, 1963), paraffin embedded and serially sectioned in either the transverse or frontal plane. Staining employed a progressive Harris hematoxylin and
eosin procedure adapted from Humason (1967). Drawings were made from
camera lucida or microprojector images of fixed and stained materials.
RESULTS
It has been shown in Rana pipiens that the appearance of the magnesiumdeficient syndrome is paralleled by the failure to accumulate magnesium from
the environment (Brown & Caston, 1962). In Xenopus, although the initial
amounts of magnesium per embryo varied widely, the same pattern can be seen
(Fig. 1). In all cases the average magnesium content of the embryos declined
between early cleavage and hatching and then began to increase steadily so
that by the onset of feeding the embryos contained as much as twice their
initial magnesium content.
Since the early stage embryos release magnesium to their environment, it was
desirable to attempt to maintain the chosen levels of magnesium in the culture
dishes. Sodium pyrophosphate was used in an attempt to control the magnesium
concentrations of the culture medium by acting as a selective chelating agent
100
J. C. MILLER AND R. LANDESMAN
Fig. 2. External morphology of stage 44-45 Xenopus embryos reared at different
temperatures and magnesium ion concentrations. (A) 10~7 M-Mg2+ 15 °C;
(B) 10-7M-Mg2+ 30 °C; (C) lO"6 M-Mg2+ 15 °C; (D) 10~6 M-Mg2+ 30 °C;
(E) > 10-5M-Mg2+15-30 °C.
for magnesium (Rubin, 1975) but had no apparent effect. In groups of embryos
reared in media of differing magnesium content it neither intensified nor induced magnesium deficiency despite concentrations of up to five times that of
the magnesium in the medium. However, when the concentration of pyrophosphate approached or slightly exceeded the sum of the magnesium and
calcium concentrations in the medium, the embryos gastrulated poorly, showing large yolk plugs which persisted to the tail-bud stage when the embryos
began to undergo cellular dissociation. Higher pyrophosphate concentrations
brought about cellular dissociation even more quickly. Consequently, magnesium concentrations were maintained until stage 35 at their desired levels in the
medium through scrupulous precleaning of the culture dishes to eliminate any
foreign magnesium and by daily changes of the culture medium to remove
magnesium lost from the embryos. After this the medium was not changed and
magnesium concentrations declined as the embryos accumulated this ion. Culture dishes with medium having magnesium ion concentrations 10~5 M or
Magnesium deficiency in embryos o/Xenopus
101
greater contained amounts of magnesium far in excess of the embryos' needs.
However, when magnesium ion concentrations fall below 10~5 M the embryos
are apparently unable to accumulate sufficient quantities for normal growth.
Morphology
The severity with which magnesium deficiency is expressed depends on both
the availability of magnesium ions to the embryo and the rearing temperature.
Embryos grown in media with magnesium concentrations of 10~2 M to 10~4 M
develop normally while those reared in media with initial magnesium ion
concentrations of 10~5 M, 10~6 M, 10~7 M, or lower show progessively more
severe symptoms of magnesium deficiency. Also, embryos reared at 15-18 °C
are less radically affected than those reared at 28-30 °C (Fig. 2). Furthermore,
the length of time the severely affected embryos survive is a function of the
rearing temperature. Magnesium-deficient embryos reared at 30 °C will perish
in a little over 2 days at about the same time that control embryos reach
stage 44 while those raised at 15-17 °C may live in their arrested condition for
as long as 2 weeks as their controls reach stage 48.
When describing the anucleolate mutant Wallace (1960) listed a number of
features characteristic of the anucleolate embryo in tabular form to illustrate
the development of the syndrome. Based on detailed observations from eight
different spawnings reared in this investigation at temperatures ranging from
15 to 30 °C this table can be expanded to demonstrate the differences between
the normal, anucleolate and magnesium-deficient conditions (Table 1).
While the anucleolate embryo is indistinguishable from its normal siblings
up to stage 39, as early as stages 35-36 the magnesium-deficient embryo can be
distinguished because its melanophores are smaller and more punctate than
normal. However, it is not until after stage 40 that the major aspects of growth
in both the magnesium-deficient and anucleolate embryo are affected. Most
conspicuous are reduced rates of tail expansion, limited coiling of the gut,
slowed head enlargement and restricted heart growth. Also, the edema common to both conditions is far more severe in the magnesium-deficient embryo.
Not only does it occur earlier but it deforms the embryos more severely. First
seen at stage 40 as a mild generalized swelling of the entire embryo it quickly
results in fluid filling the pericardial and peritoneal cavities by stage 42 and by
the time the embryos die they have become distorted by numerous fluid filled
blebs and blisters.
Active corpuscular circulation which began normally at stage 37 persists in
the magnesium-deficient embryo until stages 42-43 when clumps of blood
cells clog some of the intersegmental vessels and the subcaudal vein. Also,
hemorrhages may appear in the head region and over the gut so that soon only
scattered blood cells may be found in the liver and pronephric sinus. Nevertheless, the heart, although small and underdeveloped, continues to beat regularly
until shortly before the embryo's death.
Ciliary motion
Muscular twitch
Regular swimming
Gulping
Eye-twitch
Heart beat
Blood circulation
Sucker secretion
Melanophores
Erythrocytes
Otolith
Head edema
Heart edema
Anal edema
% On survival
% + n survival
% m survival
Nieuwkoop stage
Days at 18-19 °C
37
40
41
42
43
44
7
9
10
11
100
100
100
.
.
.
.
.
.
.
o+m
o+m
o+m
o+m
o+m
o+m
m
m
om
o+m
o+m
o+ m
o+ m
o+m
o+ m
m
m
om
100
.
100
.
*
o+m
o+
o+m
o+m
o+m
o+m
m
m
om
o+m
o+
o+m
o+m
o+m
o+m
m
om
om
.
.
*
o+ o+ o +
o+ o+ o +
o+
o
o
o+ o+ o +
o+ o+ o +
o+ o+ o +
.
.
o
o
o
o
o
o
o
.
.
100
.
.
100
o
* % survival varies from one spawning to the next.
.
.
.
o+ m
o+ m
o+ m
o+m
o+m
o+ m
m
12
13
100
o
o
56
o
o
o
o
84
o
o+
o+
o+
o
o+
o+
o+
o+
o
o+
o+
o+
o
o
o
96
o
O
o + o+
o+
o+
o+
o+
o+ o +
o+ o +
o
o
o
o
o
o
o
o
o
96
100
o+
o+
o+
0
o+
.
o+
o
o+
.
o+
o
.
o+
t/3
w
z
o
r
f"1
+ W
o + o + o+ +
o + o+ o+ +
o+ o + O+ +
o
o
o
o
o
o
o
o
o
20
12
4
0
100
o
o+
.
o+
45 46 46 46 46 47 47 47 47 47 47 47 47 £
8
o+m o+m
o+m
o+ m o+ m o+ m o+ m
o+m o+ m
o+ m
35
6
.
o+
33
5
o+ m o+ m o+ m o+ m
.
.
.
.
.
.
.
.
.
.
.
.
.
o + m o + m o + m o + m o + m o + m o + m o + m o + m o+ o+ o+ o+ o+ o+ o+
32
4
(o = present in On; + = present in + n ; m = present in magnesium-deficient larvae)
Table 1. Comparison of characters of On, control, and magnesium-deficient larvae from observations at 12 h intervals
Magnesium deficiency in embryos o/Xenopus
103
A
0 1 mm
Fig. 3. Somite structure from normal and magnesium-deficient embryos. (A)
Stage-31 normal or magnesium-deficient. (B) Stage-42 normal. (C) Stage-42 magnesium-deficient, e, Epidermis; ens, neural tube; sg, spinal ganglion; m, mesenchyme;
5, fifth post-otic somite; 6, sixth post-otic somite.
104
J. C. MILLER AND R. LANDESMAN
10-5M-Mg 2 +
n
17=19
.v=53-2
16-18 "C
«=15
.Y = 52-4
S.D. = 2-82
1
60
n
21-25 X
50
40
60
50
40
n=\l
.v = 48 0
S.D. = 3 I 1
/i=14
.v = 51-6
S.D. = 4 0 5
_•
60
50
40
•
60
U.
A = 51-6
S.D. = 4 0 8
n
40
.Y = 42-9
S.D. =
l_Bi
50
Somite number
•J
50
/;=12
/;=16
28-30 °C
60
40
60
50
311
•_•k ^
40
Somite number
Fig. 4. Variation in somite number in Xenopus embryos in response to the temperature and magnesium ion content of the rearing medium. (Post-otic somites plus
segmented mesoderm to the tip of the tail.)
Variations in the concentration of the Holtfreter's solution rearing medium
had only slight eifect on the expression of the magnesium deficiency syndrome,
the only difference being a slight reduction in the extent of the edema in those
embryos reared in the 80-100 % concentrations. However, addition of a magnesium chloride solution to restore magnesium ion levels to 10"4 M will bring
about a reversal of the syndrome if this is done before stage 42. In which case
growth resumes until normal proportions are attained, previously failing
corpuscular circulation is restored, paralysis is overcome and the extensive
edema subsides. This compares favorably with the reversibility of this syndrome reported in Ranapipiens (Brown, 1961), but the reversal is not complete
in the tadpoles since pigmentation is not restored to normal levels on the dorsal
surface of the body and in the pigmented layer of the eye.
Histology
Tissue differentiation in the magnesium-deficient embryo becomes arrested at
a level which corresponds to stage 42 in normally reared embryos. Despite some
aberrations caused by edema, the magnesium-deficient embryo is histologically
distinct from the anucleolate. The extensive pycnosis seen in the central nervous
system, optic cup, otic vesicle, olfactory placode, ectomesenchyme and pharyngeal floor of the anucleolate mutant during stages 35-40 (Wallace, 1960) does
not occur in magnesium deficiency. Although the mesodermal tissues are retarded in both syndromes, the somites of the magnesium-deficient embryo are
particularly severely affected. The development of the myotubes ceases as they
Magnesium deficiency in embryos o/Xenopus
105
A
PS
0-3 mm
Fig. 5. Eye-level cross-sections through stage-46 Xenopus embryos reared at 18 °C.
(A) Normal; (B) magnesium-deficient, c, Cartilage; ms, mesencephalon; le, lens;
pg, pigmented layer;/?, pharynx; /, tongue.
appear to become detached at their ends and come to lie randomly oriented in
vacuolated spaces lateral to the neural tube (Fig. 3). Another effect of magnesium deficiency on somite formation occurs when there is only a marginal
deficiency condition (5 x 10~6 M to 10~5 M Mg 2+ ). In this situation embryos
reared at 27-30 °C have a lower average number of somites than those reared
at 15-18 °C (Fig. 4). Although the effect is difficult to achieve in all spawnings,
it is reminiscent of temperature-induced meristic variation in fishes reviewed
106
J. C. MILLER AND R. LANDESMAN
A
acv
Magnesium deficiency in embryos o/Xenopus
107
by Fowler (1970) and is supported by recent observations on the heat sensitivity
of somitogenesis (Elsdale, Pearson & Whitehead, 1976).
Figures 5-7 show some of the effects of magnesium deficiency on embryonic
development. In Fig. 5 a transverse section at the level of the eyes shows that
the edema has caused the collapse of the poorly pigmented optic cup. The incomplete formation of the pigmented layer in the eye is one of the first recognizable features of magnesium deficiency and apparently subject to a critical
period because if magnesium deficiency is reversed at stage 42 it will not be
restored to normal density. Another characteristic feature of magnesium deficiency seen in this region is the limited differentiation of the cartilages which
support the tongue and branchial apparatus leading to the lateral expansion of
the head. Similarly, the tall columnar epithelium of the tongue fails to form.
A section through the embryo at the level of the otic vesicles (Fig. 6) shows
the small bloodless heart surrounded by a fluid filled space. Also the poor
formation of the gill chamber is particularly conspicuous as its epithelium
remains thick and undifferentiated. More posteriorly (Fig. 7) the lack of differentiation in the gut endoderm is especially clear since the gut in the magnesium-deficient embryo is often a simple S or a single loop instead of the three
loops commonly seen in the stage-46 control. Similarly affected are the lung and
liver primordia which remain as small thickened masses in the magnesiumdeficient embryo. Furthermore, the edema associated with magnesium deficiency has resulted in fluid filling the peritoneal cavity and causing the distension of the pronephric tubules and sinuses. Finally, the severe disruption of the
somites seen in Fig. 3 can this time be seen in cross-section, explaining the near
paralysis of the magnesium-deficient embryo since most of the somites are
similarly affected.
Figures 5-7 show clearly that the tissues which undergo the greatest part of
their growth and differentiation after hatching are the most severely affected by
magnesium deficiency. This fact is demonstrated by the fact that the central
nervous system which differentiates early is morphologically unaffected by the
syndrome. Also, in other tissues some of the morphological changes may be in
part secondary effects. For example, the somites which were well organized
at stage 31 in the magnesium-deficient embryo are severely disorganized by
stage 42 (Fig. 3). Or, perhaps the severe distension of the pronephric tubules
seen in the later stages of magnesium-deficient embryos reared below 18 °C is
partly artifactual since the tubules are far less distended in magnesium-deficient
embryos reared at 28-30 °C despite the fact that the embryos are equally
Fig. 6. Cross-sections through the level of the heart and otic vesicle in stage-46
Xenopus embryos reared at 18 °C. (A) Normal; (B) magnesium-deficient, a, atrium;
b, erythrocytes; ec, endocardium; me, myocardium; v, ventricle; o, otic vesicle; n,
notochord; t, tongue; g, gill chamber; p, pharynx; r, rhombencephalon; acv,
anterior cardinal vein.
108
J. C. MILLER AND R. LANDESMAN
A
da
P'
0-3 mm
Fig. 7. Cross-sections at the gut level of stage-46 Xenopus embryos reared at
18 °C. (A) Normal; (B) magnesium-deficient and (C) stage-45 magnesium-deficient
embryo reared at 30 °C. b, Erythrocytes; n, notochord; s, somite; ens, neural tube;
/, liver; Ip, lung primordium; h, intestinal coil; d, duodenum; da, dorsal aorta;ps,
pronephric sinus;/?/, pronephric tubule; pd, pronephric duct; pev, posterior cardinal
vein.
edematous. In no case did this investigation demonstrate the mechanism by
which these morphological changes occur. What is shown is what effects will
occur if the Xenopus embryo is not able to take up magnesium from its
environment.
Biochemistry
The isotope incorporation patterns of whole embryo and ribosomal RNA
from normal and magnesium-deficient embryos are presented in Fig. 8. Since
the quality and the quantity of the RNA extracted from normal and magnesium-deficient embryos is identical, there is little doubt that ribosomal RNA
synthesis, processing and transport are similar in both the magnesium-deficient
and the normal embryo. While the use of labeled methionine provides a more
Magnesium deficiency in embryos o/Xenopus
109
0-3 mm
Fig. 7B. For legend see facing page.
discrete indicator of ribosomal RNA synthesis (Landesman & Gross, 1969)
comparable results were also obtained using labeled uridine. Due to the
fundamental rRNA difference between the magnesium deficiency syndrome and
the anucleolate mutant (Brown & Gurdon, 1964) these data suggest that the
magnesium-deficient syndrome may be brought about by translational or posttranslational lesions. Further evidence for this has come recently from preliminary experiments in this laboratory. After isolating nuclei from normal
and magnesium-deficient embryos according to the methods of Theriault &
Landesman (1974) the nuclei and the soluble cytoplasmic proteins were
compared with respect to numbers of nuclei per embryo and the amounts of
protein per embryo and per nucleus. The number of nuclei per embryo according
to these methods may be reduced by as little as 9 % while the amount of protein
per nucleus is reduced by 15-18 % from the normal. However, the soluble cytoplasmic protein levels are 30% lower in the magnesium-deficient embryos.
When the SDS-polyacrylamide electrophoretic patterns of these proteins are
8
EMB
39
10
J. C. M I L L E R A N D R. L A N D E S M A N
C
0-3 mm
Fig. 7C. For legend see page 108.
compared there are selective reductions of some protein species during the later
stages (39+) of magnesium deficiency. These differences can be abolished if the
magnesium deficiency is reversed prior to stage 42.
DISCUSSION
The purpose of this investigation was to examine and characterize the effects
of magnesium deficiency on the development of Xenopus embryos. There is no
doubt that there is an absolute magnesium requirement for normal embryoFig. 8. Sedimentation profiles of RNA extracted from stage-42-44 normal and
magnesium-deficient embryos. (A) Comparison of whole embryo RNA; (B) comparison of ribosomal RNA. Optical density (
) labeling: normal (—O—),
magnesium-deficient (—•—).
Magnesium deficiency in embryos o/Xenopus
A
K
7000
6000
5000
- 2-0
1 4000
3000
1
2000
1000
i
i
i
1
5
10
Fraction number
15
20
700
A
B
AI V 1•
600
500
- 20
[ 400
o
f/
300
200
100
a^* J
„
^-^
1
5
1
1
10
15
Fraction number
1
20
111
112
J. C. MILLER AND R. LANDESMAN
genesis, and that the tissues which differentiate after hatching are the most
severely affected. Although it is most unlikely that a natural environment
would exist which could evoke magnesium deficiency syndromes in the amphibians inhabiting it, this condition cannot be dismissed merely as a laboratory
curiosity because developing amphibian embryos respond differently to various
levels of magnesium. Manipulation of this ion may provide means by which
some aspects of growth and differentiation can be investigated. In fact, the
arrest of growth and differentiation of induced pigment cells has been reported
in ectodermal explants from Rana when reared in a medium without magnesium
(Barth & Barth, 1974). Similarly, the manipulation of the rate of DNA synthesis in cultured fibroblasts through alterations in available magnesium ion
levels in the culture medium suggests a possible 'central role for magnesium in
coordinate control of metabolism and growth in animal cells' (Rubin, 1975).
While the mechanism for the uptake of magnesium by the embryo is still
unknown, one might suspect that an enzymic transport mechanism may exist
similar to that reported for phosphate in sea urchins (Griffiths & Whitely, 1964).
Similarly, those protein species which are reduced in the nuclei and the cytoplasm of the magnesium-deficient embryo have yet to be identified and the
possible enzymic changes associated with these reductions remain unknown. This
leads to the obvious question of why some protein species are apparently reduced and not others. Consequently, we feel that control of magnesium ion
availability to differentiating systems may help to provide a means by which
biochemical hierarchies and temporal sequences in growth and differentiation
may be clarified. Work to this end is now in progress.
We wish to thank Dr C. William Kilpatrick for his helpful comments and review of the
manuscript. This research was supported in part by a University of Vermont Institutional
Grant.
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(Received 17 August 1976, revised 20 December 1976)