ABSTRACT
‘Immobile’ (im) is a recessive lethal mutation discovered in the F3 of a Xenopus (Xenopus laevis laevis) originating from a mesodermal nucleus of a neurula transplanted into an enucleated egg. The im embryos do not contract after mechanical stimulation nor do they present any spontaneous contraction from the neurula stage onwards. Development proceeds normally during the first days after which deformation of the lower jaw and tail are observed. The im tadpoles die when normal controls are at the feeding stage. Nervous and muscular tissues are histologically normal in the mutant tadpoles; at advanced stages, however, an irregularity in the path of the myofibrils is observed which is especially conspicuous in the electron microscope. Cholinesterases and ATPase are present in the mutant muscles.
Parabiosis and chimerae experiments have shown that parabionts and grafts behave according to their own genotype. Cultures of presumptive axial systems with or without ectoderm lead to the conclusion that, first of all, the abnormality is situated in the mesodermal cells and secondly that the first muscular contractions in normal Xenopus laevis are of myogenic origin. The banding pattern of the myofibrils is normal as was shown by obtaining contractions of glycerol extracted im myoblasts with ATP. It seems therefore that in this mutation, the abnormality is situated in the membranous system of the muscular cell, sarcoplasmic reticulum and/or tubular system as is probably the case in the mdg mutation of the mouse.
INTRODUCTION
Muscular contraction and nerve-muscle relationships have been extensively studied in mammals and amphibians, but only a few studies are concerned with the ontogenesis of these events. Here we present a mutation in which contraction and movement of the embryo and young tadpole of Xenopus laevis do not occur and which may help to elucidate the genesis of muscular contraction in normal development. The morphology of the mutant is described in this paper as well as some experiments which show that the abnormality resides in the muscles.
MATERIAL AND METHODS
Nuclear-transplant frogs (Xenopus laevis laevis) as well as laboratory stock animals were used. Spawning and fertilization were provoked according to Gurdon (1967). Two hundred blastulae per cross were preserved, separated in groups of ten in Petri dishes containing aqualab (dechlorinated water) at room temperature and observed daily. After 7 days, the normal tadpoles were fed with nettle powder and transferred into large containers (101). The developmental stages were determined according to Nieuwkoop & Faber (1956).
For light microscopy, the tadpoles were fixed with Zenker’s or Gomori 1 –2 – 3 fluid and stained with hemalum –eosin or fuchsin –orange G-fast green (Humason, 1972); for nerve histology they were fixed in alcoholic saturated picric acid –formaldehyde and impregnated with silver nitrate according to Bodian modified by Fitzgerald (Gabe, 1968). Transverse, sagittal and longitudinal sections were cut at 7, 10 and 15, μm. For electron-microscopic examinations the embryos were fixed in a solution of glutaraldehyde μformaldehyde μ acrolein in cacodylate buffer, post-fixed in osmium tetroxide, dehydrated with acetone and embedded in Epon (Kalt & Tandler, 1971); they were cut with a Porter μBlum ultramicrotome and observed in a Philips 300 electron microscope. The histochemical reaction for cholinesterases was carried out on whole embryos using the azo-dye technique of Lewis (1958). The ATPase was revealed by the method of Wachstein & Meisel (1957) on frozen sections of tails of stage-46 tadpoles, obtained by using a Cryostat freezing microtome after incubation in the ATP medium at 23 °C at different times varying from 5 to 60 min. Parabiosis was performed and chimerae were made by operation on neurulae (stage 20 μ22) in standard Holtfreter solution.
For cultures of the presumptive axial systems, normal and presumptive im embryos of stage 14 were liberated from their jelly coats and after removal of the endoderm the dorsal part of the embryo was put in Ca-Mg-free solution for about 30 min. The presumptive neural and epidermal ectoderm was then peeled off and the remaining mesoderm with some underlying endodermal cells was cultured for 48 h in standard Holtfreter solution. The tissues formed balllike structures which, after a few hours, could be tested with a hair-loop for contraction. Controls were treated similarly, apart from the ectoderm which was left undisturbed. In these cases the tissues developed into normal tadpole backs with complete axial systems and were also tested for motility.
The in vitro effect of ATP was tested with the method of Holtzer & Abbott (1958) which applied the Szent-Gyorgi model (1949) to chick embryo myoblasts. Stage-46 tadpoles were prepared by removing head, gut and preferably skin. The remaining part composed of trunk and tail was extracted with 50 % glycerol at 0° for 24 h and μ20° for 48 h or more. Clusters and isolated myoblasts were then obtained by teasing bits of the extracted trunk or tail somites on a slide in a drop of 25 % iced glycerol; the slides were covered with a coverslip, maintained in the freezer for a short time and observed under a phase-contrast microscope. Two or three myoblasts per preparation were usually measured with a millimetric ocular; a few drops of an ATP solution (disodium salt, 5 niM. pH 6 ·8) were then added under the coverslip. After about 1 min the myoblasts were seen contracting. They were measured again at the end of contraction and the percentage of contraction was calculated on the basis of these two values.
OBSERVATIONS
Heredity of the mutation
This mutation has been found in the third generation (F3) of a male ( ♂70) resulting from a mesodermal nuclear transplantation of a young neurula into an enucleated egg (Fig. 1). The parents of the donor embryo are unknown. The mother of the F2 (♀72) crossed with her heterozygous sons ( + im) possesses the wild-type genotype +/ +, the father (♂23) originating from the laboratory stock could not be tested. Eleven out of the 28 individuals (39 · 3 %) tested in the F2 are heterozygous; several individuals of one F3 have already been crossed and four out of nine of them are heterozygous.
Genealogy of the ‘family’ in which the mutation im was discovered. D = unknown parents of the donor embryo; ⟶○= nuclear transplantation; ♂70=♂resulting from the nuclear transplantation; >, ◧= ♀and ó’ heterozygous for im; ▴= im/im tadpoles.
In the F2, 12 crosses have been carried out between heterozygous individuals: 550 homozygous (im/im) tadpoles have been obtained out of 2242 embryos giving a percentage 24 ·5%, thus showing that the mutation is inherited as a mendelian recessive and that the penetrance is complete.
Some other mutations have been obtained in this same family - ‘oedema’ (oe) (Uehlinger & Beauchemin, 1968), ‘dwarf 11’ (dw-Il) and ‘precocious oedema’ (p.oé) (Droin, 1974) - but no linkage tests have been effected.
Description of the phenotype
External morphology
No difference can be observed between normal and presumptive ‘immobile’ embryos from fertilization up to the neurula stage. At the pre-motile stages (22 –24), normal embryos respond to mechanical stimulation with a neck contraction. From stage 25 (flexure and coil stage) they contract spontaneously whereas ‘immobile’ embryos show neither flexure after stimulation nor any spontaneous contraction. When stimulated for a long time, however, the im tadpoles sometimes exhibit a very slow bending movement which leaves them bent at a right angle for a few minutes.
Except for the lack of motility which leaves the mutant tadpoles lying on the bottom of the dish, general development, heart beating and hatching proceed normally up to stage 42. From this stage onwards the head becomes narrower than that of the normal tadpoles due to a deformation of the lower jaws which either protrude or are folded inside such as are seen in the mutation ‘folded jaw’ (Droin, Reynaud & Uehlinger, 1968); no jaw movement is observed.
At stage 43, the chromatophores contract and at stage 44, an elongation of the back and the tail takes place, giving the mutants a white appearance and a ventrally curved aspect (Fig. 2).
The number of somites in the im tadpoles is the same as in the normal ones but in these advanced stages they appear wider and more pointed, the angle of the chevron being more acute (cf. Fig. 8). The eyes are fully developed but do not move; the pharynx, branchial chamber and gut develop normally but the tadpoles cannot absorb food. They die between the 8th and the 10th day when the normal tadpoles are at the feeding stage.
Internal morphology
(a) Histology. Light microscopy does not reveal any obvious difference, during early developmental stages, between the muscle cells of normal and those of im individuals. However, from stage 38 a slight disorganization is observed in the myotomal arrangement of the im somite. The extremities of the myoblasts are more loosely packed at the level of the myocommata as compared with the tightly packed normal ones. Moreover, inside the myoblasts, several of the ribbon-like strands formed by the myofibrils take a slightly winding course whereas the myofibrils of the normal muscles are always arranged in straight rows (Figs. 3, 4).
With the Bodian silver impregnation technique the brain appears normal and the pattern of innervation of the motor and sensory fibres of the mutant is quite similar to the normal one. The motor fibres situated laterally to the neural tube and forming the motor roots innervating the myotomes can be seen, as well as the oblique fibres traversing the lower part of the neural tube (Figs. 5 –7). The Mauthner neurons which are thought to be involved in swimming coordination develop normally in the im rhombencephalon.
Longitudinal section of the muscles and neural tube of a stage-42 normal tadpole (silver impregnation).
Longitudinal section of the muscles and neural tube of a stage-42 im tadpole showing the motor and transverse nerve fibres. The irregularity of the muscle fibres is very conspicuous as compared with the regularity of normal ones (cf. Fig. 5) (silver impregnation).
Longitudinal section of the muscles and neural tube of a stage-42 im tadpole showing the motor and transverse nerve fibres. The irregularity of the muscle fibres is very conspicuous as compared with the regularity of normal ones (cf. Fig. 5) (silver impregnation).
Sagittal section of the muscles of a stage-38/39 im tadpole showing the innervating nerve fibres (silver impregnation).
Normal Rohon-Beard cells, constituting the early sensory system of the tadpoles, are observed in the nervous system of the mutant. These cells are easily recognized by their large size and the reddish colour of the cytoplasm when stained with eosin; they are situated in the dorsal part of the spinal cord; their sensory fibres leave the spinal cord and pass at the level of the myocommata to innervate the epidermis.
(b) Histochemistry. The histochemical reaction for cholinesterases (Lewis, 1958) was performed on skinned embryos. The reaction becomes visible from stage 31 and is much more conspicuous in the anterior part of the embryo than in the posterior part. The presence of cholinesterases, which manifests itself by a brown colouring, is localized in the myocommata where nerve plexuses are formed (Lewis & Hughes, 1960). The im tadpoles exhibit this reaction as well as the normal ones indicating that cholinesterases are present in the mutants (Fig. 8).
Cholinesterase reaction in whole skinned and eviscerated stage-46 normal (above) and im tadpole (below). The larger width and the more acute angle of the im somites are especially conspicuous.
Brown deposits revealing the presence of ATPase are also visible in the mutants’ tails. They are scattered along the myofibrils and exhibit the same pattern as in the normal tails.
(c) Electron microscopy. At an ultrastructural level no difference is observed before stage 33. The banding of the fibrils as well as the sarcoplasmic and tubular systems appear normal in the im muscles. From this stage on, the arrangement of the fibrils is not as regular in the im fibres as in the normal ones; slightly irregular branching and splittings of grouped filaments can be observed (Figs. 9, 10).
Electron micrograph of myofibrils in the tail of a stage-38 normal tadpole showing an orderly array of myofilaments.
Electron micrograph of myofibrils in the tail of a stage-38 im tadpole showing a normal ultrastructure with the exception of a slight disarrangement of certain myofilaments.
At stage 42 these splittings and the deviation of the fibrils are more conspicuous. Often the fibrils are displaced around a nucleus or around yolk platelets which appear to be more numerous than they are in the controls. The myofibrils are sometimes cut, in the same section, both obliquely and transversally, indicating what appears to be a rather haphazard arrangement. The banding of the acto-myosin filaments still shows a normal structure. The sarcoplasmic reticulum and T-system do not present any major discrepancy in their structure (Fig. 11).
Electron micrograph of myofibrils in the trunk of a stage-42 im tadpole. Re-routing of myofibrils around a nucleus and a yolk platelet can be seen. Note occasional haphazardly located myofilaments (arrows).
At stage 45 the fibrils in the trunk of the mutant still keep their normal appearance along with the sarcoplasmic reticulum and T-structure. The muscles of the tail region however, are more affected than those of the trunk region. Isolated myofibrils may be separated by vacuolated spaces of disintegrating sarcoplasm. When the sarcoplasmic reticulum and T-structure finally break down, a degeneration of the acto-myosin filaments follows (Fig. 12).
Electron micrograph of myofibrils in the tail of a stage-45 im tadpole. The sarcoplasm and mitochondria are undergoing degeneration, followed by the sarcoplasmic reticulum and T-structure (thin arrows) and finally the myofilaments (thick arrow).
EXPERIMENTAL RESULTS
The above observations were completed by a series of experiments devised to determine the level of gene action.
Parabiosis
Ten successful parabioses were performed on neurula stages between wildtype and mutant embryos. Both partners react according to their own genotype, the wild-type parabiont being mobile and the mutant one remaining immobile and exhibiting the typical abnormalities of the advanced stages. The only change observed in the ‘immobile’ partner is the lack of melanophore contraction and a prolongation of survival.
Chimerae
Chimerae were formed at neurula stages before the ‘immobile’ phenotype could be recognized, the combination being haphazard. The embryos were transversally cut through the middle and the halves exchanged. Of a total of 122 grafts, 29 mixed combinations were obtained. They were easily recognizable because each half behaves according to its genotype. When stimulated, the head and the anterior part of the trunk moved, the posterior part and tail remained immobile and vice versa. When the head was immobile it showed the typical deformation of the mutant and when the tail was immobile it was usually curled up. With the light microscope one could observe that the neural tube connexion had been re-established, thus indicating the probable continuity of the nervous tissue. In these chimerae there was no clear limit of melanophore contraction.
Cultures of presumptive axial systems
The eggs used for this experiment came from two kinds of crosses. In the first three series there were only wild-type embryos; in the other series the embryos were either wild-type (homozygous or heterozygous) or homozygous mutants (Table 1). As the preparations were made at stage 14, the embryos were taken at random. The results show that in the first series all the normal expiants with or without ectoderm exhibit contractions after mechanical stimulation whereas in the second series composed of +/ +, + /im and im/im embryos, the proportions of the cultures that did not react to stimulation are respectively 28 ·6 % with ectoderm and 24 ·4 % without ectoderm. These percentages correspond to the mendelian percentages of the homozygous mutants.
After sectioning the wild-type- and the mutant-cultured expiants without ectoderm, we observe that differentiation has taken place. The notochord is developed and vacuolated and the myoblasts are arranged metamerically, forming somite-like units (Fig. 13). In the myoblasts, the myofibrils are also differentiated and the striation is visible in the more advanced expiants.
Section through an im presumptive axial system explant (without ectoderm) aged 48 h showing the beginning of notochord and metameric myoblast differentiation.
Effect of ATP
The normal isolated myoblasts obtained for contraction assays are very regular elongated cells. The banding of the myofibrils is recognizable as an alternating pattern of light and dark zones. This banding pattern in the im myoblasts is visible as well as the internal disorganization of these cells (Fig. 14 A). After addition of ATP, however, the im myoblasts contract in the same manner as the normal ones (Figs. 14B-D). The average length of the myoblasts is about 150 μm and the width 30 μm. On contraction they shorten and become wider. Only the shortening was measured. The percentages of contraction as well as their variability referred to in Table 2 are quite similar in normal and in im tadpoles. Each percentage corresponds to the mean value of two or three isolated myoblasts measured for each preparation; two to three preparations are made per tadpole. The measurements were made in a comparative way preparing successively, whenever possible, one normal and one mutant tadpole. The variability of the results can be explained by several factors: firstly myoblasts very often adhere to the slide or the cover-slip, thus inhibiting a full contraction; secondly, with the change of the refractive index of the medium which occurs after the addition of the ATP solution, the measurements are not always very precise; thirdly, the maintenance of the temperatures of the preparation before contraction may vary slightly; the capacity of contraction diminishes with higher temperatures.
(A) An im glycerol-extracted myoblast prepared for contraction test; B-D, three stages of contraction of the same myoblast after addition of ATP (phase-contrast microscope).
A small series of controls has been made using distilled water as the extraction medium instead of glycerol. Contractions also take place after addition of ATP but they are less strong than after glycerol extraction. In this case again, the reaction of normal and im myoblasts is quite similar.
DISCUSSION
The cultures of the presumptive axial systems allow us to draw two main conclusions. The first one is that the prospective inability for contraction expressed by the mutant gene is laid down intrinsically in the mesodermal cells at the end of gastrulation. The results show on the one hand that the lack of contraction of the mesodermal explants depends indeed on the genotype of the cells. On the other hand, the presence of nerve cells cannot induce contraction in the ectomesodermal explants of im/im embryos.
The second conclusion is that the + / + or +/im mesodermal explants can contract in the absence of nerve cells. This last result raises the problem of the nervous or muscular origin of the first contraction responses in lower vertebrates. In fishes it is known that the first contractions have a myogenic origin (Leghissa, 1941 ; Sawyer, 1944; Harris & Whiting, 1954) but in Amphibians this is still a subject of controversy. On the one hand, Corner (1964) making explants of neural plate in a jacket of ectoderm and mesoderm of Xenopus ascertained that no contraction could be evoked after mechanical stimulation when there was no nervous tissue associated with mesoderm; he concluded that the movements were of non-myogenic origin. On the other hand, Hughes (1959) and Muntz (1964) in a detailed description of the differentiation of the motor system in Xenopus stated that no motor fibres leave the neural tube at the premotile stage (stage when the first body contractions occur after stimulation) and Muntz (1964) postulated that it is the direct action of the skin on the muscle cell which elicits the contraction response. This statement was confirmed by Macklin & Wojtkowski (1973), who showed, in measuring the electrical activity of Xenopus embryos, that the first rhythmical spikes have a myogenic origin and are derived from the direct relation between skin and muscles. This relation was also confirmed by Roberts & Smith (1974), who demonstrated the excitability of the skin before innervation, thus allowing the first responses of the embryo after stimulation. The results obtained from the cultures of the axial systems confirm the myogenic origin of the first contractions in Xenopus embryos which may happen even in the absence of skin.
The last experiment (effect of ATP) leads to the third conclusion, namely the normality of the contractile apparatus of the im myoblasts. Besides the normal banding pattern of the fibrils observed in the micrographs, the ATPase-positive reaction and the contraction response of the im glycerinated myoblasts to ATP, entirely comparable to the response of the normal myoblasts, confirm the normal potentiality for activity and behaviour of the contractile proteins.
The site of the gene expression must then be looked for in the other cell components of the myoblasts. Among the numerous reactions ending in the contraction of the muscle cell, two important steps are concerned with the membranous cell components. The first is the membrane depolarization which spreads through the tubular system on to the sarcoplasmic reticulum; the second is the release by the sarcoplasmic reticulum of Ca2+, which is then fixed to the contractile apparatus, thus allowing the mechanical reaction of contraction to take place (Ebashi & Endo, 1968; Sandow, 1970; Huxley, 1971). Caffeine is known to act upon the sarcoplasmic reticulum by increasing the release of Ca2 +and therefore the contraction reaction (Weber & Herz, 1968; Ebashi & Endo, 1968). An attempt was made to rear normal and immobile embryos in caffeine solutions of 2 –3 and 5 –8 mM. In normal individuals, contractions were increased with the tadpoles becoming restless and twitching continuously. In the im tadpoles the slow bending movements occurring irregularly after stimulation were more pronounced in low concentrations while in higher concentrations, they occurred spontaneously leaving the tadpoles in a bent state. These preliminary results indicate that the im muscles may react to caffeine. It is then tempting to put forward the hypothesis that the sarcoplasmic reticulum and/or the tubular system are the sites of the primary gene action. To test this hypothesis, further studies are needed, especially an electrophysiological one in order to show whether or not the resting and the action potentials of the cell membrane are normal, a detailed analysis of the Ca2+ regulation in the myoblasts during differentiation and a biochemical analysis of the membranous cell components.
The same hypothesis of the abnormality of the membranous apparatus of the muscle cells was also postulated in the case of the mutation muscular dysgenesis (mdg) of the mouse. It is interesting to compare the mdg with the im mutation as they have several features in common, particularly the inability of the mutant newborn mouse to move, due to defective muscle cells (Pai, 1965 a, b). The phenotypic abnormalities, however, are more conspicuous in the mdg mutation, especially the skeletal ones, which may be compared to a certain extent to the deformation of the lower jaw of the im mutant. The cytological degeneration of the mdg myoblasts (abnormal and dilated sarcoplasmic reticulum, atypical pattern and atrophy of the myofibrils) constitutes another noteworthy aspect of this mutation (Platzer & Gluecksohn-Waelsch, 1972). When the mdg myoblasts are analysed in monolayer cell cultures the differentiation proceeds normally with respect to the development and rate of maturation but they fail to contract or exhibit partially abnormal, slow contractions. When exposed to caffeine, a few mutant cells contract normally but localized contractions are the predominant responses. The normality of the contractile apparatus was confirmed as well by the contraction response of glycerinated myoblasts to ATP after 8-14 days of culture (Bowden-Essien, 1972). In both mutations (mdg and im) the functional differentiation of the muscle cells seems to be impeded primarily.
RÉSUMÉ
Immobile (im) est une mutation récessive létale trouvée dans la descendance d’un Xenopus (Xenopus laevis laevis) issu de la greffe d’un noyau mésodermique de neurula dans un oeuf énucléé. L’embryon im ne présente aucune contraction musculaire après stimulation mécanique ni contraction spontanée dès le stade de neurula. Le développement est normal pendant les premiers jours puis des déformations apparaissent dans la mâchoire et la queue. Les têtards meurent au moment où les têtards normaux commencent à se nourrir. Le système nerveux et le système musculaire sont histologiquement normaux chez les mutants; on observe cependant, dans les stades avancés, une certaine irrégularité dans le trajet des myofibrilles, particulièrement visible au microscope électronique. Les cholinestérases et l’ATPase sont présentes dans les muscles des têtards immobiles.
Les expériences de parabiose et de chimères ont montré que les parabiontes et les greffes se développent selon leur génotype respectif. Des cultures de systèmes axiaux présomptifs, avec ou sans ectoderme, ont permis de conclure, d’une part, que l’anomalie se situe dans les cellules mésodermiques et, d’autre part, que les premières contractions musculaires chez les Xenopus normaux sont d’origine myogénique. En outre, les myoblastes des têtards immobiles extraits à la glycérine peuvent se contracter sous l’effet de l’ATP démontrant ainsi la normalité de la striation fibrillaire. Il semblerait donc que le gène exerce son effet au niveau du réseau membranaire de la cellule musculaire, réticulum sarcoplasmique ou système tubulaire comme ce serait le cas dans la mutation muscular dysgenesis (mdg) de la souris.
ACKNOWLEDGEMENTS
We thank Professor M. Fischberg, who put the material at our disposal, for his interest and criticism and the ‘Département d’Ophtalmologie de l’Hôpital cantonal’ for the use of the electron miscroscope. We are indebted to Professor Huggel and Dr Benzonana for their critical reading of the manuscript.
This work was supported by the ‘Fonds national suisse de la Recherche scientifique’ (no. 3.60.68).