Action of rat submaxillary gland extracts on neural tube growth in organ culture

Transcription

1 J. Embryol. exp. Morph., Vol. 14, Part 3, pp , December 1965 Printed in Great Britain Action of rat submaxillary gland extracts on neural tube growth in organ culture by RUBEN ADLER and ROBERTO NARBAITZ 1 From the Instituto de Anatomia General y Embryiologia, Facultad de Medicina, Buenos Aires. WITH ONE PLATE INTRODUCTION FOR a long time embryologists have been interested in mechanisms which control growth of the neural tube. The results of experiments made on amphibian and chick embryos suggested that the differential growth of different portions of the neural tube is dependent both on genetic factors and on the degree of development of peripheral tissues (see Weiss, 1955). The mechanism of this peripheral action has not yet been elucidated. It has been attributed to diffusible substances from peripheral tissues acting on the growing neural tube. However, according to Hamburger (1958), the junction between neurons and peripheral tissue is necessary for this growth effect to occur. Studies on a neural growth factor found in mouse sarcoma 180 tissue, snake venom and mouse and rat submaxillary glands (Levi Montalcini, 1959) has renewed interest in this problem. However, this factor acts only on sympathetic and spinal ganglia and not on neural tube derivatives. Recently organ culture techniques have been used with increasing frequency by embryologists, and their use has broadened our understanding of many aspects of morphogenesis (see Borghese, 1958). However, to the best of our knowledge, these techniques have not so far been used with success in the analysis of neural tube growth. In the present work, organ cultures of portions of chick embryo neural tube together with surrounding tissues, were made to test the action of other tissues in nearby culture, or of organ extracts added to the medium. MATERIALS AND METHODS White Leghorn embryos were used in all our experiments. The explants were removed at stages 11 to 13 (Hamburger & Hamilton, 1951). Seven series of experiments were made, as described in Table 1. As shown in the plate, Fig. A, the 1 Authors' address: Instituto de Anatomia General y Embriologia, Facultad de Medicina, Paraguay 2155, Buenos Aires, Argentina.

2 282 R. ADLER and R. NARBAITZ ' simple explants' were four somites long and the ectoderm and endoderm were cut laterally at the external limit of the somites. In the' combined explants', wing or leg primordia were dissected from embryos in stages 18 to 19 (Hamburger & Hamilton, 1951), sectioned into small pieces and cultured in apposition to neural tube explants. Isolated limb bud pieces were cultured as controls. TABLE 1 The different series of experiments Series Type of explant No Media Duration of Culture Combined Limb bud * A,B A A plus submaxillary gland extract (S.G.E.) A plus S.G.E. treated with iodine A plus extracts of rat brain,] lung, heart, liver and kid- [ ney, and chicken brainj A plus purified ' nerve growth factor' A hr hr hr hr. 72 hr. 72 hr. 72 hr. * 74 with each kind of organ extract. Cultures were made following the technique of Wolff & Haffen (1952), using embryological watch glasses. The medium was made solid by the addition of 1 per cent agar. The following two media were used: A. 1 per cent agar in Hanks' solution, 10 ml.; E.E. 50, 5 ml.; Eagle's basal medium, with double amount of dextrose, 5 ml.; Sodium penicillin, 20,000 U. B. 1 per cent in Hanks' solution, 10 ml; Horse serum, 1 ml.; Eagle's basal medium with double amount of dextrose, 9 ml.; Sodium penicillin, 20,000 U. In both cases these amounts were enough for ten cultures. Extracts from chicken brain, and from rat brain, liver, submaxillary gland, kidney, heart and lung were all prepared in the same way. The tissues were homogenized with Hanks' solution in a cool Omni-mixer; homogenates were then centrifuged at 4000 r.p.m. for 15 min. and supernatants were added in a proportion of ml./ml. to the media. Both chicken and rat brain extracts were also tested at a higher concentration (0-14 ml./ml.). In one of the series iodine was added to submaxillary gland extract up to a final concentration of to M as suggested by Sreenby, Meyer & Bachem (1955) in order to inhibit proteases

3 J. Embryol. exp. Morph. Vol. 14, Part >x FIG. A. Drawing of a twelve somites embryo. The pieces which were used as' simple explants' are indicated. FIG. B. ' explant' cultured during three days. H.&.E. 180x. FIG. C. 'Combined explant'cultured during three days. H.&E. 100x. FIG. D. 'Combined explant' cultured during six days. H.&.E. 100 x FIG. E. 'explant' cultured during six hours. Medium with submaxillary gland extract. H.&E. 180x. FIG. F. ' explant' cultured during three days. Medium with submaxillary gland extract. H.&E. 230 x. RUBEN ADLER and ROBERTO NARBAITZ (Facing page 283)

4 Neural tube growth in organ culture 283 contained in the extract. This iodine treated extract was left half an hour at room temperature before medium preparation. Purified 'nerve growth factor' (kindly supplied to Dr De Robertis by Dr Levi Montalcini) was added to the media in amounts varying from to 16-6y/ml. Cultures were maintained at 37 C. being observed under a dissecting microscope at various intervals. The explants were fixed in Bouin's fluid and prepared with routine histological procedures. An approximate measure of the volume of neural tissue cultivated was obtained by projection and drawing of the 10/A serial sections on thick paper and weighing the resulting paper images (paper weight). In order to establish the extent to which peripheral tissues were innervated several explants were stained with a silver technique (Cajal & De Castro, 1933). RESULTS ' explants' After 3 days of culture, the ectoderm and endoderm fuse forming a continuous layer of cubical cells around the explant. Neural tubes differentiate normally and the three layers (germinal, mantle and marginal) characteristic of normal neural tube development are present (Plate, Fig. B). After 6 days of culture, neural tubes continue to grow keeping the same type of organization. In some of the explants the mantle layer looses its uniform width showing thickenings which simulate the ventral horns of the spinal cord. In very few explants fibre bundles were seen to arise from the neural tube. After 3 days culture somites lose their organization forming a mesenchymal type of tissue which fills the whole explant. Myoblasts are seen arranged in tight groups at both sides of the neural tube; some of them fuse forming multinucleated masses. After 6 days culture, myoblasts have continued differentiating and typical spontaneous, irregularly spaced contractions are detected. Medium l A' gave the most consistent results. Explants cultured in medium' B', in which embryo extract was replaced by horse serum failed on several occasions to develop. However, in other cases the culture succeeded in a similar way as with medium 'A'. ' Combined explants' In experiments in which pieces of limb bud and' simple explants' were cultured together, both explants fused completely and became surrounded by a single layer of cuboidal cells. The mesoderm of the limb bud after three days culture became transformed into a mass of closely packed pre-cartilaginous cells (Plate, Fig. C). After 6 days, large masses of cartilage were observed (Plate, Fig. D). Nerve fibres connecting the neural tubes with the added limb bud tissues were never found. The volume of neural tissue developed in the 'combined explants', as judged by the paper weight, is expressed in Table 2. The difference from that developed in the 'simple explants' was not statistically significant.

5 284 R. ADLER and R. NARBAITZ Addition of organ extracts to culture media The addition of submaxillary gland extract produced the dorsal opening of neural tubes during the first 6 hours of culture (Plate, Fig. E). After 3 days, neural tubes showed an exuberant and irregular growth (Plate, Fig. F). As showed in Table 2 neural tissues volume as judged by its paper weight is greater in these explants than in the simple ones, this difference being statistically significant. The mesodermal tissue showed a comparatively poor development, the bulk of the cultured explants being formed by neural tissue. TABLE 2 Mean paper weight of neural tissue* Type of explants Combined Medium A No 20 Paper weight l-05g.-^ Difference > 013g.f A g.< > l-45g.j A plus S.G.E g.^ * Weight of paper used:20-4 mg./sq.c.; magnification: 125 x. t Statistically non significant. j Statistically significant (p < ). The addition of chicken and rat-brain extracts, even at high doses did not produce morphological changes in the development of the explants, and the same negative results were obtained with other organ extracts such as kidney, heart, lung and liver. Explants cultured in media containing purified nerve growth factor showed no morphological difference from the controls. DISCUSSION Our results show that the neural tube of the chick embryo is capable of growth and differentiation while kept in organ culture. However, both processes progress at slower pace than in ovo. The fact that for several days the tissue keeps its typical shape and organization is an important advantage over histotypical cultures for the study of morphogenesis of the neural tube. Hamburger & Keefe (1944) showed that the addition of an extra limb bud to chick embryos produces a rise in the mitotic index of neural tube cells. Nevertheless, later results from the same laboratory (Hamburger, 1958) cast some doubt on that conclusion by demonstrating that the action of the limb bud on the size of the neural tube depends more on prevention of late cell degeneration than on early growth stimulation. This author believes that the junction between neurones and peripheral tissues is necessary for this action to take place. This would explain the absence of peripheral action on neural tubes in our combined explants in

6 Neural tube growth in organ culture 285 which innervation does not exist, at least in the period in which our experiments were performed. Our most interesting observation is undoubtedly the powerful action that the submaxillary gland extracts exert on the neural tube. Two main effects of these extracts were observed: an early dorsal opening of neural tubes and a marked increase in neural tissue volume. The assumption that this greater volume in the explants treated with submaxillary extracts is indicative of an increased number of cells is acceptable only if it is known that cellular density is similar in both types of explants. Although no cell count was made, an examination of all the explants indicated that if there is a difference in cellular density those cultured with submaxillary extracts are denser, in which cells appear to be more closely packed than those in control explants. Thus, it appears to us that increase in paper weight observed in submaxillary extract treated explants indicates a real increase in cell population and not merely cellular dispersal. Nevertheless, it remains to be demonstrated that cell proliferation is an independent phenomenon and not a consequence of early neural tube opening and disorganization. Cohen (1960) found in submaxillary extracts a factor which stimulates growth of sympathetic and spinal ganglia, and could isolate a protein fraction in which the activity was highly concentrated. Levi Montalcini (1964) postulated that this nerve growth factor is specific for the neurons of the ganglia, and has no action on neural tubes derivatives. This is in agreement with our experiments in which 'nerve growth factor' showed no action on neural tube development. The action over neural tubes found by us, would then be dependent on some component of the submaxillary extract different from'nerve growth factor' and also from submaxillary protease activity, as suggested by the fact that iodine, that was showed (Sreenby, Meyer & Bachem, 1955) to be an inhibitor of those proteases did not inhibit the action of submaxillary extracts on neural tubes. Cohen (1962) has isolated from mouse submaxillary extract another factor with an action on incisor eruption and eyelid opening in the newborn mouse. The existence of such a variety of substances with morphogenetic action in rat and mouse submaxillary glands is difficult to understand. It might be that these substances are unrelated to normal development, but some of them may prove to be important tools in the investigation of the mechanisms of neural tube growth and differentiation. In this respect the action studied by us may be of interest in relation to the understanding of mechanisms involved in neural tube closure and its abnormalities. SUMMARY 1. Organ cultures of portions of chick embryo neural tube together with surrounding tissues, were made in order to test the action of other tissues in nearby culture, and of organ extracts added to the medium. 2. Explants four somites long, dissected from embryos at Stages 11 to 13 (Hamburger & Hamilton), developed and differentiated normally in vitro. Pieces

7 286 R. ADLER and R. NARBAITZ of limb bud from older embryos, added to the explants, fused with them and differentiated as usual. The addition of the limb bud pieces did not influence the size of the cultured neural tube. 3. The addition of submaxillary gland extract to the medium produced during the first 6 hours of culture a dorsal opening of the neural tubes and, thereafter, an exuberant and irregular growth of them. Other organ extracts, purified 'nerve growth factor' and iodine treated submaxillary gland extract failed to produce the same action. 4. The presence in submaxillary extracts of an active factor independent of 'nerve growth factor' and protease activity is deduced from our results. RESUME Action d'extraits de giande sous-maxillaire de rat sur la croissance du tube nerveux en culture d'organes. 1. Pour eprouver l'action d'autres tissus en culture avoisinante, et celle d'extraits d'organes ajoutes au milieu, on a realise des cultures de tube nerveux d'embryon de poulet et de tissus environnants. 2. Des explants correspondant a la longueur de quatre somites, preleves sur des embryons aux stades 11 a 13 (Hamburger et Hamilton), se sont developpes et se sont differencies normalement in vitro. Des fragments de bourgeon de membre d'embryons plus ages, ajoutes aux explants, se sont fusionnes avec eux et se sont differencies selon le mode habituel. L'adjonction de fragments de bourgeon de membre n'a pas influence la taille du tube nerveux cultive. 3. L'adjonction d'extrait de glande sous-maxillaire au milieu de culture a provoque pendant les six premieres heures de culture un percement dorsal et par la suite une croissance exuberante et irreguliere des tubes nerveux. D'autres extraits d'organes, du 'facteur de croissance nerveuse' purifie, et de l'extrait de glande sous-maxillaire traite a l'iode n'ont pas eu la meme action. 4. Nous deduisons de nos resultats la presence dans les extraits sous-maxillaires d'un facteur actif independent du ' facteur de croissance nerveuse' et de l'activite proteasique. ACKNOWLEDGEMENTS The authors thank Dr E. De Robertis, who read the manuscript and made important suggestions. This work was supported by a grant of the Consejo Nacional de Investigaciones Cientificas y Tecnicas de Argentina. Dr Adler holds a Fellowship and Dr Narbaitz a permanent position in the same Institution. REFERENCES BORGHESE, E. (1958). Organ differentiation in culture. In 'Chemical basis of development'. (Eds. W. McElroy & B. Glass), pp Baltimore: Johns Hopkins Press. CAJAL, RAMON T. S. & DE CASTRO, F. (1933). "Elementos de tecnica microgrdfica del sistema nervioso\ pp Madrid: Tipografia Artistica.

8 Neural tube growth in organ culture 287 COHEN, S. (1960). Purification of a nerve growth promoting protein from the mouse salivary gland and its neurocytotoxic antiserum. Proc. natn. Acad. Sci. U.S.A. 46, COHEN, S. (1962). Isolation of a mouse submaxillary glandproteinacceleratingincisoreruption and eyelid opening in the newborn animal. J. biol. Chem. 237, HAMBURGER, V. (1958). Regression versus peripheral control of differentiation in motor hypoplasia. Am. J. Anat. 102, HAMBURGER, V. & HAMILTON, H. L. (1951). A series of normal stages in the development of the chick embryo. /. Morph. 88, HAMBURGER, V. & KEEFE, E. L. (1944). The effects of peripheral factors on the proliferation and differentiation in the spinal cord of the chick embryo. /. exp. Zool. 96, LEVI MONTALCINI, R. (1964). The nerve growth factor. Ann. N.Y. Acad. Sci. 118 (art. 3), SREENBY, L. M., MEYER, J. &BACHEM,E. (1955). Characteristics of a proteolytic enzyme in the submaxillary and sublingual glands of the Albino Rat. J. dent. Res. 34, WEISS, P. (1955). Nervous System (Neurogenesis). In Analysis of development (Eds. B. H. Willier, P. Weiss and V. Hamburger), pp Philadelphia and London: W. B. Saunders Co. WOLFF, ET. & HAFFEN, K. (1952). Sur une methode de culture d'organes embryonnaires in vitro. Texas Rep. Biol. Med. 10, {Manuscript received 26th March 1965; revised 17th June 1965)