FORMATION OF CELL MASSES IN THE MYELENCEPHALON OF THE CLAWED FROG, XENOPUS MUELLERI

Flora Stephano Department of Zoology and Wildlife Conservation, University of Dar es Salaam P.O Box 35064 Dar es Salaam E-mail: [email protected]

ABSTRACT An important process in the organization of developing nervous system is the clustering of neurons with similar properties to form nuclei. The development of myelencephalon of Xenopus muelleri, a pipid frog that retains a lateral line system throughout life, was studied in Nissl stained serial sections. The results showed that density of neurons increases as the animal develops. Cell masses were formed in the latter half of larval stages (stage 48 to 59). Large neurons migrate first before small neurons. Raphes and reticular formation nuclei and Mauthner cells were the earliest neurons that could be distinctly recognized on the ventral part of the myelencephalon. By stage 54 out of 66 stages, the structure of the myelencephalon resembled that of the adult frogs.

INTRODUCTION investigating the development of the The course of neural development can be myelencephalon from neurogenesis through traced through four overlapping processes; the definite appearance of recognizable cell cell birth (neurogenesis), migration, masses (nuclei) and secondly demonstrated formation of connectivity (including how the observed pattern of development elaboration of processes, synapse formation, complies with the known principles of cell death, and axonal regression), and neurogenesis and neuron migration. myelination. The time when specific neurons are born can be detected using 3H- MATERIALS AND METHODS thymidine autoradiography (Altman and Collection and Housing of Adult Frogs Bayer 1980). These studies in which labelled Adult Xenopus muelleri, were obtained from thymidine is permanently incorporated in the ponds using seine net and reared at DNA of dividing cells, have shown several normal room temperature (25 °C). The frogs principles of neurogenesis. were acclimatized in the laboratory for five weeks before they were allowed to breed. In a given region, there is characteristic They were fed beef liver cut into small block “inside-out” pattern of origin, i.e., neurons twice a week and aquarium water was that are originate later migrate past those replaced before feeding. The aquaria were born earlier to attain the most peripheral maintained at a diurnal light: dark cycle of location. Large neurons are produced before approximately 12:12 hours, which is natural small neurons, motor nuclei originate before equatorial cycle. sensory nuclei (at the same level of neuraxis) (Nadarajah and Parnavelas Breeding 2002).While a number of studies have been Spawning was induced by subcutaneous undertaken to explain cell masses of brain in injection of Human Chorionic adult frogs (Opdam et al. 1976, Matesz and Gonadothrophin Hormone; Pregnyl Szekely 1978, ten Donkelaar et al. 1981, (Organon, Oss, Netherlands) into the thigh. Nikundiwe and Nieuwenhuys 1983, ten For a mating pair the male was given a daily Donkelaar 1998), the present study aimed at injection of 300 I.U. for two consecutive Tanz. J. Sci. Vol 39 2013

days, while the female was administered 100 RESULTS I.U as a primer dose and 500 I.U five hours In the Nissl stained sections and in semi thin later (on the second day only). The mating (2 µm) epoxy sections, growth, pair was placed in a different darkened differentiation and migration of the neurons breeding aquarium with twigs for were observed. oviposition and left undisturbed until mating was over. Stage 22: At this early stage of development, neurons (N) in the myelencephalon were Care, Rearing and Staging of Tadpoles darkly stained, oblong in shape with an Eggs were hatched within three days after average diameter of (9.7 µm). They were mating, and feeding started on the fifth day. distributed throughout its mass and attached Tadpoles were fed with powdered to the dense meshwork processes, which are Amaranthus sp. “mchicha” daily and water radially oriented from the ependymal layer was changed just before feeding. After in the ventricular zone to the peripheral area. metamorphosis, animals were fed with These cells seem to be migrating in these Lumbricus (earthworms) and later they were processes. The whole structure of switched to beef liver blocks. The myelencephalon which, is normally developmental stages of the tadpoles were observed at the level of otic vesicle (OT), determined using standard tale of was covered by an epidermis layer (EP). An Nieuwekoop and Faber (1956). embryonic brain cavity, the neurocoel (Ne) could also be observed (Fig. 1A). Histological Techniques Perfusion and Fixation of the Animals Stage 25: At this stage, the myelencephalon To investigate the development of the anlagen was larger than in stage 22 tadpoles myelencephalon, tadpoles of various stages with the broader neurocoel, causing the were selected and anesthetized in a 0.025% lateral lips to bulge out more. Neurons solution of MS.222 (Sandoz). During occupy the whole of this area connected in processing the brains of stages 22 up to 44, the meshwork of processes like in the the whole animals were fixed in 3% previous stage but with many vacuoles (V). glutaraldehyde, embedded in epoxy resin, The density of neurons had increased which sectioned by an ultramicrotome (Reichert probably imply that new cells were still OmU3) at a thickness of 2 µm and stained being produced. The shape and size of with Toluidine blue O. From stage 45 up to individual neurons was similar to those of 59, whole heads were taken and fixed stage 22 (Fig. 1B). directly in 10% formalin and then fixed secondarily in Boun´s fluid for paraffin wax Stage 33: There was a notable difference embedding technique. Tissues were stained between this stage and the previous ones. with cresyl violet for Nissl substances. Size The gross external shape of the of cells was obtained by averaging the myelencephalon anlagen consisted of a very diameters of their somata which were thin roof and very thick lateral parts with a measured in two directions perpendicular to fourth ventricle (4thV) in-between. This thin each other. The calibration was made at and delicate rhombencephalic roof was magnification of X 200 where the intact and consisted of a single-celled layer multiplying factor was 4.84 approximately 5 of ependymal (E). Neurons were still µm for each one unit of the eyepiece connected to a meshwork processes but the graticule. Photomicrographs of sections latter was not as conspicuous as in the were made by a digital camera (FinePix previous stages. Some neurons, especially 28000zoom model). those in the ependymal area were darker and

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smaller in size than those at the ventro- in the ventricular zone. The lateral parts in lateral parts. In the ventral parts, some of the the basal plate were devoid of neurons. neurons had already differentiated and Vacuoles were still present between the cells become round/oval in shape and were (Fig. 1.C) approximately two times bigger than those

Figure 1: Developmental stages of the myelencephalon of the Xenopus muelleri tadpoles. (A-D) Show developmental stages (22, 25, 33, and 36) X 400, semi-thin sections stained with toluidine blue O. (A) Shows stage 22, in which neurons (N) are distributed in the entire myelencephalon surrounding the neural embryonic cavity, the Neurocoel (N). The Myelencephalon is located at the level of otic vesicle (OT) and is covered by an epidermis layer (EP). (B) shows developmental stage 25, with (V) representing the vacuoles. (C) show developmental stage 33, where the Neurocoel has developed to the 4th ventricle, which is lined by a single-celled layer of Ependymal (E). (D) Shows stage 36, where three quarter of the whole myelencephalon is occupied by the gray matter with the outer white matter devoid of neurons.

Stage 36: The gross shape of the whole myelencephalon was occupied by the myelencephalon anlagen was quite similar to gray matter with the outer white matter that of the previous stage but the notable remaining empty without cells. Vacuoles difference was that the neurons assumed a were still present between the cells (Fig. more elaborate oval shape and their size had 1.D). increased to 12 µm. Three quarters of the 74 Tanz. J. Sci. Vol 39 2013

Stage 40: At this stage the ependymal layer neurons to differentiate, was observed at this covering the roof of fourth ventricle was still stage. It is a large neuron with the average intact but flat. Vacuoles have disappeared diameter of 30 µm (Fig. 2B). The sulcus and most neurons were round in shape with medianus inferior was the only sulcus that the average diameter of (12.7 µm). Apart was clearly distinguishable along the entire from the increase in size, there was a ventricular margin (Fig. 2A-C). remarkable increase in number of cells. As noted in the previous stages, cells in the Stage 47 was notably different from the ependymal area were stained darker and previous stages. The area of the smaller than those cells in the peripheral myelencephalon that was occupied by cells area, which tend to be larger and paler. The was large due to the spread or migration of increase in number and size of cells was these cells from the ependymal area in the gradual and apparently due to mitosis and ventricular zone to their destination areas. growth from a proliferation zone at the Few cells could be recognized at this stage; ventricular surface. Neurons were observed nucleus dorsalis nervi octavi (VIIId) was aggregating in groups limiting their spread situated in the dorsal lateral of the alar plate. to the core of the gray matter. The peripheral The size of these cells was 7.3 µm. Nucleus surface area remained without cells, thereby dorsalis nervi octavi was formed by loosely constituting the neuropil. The sulcus aggregated cells that migrate in a ventro- medianus inferior (smi) could also be lateral arc from the marginal zone of the alar distinguished as an indentation on the mid- plate to the peripheral zone (see arrow Fig. ventral margin of the 4th ventricle. Below it 2C). Other cell masses were nucleus there was well observed mass of round cells, motorius nervi facialis (VIIm) and nucleus which presumably constitute neurons of reticularis medius (Rm). Mauthner cells had nucleus raphes (RP) of the median reticular increased in size from their previous status formation (Fig. 2A). by attaining a diameter of 36.6 µm. Another notable difference was the presence of the Stage 44: At this stage the rhombencephalic sulcus limitans of His (slH), which separates roof is still intact but ependymal layer was the dorsal alar plate from the ventral basal stretched upward making the gross plate of the myelencephalon. The myelencephalon look like a lady’s handbag. primordium of the choroid plexus (Cp) also The arachnoid (A) surrounding the expands and forms invagination into the rhombencephalon could be traced as a thin fourth ventricle. The dorsal island (di) was black, delicate membrane (Fig. 2B). The clearly differentiated from the rest of the structure of myelencephalon in general was myelencephalon as a clear area on the latter more defined where the gray matter occupies part of the alar plate. The dorsal island two thirds of it while the remaining one third represents a neuropil, i.e. an area which is was occupied by white matter. Neurons were occupied by a framework of interlacing still in the differentiation and migration dendrites and axonal terminal ramifications. stages. However, no well defined cell Fig. 2D shows the level at which nerve masses were formed yet. Many of the large number eight (nVIII) entering the brain and cells that were away from the ependymal few cells of nucleus dorsalis nervi octavi area have assumed an oval shape with the (VIIId) were also observed. average diameter of 13 µm. Mauthner cell (Mc), which is believed to be among the first

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Figure 2: Developmental stages of the myelencephalon of the Xenopus muelleri tadpoles. (A-B) Show developmental stages (40 and 44) X 400, semi-thin sections stained with toluidine blue O. (A) Shows stage 40 with a small groove at the mid-ventral of the 4th ventricle (4th V), called sulcus medianus inferior (smi). Below the smi is a group of rounded neurons of the Raphe nucleus (RP). Ependymal layer (E) is seen as a flat membrane covering the 4th ventricle. (B) Shows stage 44. At this stage a Mauthner cell (Mc) is shown. Dorsal to the ependymal layer is a delicate membrane, the Archanoid (A). (C-D) Show Nissl stained sections of stage 47. Di represents the dorsal island. The black arrows show the migratory route of the nucleus dorsalis nervi octavi (VIIId). Lateral to the VIIId is nerve number eight (nVIII). Nucleus motorius nervi facialis (VIIm) and nucleus reticularis medius (Rm) are other structures, which could be identified. Lateral to the smi is sulcus limitans of His (slH) whereas the choroid plexus (Cp) is located on its dorsal side.

Stage 49/50: There was a continuous layer was practically devoid of cells and a of cells from the ventricular zone to the continuous zone of central gray; stratum mantle and marginal zones in the previous griseum where majority of the neuronal stages of development (Fig. 1 & 2). At perikarya are located. The peripheral half stages 49/50 there was a notable difference wall of the rhombencephalon is supposed to where the myelencephalon was segregating be largely occupied by myelinated fibers into layers (Fig. 3A-B). A conspicuous layer without cells. However, at these stages, few of columnar ependymal cells occupies the cells of the olivary superior nucleus (Ols) first inner layer. This was followed by a and dorsal nucleus of nerve number eight narrow strip (stratum subependymal) which (VIIId) were still scattered in this layer (Fig. 76 Tanz. J. Sci. Vol 39 2013

3A). In the alar plate, while the VIIId and approaching the adult form. The migration nucleus caudalis nervi lineae lateralis (CII) and differentiation processes, however, were were distinct nuclear masses, that can be not yet over since some cells had not yet hardly said of ventral nucleus of eight settled at their respective nuclei. Sensory (VIIIv). This observation implies that at fibers of nerve eight were seen entering the these stages, the myelencephalon was brain as in the previous stage (Fig. 3A).

Figure 3: Developmental stages of the myelencephalon of the Xenopus muelleri tadpoles. (A-D) show Nissl stained sections of stages (49, 52, 54 and 59) X 200. Nomenclature of cell masses (nuclei), grooves and nerves are given. (A) Shows stage 49, whereas stage 52 is shown in (B), and stages 54 and 59 are shown in (C) and (D), respectively. The nuclei observed at these stages were; the nucleus dorsalis nervi octavi (VIIId), the ventral nucleus of eight (VIIIv), the nucleus motorius nervi facialis (VIIm), the nucleus reticularis medius (Rm), the olivary superior nucleus (Ols) and the nucleus caudalis nervi lineae lateralis (CII). Other structures include; the nerve seven (nVII), the nerve eight (nVIII), the sulcus limitans of His (slH), the sulcus medianus inferior (smi) and the Mauthner cell (Mc).

In Fig. 3B, a similar pattern as of stage 49 Ventral nucleus of eight (VIIIv) somehow was seen in the myelencephalon of stage 52. could be delineated from the 77 Stephano, F. Formation of cell masses in the myelencephalon…..

undifferentiated central gray. It seems that of yolk granules and vacuoles during larval neurons that form the Ols broke away from development of Xenopus laevis. In all the neuronal population that forms nucleus tadpoles, the rhombencephalic roof was reticularis medius. In addition, many cells intact. It was covered by a thin delicate can be seen migrating to aggregate into single-celled ependymal layer. Brocklehurts respective nuclei. (1976) found the same region intact in tadpoles of Rana temporaria. Stage 54-59: From stage 54 to 59 (Fig. 3C- D), there were no significant changes Neuronal Migration and Formation of compared to stage 52 (Fig. 3B). What could Cell Masses be observed was the fact that the cellular The density of neurons increased as the larva organization of the myelencephalon was developed. This suggests that dividing nerve more or less similar to that observed in the cell precursors continuously add new adult frog. The empty stratum neurons to the pre-existing ones, during subependymale was clearly defined, leaving larval stages. Recognition of presumptive the inner ependymal layer with columnar nuclear mass started at stage 40 during ependymal cells. The nuclei were more which time neurons in the ventral part of the organized. This observation implies that myelencephalon begin to segregate to form from stage 54 (Fig. 3C) the cell masses in the presumptive raphes nucleus and nuclei the myelencephalon were completely of the reticular formation. These formed. What remained was the later observations were supported by similar development, consisting mainly of findings Van Mier, (1986) in Xenopus maturation and some further cytological laevis. One of the earliest neurons that can differentiation. be distinctly recognized from the mantle are the two-Mauthner cell, each of which was DISCUSSION located in the ventro-lateral half of the rhombencephalon. In the dorsal part of this General Features of Myelencephalon area of the brain the first to be identified Development were neurons that will later form the dorsal The earliest tadpoles that were studied nucleus of VIIIth. In general, neurons in the correspond to developmental stages 22 to 47 mantle area were larger compared to those (Nieuwekoop and Faber 1956). At these late of the ependymal area. It was difficult, embryonic stages, no specific cell masses however to recognize in a given larval cell were distinguishable. Cell masses were population the forerunner of an adult formed in the latter half of larval stages structure. In spite of these problems, it was (stages 48 to 59). Through metamorphosis possible to identify several components of especially from stage 59 onwards, the size the myelencephalon anlagen with the adult and shape of neurons resembled those seen counterparts on the basis of a similar in adult animals (Fig. 3D). During the later position. stages the neurons seemed to settle on their respective nuclei. Hence, change of shape The myelencephalon anlagen of the newly and size of neurons was a gradual hatched tadpole stages 22-33 consists of post development process and did not coincide mitotic neurons, which were oblong in with large morphological and physiological shape. The neurons were distributed changes that took place at metamorphosis. throughout its mass, attached to dense Vacuoles that were observed in developing meshwork processes, which are radially myelencephalon disappeared at stage 40. oriented. These cells presumably are Van Mier (1986) observed the disappearance migrating in these processes. But it was

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difficult to establish exactly if these something else. Probably they depend on the processes were glia fibers which, extend interactions between neurons of the same from ventricular zone to the pial surface. group, which enables them to reach their Also it was difficult to tell exactly which, final destinations and hence to form the mode of migration these neurons played nuclei. Based on histological techniques, whether it was radial glia or nuclear large neurons migrate first before small translocation into preformed pial processes. neurons and motor nuclei migrate before The “inside out” pattern of origin, which sensory ones during the development. By normally is observed in other structures of stage 54, the structure of myelencephalon the Central Nervous System (CNS), was not resembled that of the adult frog. What observed in the myelencephalon. This remained in later development constituted suggests that this mode of migration was mainly of maturation and some further probably specific for layered structures of cytological differentiations. In order to yield CNS (cerebellum and cerebral cortex). more information about the origin, migration and the destination of nuclei, combination of In order to demonstrate a freely migrating normal histology and silver staining cell in the developing nervous system, one techniques will be the best option. The would have to label the cell and follow it growth and differentiation of individual sequentially with reference to some fixed neurons in such nuclei, in successive stages point, which was out of scope of this study. of development can be studied over time to These results suggests that in earlier stages record when and how the adult form is of the development of the myelencephalon attained. of Xenopus muelleri, the neurons migrate from the ependymal layer in the ventricular ACKNOWLEDGEMENTS zone either along the radial glia fibers or by The author thanks Sida/SAREC under perikaryal translocation to the Directorate of Postgraduate Studies for mantle/marginal zones in the peripheral financial support. I am grateful to the late areas. Various hypotheses have been Prof. A.M Nikundiwe for his valuable ideas proposed to describe the migration of and supports, May Our Almighty God rest immature neurons. The most widely his soul in peace, Amen. I also thank Mr. E. accepted is the radial glia hypothesis (Rakic Urasa of Electron Microscope Unit, 1971, 1972 and 1990). According to this Department of Zoology and Wildlife theory, newly post mitotic neurons acquire a Conservation of the University of Dar es bipolar shape and lose their attachments to Salaam for his assistance in processing and both the ventricular and pial surfaces. sectioning of semi thin sections using ultra Instead they contact processes of radial and microtome. migrate along them to their final destinations. Thus, radial glia serves both as REFERENCES guides and as substrates for neuronal Altman J and Bayer SA 1980 Development migration. of the Brain Stem in the Rat: Thymidine-Radiographic Study of In the later stages of development, the the Time of Origin of Neurons of the meshwork type of fibers was no longer Lower Medulla. J. Comp. Neurol. visible but the neurons were observed to 194: 1-36. continue with the process of migration to Brocklehurst G 1976 The structure of their final destinations in their respective Rhombencephalic Roof in the Frog. nuclei. This implies that these neurons were Acta Neurochirurgica 35: 205-214. no longer depending on fibers but rather on Matesz C and Szekely G 1978 The Motor

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