L'ONTOGENESI DEL SISTEMA OLFATTIVO E VISIVO DEI VERTEBRATI
Morgillo, Antonietta (2010) L'ONTOGENESI DEL SISTEMA OLFATTIVO E VISIVO DEI VERTEBRATI. [Tesi di dottorato] (Inedito)
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The mechanisms of differentiation within the nervous system including proliferation and differentiation, migration cell growth and development of axonal and dendritic synaptic connections. Are fundamental processes that generate a remarkably complex neuronal and, to a large extent, still poorly understood. As drafted by the neurotrophic theory, another important process involved in determining the morphology and physiology of neural "programmed cell death (PCD) or apoptosis, which acts both on different types of neuronal and glial cells (Kuan et al. , 2000; Roth and D'Sa, 2001; Davies, 2003, Buss et al, 2006). Although recently this early stage of cell death is the subject of numerous studies, the cell populations involved, the regulatory mechanisms involved, its scale and its functional implications are still far from being characterized as appropriate. The retina is an easily accessible part of the central nervous system and therefore has become a classic model for studies of neural development and apoptosis (Cepko et al., 1996, Harris 1997, Marquardt and Gruss, 2002; Adler, 2005) . In fact, the visual system, while preserving the intense proliferative activity ontogenetic, passes through a number of significant apoptosis (Cook et al 1998; Biehlmaier et al., 2001; Candal et al., 2005; Alunni et al., 2007) . The development of the olfactory system in vertebrate species studied morphogenetic level requires an intense proliferative activity, as witnessed by strong business PCNAsica (Ino et al., 2000) which tends to decline proceeding with development. Likewise, the olfactory mucosa presents events of apoptosis, but as a pattern appears to differ from those that occur in the eye (Ohsawa1 et al., 2008). Of course for each group of vertebrates on this basic scheme, is part of the variants characteristics that determine a unique pattern of development. The data concerning the differentiation of embryonic olfactory and visual system of vertebrates are uncommon and those conducted as in Bufo bufo and X. laevis, do not provide data on the numbers of cells in apoptosis and mitosis (Mathis et al., 1988; Straznicky and Nguyen, 1989; Stiemke and Hollyfield, 1995). The counts of apoptotic cells and mitotic figures are estimates of the degree of remodeling of the organ. Then from these estimates can be inferred, first functional correlations of the other, comparing the data with those in letteraura, including the evolving nature of information. For this reason, in this work were studied in detail the various stages of development of some vertebrates (Cichlasoma nigrofasciatum, Xenopus laevis, Rana esculenta, Bufo bufo, Coturnix coturnix) in order to broaden knowledge on the morphogenesis of the eye and smell and try to get a picture on the evolutionary mechanisms underlying the events of proliferation and remodeling of these organs by comparing related species also from the point of evolution, but have a different ecology (for example, animals such as Xenopus laevis, Rana esculenta and Bufo bufo, which all belong to the same order of amphibians, but have a different degree of hydrophilicity). For this purpose, were stained with hematoxylin sections of these organs during their differentiation to detect and count apoptotic and mitotic figures as indicators of proliferative activity and rearrangement of the structures. Also, has also been used for PCNA immunohistochemistry to consolidate the data on observations of cells in active proliferation, and to confirm the apoptotic peaks observed through the reading of histological sections was evaluated nuclear protein level of PARP fragmentation. The most innovative aspect of this research is the attempt to correlate a system during development undergoes a profound and continuous rearrangement, as in the visual system, a system with a bit less dynamic as the olfactory organ. The comparison of results obtained in this study with those produced by other authors is complicated by the fact that the ontogeny of different vertebrates, as such, follows patterns that are fairly typical of the group. In Cichlasoma nigrofasciatum eye development starts very early, starting 2 days after fertilization, the eye in this stage of development consists of a cluster of cells in proliferation, where the lens is separated from the start of the retina formation of the vitreous humor. The exponential growth of the eye throughout its development. The retina has this trend to a standstill on the seventh day after fertilization, by which time the animal has almost completed its anatomical development. The number of mitoses found in the retina of the animal is always high with the occurrence of a strong proliferative activity especially during the early development stages of fish, at 4days 3 days after fertilization. In addition, during the development of retinal Cichlasoma nigrofasciatum two explosions occur apoptotic at 2 days and 5 days after fertilization (as demonstrated by the total counts of apoptosis made and confirmed by the presence of two apoptotic indices in these days of development). The presence of the first explosion apoptosis may be explained by the need to eliminate excess cells that were formed during the earliest phases of development of the eye of the fish (in fact, these days also occur strong proliferative phenomena) Approximately 5 days after fertilization, the eye of Cichlasoma nigrofasciatum is the stratification of the retina, and this event is likely to induce a second wave of apoptosis in the tumor with the plexiform layers of cells in excess are induced programmed cell death. In Xenopus laevis has been studied the development of both sensory systems. Xenopus laevis eye development starts from the stage 29; at this stage the eye is booming, with the presence of a strong proliferative activity and a crystal clear just sketched. Throughout the development of the eye grows exponentially. Analyzing the graphs of retinal growth and the number of cells observed, there was a growth of the retina from the stage 29 to stage 59 and then later in a slight decrease. In parallel there was an increase in the number of total cells during development except for two drops that occur after stage 42 and stage 63, a stage next to the metamorphosis. This decrease can be attributed to its peak apoptotic stage 42. This hypothesis is strengthened by the observation of cell density, which decreases in the retina following the events of cell death. Instead, the eye of Bufo bufo follows the pattern of differentiation typical of other vertebrates, of course, with a timetable that is characteristic of the species. The early stages of development studies covered newly hatched embryos: looking at the growth chart is also comparable to the increase in the number of cells counted in sections, it is observed that the retina does not undergo a substantial increase of the size of the stage between 10 and 13. Starting from the stage 15, however, the eye grows gradually considering that at this stage begins the secretion of the vitreous humor. In parallel, there is also increasing the number of cells in the retina was also confirmed by the intense mitotic activity occurred between stage 13 and 15. Thus, this figure contrasts with the literature which shows the increase in size more important in the early stages of development. However, proceeding in the development, the retina resumes its normal growth until the stagnation observed at the stage of metamorphic climax. Regarding the proliferative activity of the retina, the neuronal elements of the eye, after an initial phase where it is produced throughout the retina, are located in a concentrated area directly facing the lens, MCZ. This phenomenon in the toad is also confirmed by the expression of PCNA, which follows the same pattern of mitosis, although it is expressed in a greater number of nuclei. In addition, during the development process of programmed cell death and cell differentiation coexist in the retina. In the common toad is an increase in the number of apoptotic stage 21 which then tends to decrease, followed by a second peak at stage 28, the figure is confirmed by both histological and biochemical analysis. The differentiation of the retina of the toad does not proceed synchronously, but follows a gradient that goes from the lens opposite (proximal region of the retina) to the region move closer to the lens, ciliary marginal zone (MCZ). The area of the retina grows progressively until stage 31, which represents the metamorphic climax. Highly significant increases are recorded at the stadium just 28 and 31. A similar trend can be observed for the number of total cells that make up the retina, but they, having achieved a significant increase in stage 28, growth arrest. This is not surprising, since growth of the retina is also related to the counterpart of fiber, of course, continue to increase until stage 31. As expected, the strong mitotic activity in the early stages of development and suffers a decrease up to the stage 21 and then increased to 28, after which suffers a terminal decline after metamorphosis in which mitotic figures become more sporadic. The minimum mitotic stage 21 does not correspond to a slowdown in growth in the number of cells, but the significant increase in the number of cells observed at the stage 28, is concordant with the second increase in mitotic index. The development of apoptosis in two waves does not leave visible signs on the growth of the retina, even if this first wave coincides with a minimum mitotic apoptosis, as evidenced also based on the graph of PARP-1 fragmentation. The second wave of apoptosis, however, shall determine, in conjunction with the fall of mitotic activity, a drastic decrease in cells of the retina. In Coturnix coturnix in the early stages of eye development studies covered an embryo after 4 days of incubation, where you look at the outline of the eye and the whole body is in full training. The eye of Coturnix coturnix has experienced rapid growth followed by a downturn towards the latter stages of development of the animal, close to hatching. The retina of Coturnix coturnix grows in size during development, with a standstill on the seventeenth day of incubation. The number of total cells in the retina observed in development has increased in numbers until the eleventh day of incubation and then decreased on days 12, 14 and 16 days after incubation, but not significant. However, these decreases are not entirely correlated with apoptotic events, because only the reduction of cells at the stage 12 is due to an apoptotic peak. The first event of programmed cell death in Coturnix coturnix occurs at the beginning of his retinal development in an embryo after 4 days of incubation and an 'other apoptotic event occurs after 11 days of incubation. The development of the olfactory system was studied in two amphibians: Xenopus laevis, Rana esculenta. To summarize briefly the most important events are found to contain an undifferentiated olfactory placode to the stage 38. The next stage sees the start of dell'invaginazione placode. During stage 42 is medially the vomeronasal organ primordium. Finally, stage 50 begins to form the side room. The later stages can only see the progressive morphological differentiation of these three rooms and the opening of the choanae, which coincides with the metamorphosis. At the beginning of differentiation of the peripheral olfactory system in all the nuclei are intense proliferative activity. Then after this activity tends to localize progressively in the basal level in the rooms smell and fall. Later in development the expression of PCNA remains more or less the same amount in the sensory part of the three rooms. What we see is that the olfactory organs appear more complex and all the sensory areas have an PCNAsica very fine, but mainly localized in basal regions of the mucosa. This obviously means that their development goes hand in hand. Even the non-sensory areas that constitute the respiratory epithelium of the rooms have many olfactory cells PCNA-ir. During metamorphosis, while the epithelium and the vomeronasal organ side of the room are substantially similar to themselves, the epithelium of the main chamber is undergoing a profound change (Hansen et al., 1998). This is reflected in the fact that this will become a hollow "air nose" in metamorphosis, where the role of "water nose" will be hired by the chamber of training later, lying side (Altner, 1952; Weiss, 1986). Regarding the study on the development of olfactory mucosa of Rana esculenta, it was observed that the stage 20, the olfactory placode is still undifferentiated and only then begins a process of differentiation through a process of invagination that starts in the stadium 23. At stage 25, the gills are covered with skin and communicate with the outside through the opening and an oral specific trap. They will be active at least until the climax metamorphic. At this stage, a first draft it identifies olfactory mucosa seems directed toward the differentiation of sensory elements, as they are already these lashes, although very short. The activity PCNAasica intense in all subdivisions of the olfactory mucosa places obviously in favor of an intense proliferative activity. The primordium of the vomeronasal appears around stage 27. In the green frog, the vomeronasal organ is clearly visible as a diverticulum which originates from the regions interior side of the mucosa. Its formation is characterized by its strong proliferative activity at the level of intussusception. In the remaining parts of the olfactory, the activity keeps ir a pattern similar to that previously analyzed. The vomeronasal pushing medially continues to increase, as evidenced by the increased proliferative activity media. Meanwhile, also appear in the structures that will be differentiated microvilli. Even the respiratory epithelium increases progressively increasing the epithelial surface affected the exchange of oxygen. Evident, in fact, the formation of a diverticulum located dorsal to the vomeronasal compared with the presence of activity. The olfactory organ takes on characteristics similar to those found in the adult stage 30, when they appear glands of Bowman. This is supported by the analysis of proliferative activity associated with them. The presence of Bowman's glands is a very important indicator that announces the start of the functionality of the aerial olfactory mucosa. The fact that it is very early in the frog may be due to a simple time difference between the two species, or the fact that the tadpole of the frog begins to use the olfactory system in ambient air before the metamorphosis. Structurally, the remaining course of development, the olfactory organ does not undergo any significant change. Ir activity is still very fine, even after metamorphosis: the olfactory system, although functional, yet increases in parallel to increasing animal body. In conclusion of this work can be said that during the development of vertebrates, all organs are subjected to the phenomenon of programmed cell death and cell differentiation coexist. The duration of both processes was analyzed in different species of vertebrates and has been observed at different times and in different regions of structures like the retina (etal Valenciano., 2009). The reason for the phenomenon of apoptosis remains an enigma. It 'been postulated that cell death occurs due to lack of sufficient targets brain areas that receive the optical fibers. In practice, if the number of retinal afferent fibers that reach is higher than the number of available sites in the roof of the optical termination, fiber "supernumerary" degenerate (Rager and Rager, 1978). One could speculate that the transition from aquatic life to the air would require extensive reworking of the visual structures, but the idea was immediately contradicted by studies on fish products, which also undergo apoptosis. What is quite certain is that during development the eye produces a strong surplus of cells, which must be eliminated. In the frog (and other amphibians studied) the second wave of apoptosis occurs when the individual is still at the stage of tadpole stage where the eye is already working. E 'possible then, that in the tadpole's vision has not yet been perfected and that it refers primarily olfactory sensory system as the main reference. This hypothesis is supported by the fact that the olfactory system does not undergo massive apoptosis during development. Summing up the comparative data is noted that all vertebrates have a restructuring early apoptotic eye and ends in reptiles and birds during embryogenesis. This could be caused by the need to have a visual organ already differentiated and fully functional at birth. In fish and amphibians, but the apoptotic phenomenon stops just before the metamorphosis, which still corresponds to the event of birth or hatching of the amniotes, as demonstrated by the study of the levels of the hormone thyroxine (Stubbe et al., 1978; Tata , 1993; Wassenaer and Kok, 2004). It is well known that these vertebrates and especially birds and fish use the view as the main body and is therefore natural that this body is fully functional even after the birth. Amphibians and fish larvae are already using the eye long before the metamorphosis, but the phenomena of rearrangement affected during this period it is clear that the functionality is not full, but as previously mentioned can apply to other sensory systems (eg system olfactory). In contrast to mammals at birth and they see little or no use to a large extent the smell in the monitoring of environmental signals that surround them (smells of parents, food ), what might be true for larval fish and amphibians. This hypothesis is further reflected in the studies on the olfactory system of Xenopus laevis and Rana esculenta, where there is a olfactory mucosa during development of the tadpole is not subject to obvious apoptosis and cell rearrangement, then proves to be a very static compared apoptotic and proliferative events which occur in the eye of vertebrates studied show that the presence of a much more dynamic system embryologically as the eye.
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