Riassunto analitico
Focus of this thesis is to provide original information on the regeneration of the eye in adult specimens of the freshwater snail, Pomacea canaliculata. Regeneration is a process which consists of regrowth, repair or replacement of cells, tissues and/or organs. In this thesis, I have applied fluorescent in situ hybridization (FISH) protocols in order to gain information about the time required for restoring the gene expression in regenerated eyes. First, FISH protocols have been performed on wholemount retina samples and tissue sections. Results between the two approaches were comparable, and we opted for wholemount protocol, that gives more information about the spatial localization of the positivity. Then, I synthesized and tested, via wholemount FISH, 432 riboprobes for a first screening on intact (i.e., not amputated) retinas. Four main pattern of positivity have been defined, on the basis of the presence and distribution of the fluorescent signal. At the end of the first screening, we singled out 78 riboprobes, mainly providing a probe-specific and clearly recognizable pattern, for further test on 1 month post amputation (mpa) retinas. The results indicated that at 1 mpa the eye is fully reconstituted in its anatomy, but the gene expression in the retina still presents important differences. Among the 78 riboprobes tested on regenerating eyes, a special attention have been dedicated to those targeting the mRNA for the neuropeptides, FMRFamide and buccalin. At 1 mpa, I have observed an almost complete recovery only for FMRFamide expression, confirming that the anatomical structure of the eye is almost completely regenerated, but eye neural components, although already present, will need longer time to complete their differentiation and maturation.
On the whole, from the data reported in this thesis it can be concluded that:
FISH protocols on retina can be performed on either wholemount isolated retina or tissue sections, but wholemount protocol allows for a better signal localization and 3D morphology reconstruction of samples.
After testing 432 probes, wholemount FISH protocol resulted in 4 patterns of positive signal. This reveals that the technique clearly discriminates the amount and distribution of the mRNA for the target genes, allowing the study of complex organs, such as the retina of P. canaliculata.
At 1 mpa the retina is still recovering the original gene expression, although the morphology of the eye is already completely recognizable. The analysis of riboprobes that mark only few cells or well defined area of the retina is especially efficient for assessing the gene expression recovery of the regenerating retina, as demonstrated from the results collected on the expression of the neuropeptides FMRFamide and buccalin.
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Abstract
Focus of this thesis is to provide original information on the regeneration of the eye in adult specimens of the freshwater snail, Pomacea canaliculata. Regeneration is a process which consists of regrowth, repair or replacement of cells, tissues and/or organs. In this thesis, I have applied fluorescent in situ hybridization (FISH) protocols in order to gain information about the time required for restoring the gene expression in regenerated eyes. First, FISH protocols have been performed on wholemount retina samples and tissue sections. Results between the two approaches were comparable, and we opted for wholemount protocol, that gives more information about the spatial localization of the positivity. Then, I synthesized and tested, via wholemount FISH, 432 riboprobes for a first screening on intact (i.e., not amputated) retinas. Four main pattern of positivity have been defined, on the basis of the presence and distribution of the fluorescent signal. At the end of the first screening, we singled out 78 riboprobes, mainly providing a probe-specific and clearly recognizable pattern, for further test on 1 month post amputation (mpa) retinas. The results indicated that at 1 mpa the eye is fully reconstituted in its anatomy, but the gene expression in the retina still presents important differences. Among the 78 riboprobes tested on regenerating eyes, a special attention have been dedicated to those targeting the mRNA for the neuropeptides, FMRFamide and buccalin. At 1 mpa, I have observed an almost complete recovery only for FMRFamide expression, confirming that the anatomical structure of the eye is almost completely regenerated, but eye neural components, although already present, will need longer time to complete their differentiation and maturation.
On the whole, from the data reported in this thesis it can be concluded that:
FISH protocols on retina can be performed on either wholemount isolated retina or tissue sections, but wholemount protocol allows for a better signal localization and 3D morphology reconstruction of samples.
After testing 432 probes, wholemount FISH protocol resulted in 4 patterns of positive signal. This reveals that the technique clearly discriminates the amount and distribution of the mRNA for the target genes, allowing the study of complex organs, such as the retina of P. canaliculata.
At 1 mpa the retina is still recovering the original gene expression, although the morphology of the eye is already completely recognizable. The analysis of riboprobes that mark only few cells or well defined area of the retina is especially efficient for assessing the gene expression recovery of the regenerating retina, as demonstrated from the results collected on the expression of the neuropeptides FMRFamide and buccalin.
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