INDEX
ABSTRACT I I
INTRODUCTION 1 1
1.1 Discovery of serotonin 1
1.2 Biosynthesis and metabolism of
Serotonin 2
1.3 Serotonin and Development 4
1.4 Serotonin Receptors 9
1.5 The 5-HT2B Receptor 12
1.6 The role of 5-HT2B receptor
during development 15
1.6.1 Relationship between RA
and 5-HT2B during development 17 1.6.2 The role of 5-HT2B during
Xenopus development 17
2. The importance of Cranial Neural
Crest Cell 20
3. Eye Development 23
3.1 Retinoic Acid guide correct eye
morphogenetic movements 27
4. Xenopus laevis as a model system 29
MATERIALS AND METHODS 33
1. Embryos handling 33 2. Embryos microinjection and manipulation 33
2.1 Capped mRNAs 34
2.1.1 Constructs used for over-expression analysis: linearization and capped mRNA
transcription 35
2.1.2 Pharmacological treatments 35 3. RNA Extraction, cDNA Synthesis and
qPCR 35
3.1 Primers used for qPCR gene expression
analysis 36
4. In situ hybridization 37
4.1 Purification of plasmidic DNA 37 4.1.1 Constructs used for gene
expression analysis: linearization and RNA transcription to generate antisense
mRNA probes 37
4.2 Synthesis of digoxigenin (DIG) labeled
Probes 38
4.3 Whole mount in situ hybridization 38 4.4 In situ hybridization on frozen tissue
section 39
5. Whole mount immunohistochemistry 39 6. Neural crest cells transplantation assay 39
7. Tunel Assay 40
8. Solutions 40
RESULTS 42 1. 5-HT2B loss of function during Xenopus
laevis ocular morphogenesis 42
1.1 Morphological analysis 42
1.2 Abrogation of 5-HT2B causes defects
in the optic nerve 43
2. 5-HT2B signaling pathway interacts with Retinoic Acid pathway during ocular
morphogenesis 46
2.1 5-HT2B abrogation results in a
disregulation of gene expression involved in
Retinoic Acid synthesis 46
2.2 5-HT2B loss of function interferes with the expression of Pitx2 and FoxC1, key
genes involved in ocular morphogenesis 49 3. 5-HT2B loss of function influences NCCs migration during ocular morphogenesis 52 3.1 5-HT2B abrogation does not influence
structures derived by the mesodermal cells
component of the POM 54
4. Validation of Morpholino results 56
DISCUSSION 60
1.1 5-HT2B role during ocular
morphogenesis 61
1.2 5-HT2B loss of function results in ocular defects due to the NNC component of
the POM 62
1.2 5-HT2B signaling interferes with the
1.3 Retinoic acid metabolism in the developing
Eye 63
1.4 5-HT2B abrogation alters the migration of the NC POM cells in the eye 65
CONCLUSIONS 66
REFERENCES 67
PUBLISHED PAPER
