The combination of severe ocular and distal limb malformations is rare. Ophthalmo-acromelic syndrome (OAS; MIM 206920) is characterised by anophthalmia with lower limb oligodactyly. To date <40 cases of this autosomal recessive disorder have been reported. Genome-wide analysis of ~10,000 SNPs typed on two apparently unrelated families - comprising a total of three affected individuals, four unaffected siblings and their consanguineous parents - identified a large region of overlapping autozygosity on chromosome 14q. Adding data from a third consanguineous family gave a combined LOD score of >5 with no evidence of locus heterogeneity. Collaborative data from a further 6 individuals refined the critical interval to a 3.4 Mb region on chromosome14:69,652,605-73,059,612 Mb. To sequence all 19 known protein-coding genes in the region, the 238 exons were ranked by evolutionary sequence conservation and divided equally between the Edinburgh and Nijmegen groups. Complete sequence coverage has been obtained for 61% of the “Edinburgh” exons but no potentially causative mutations have been identified. Further mutation analysis of the OAS locus is on-going. Mice homozygous for the X-ray induced Mp mutation were reportedly anophthalmic with hind limb oligodactyly and thus represented a potential model for human OAS. This line was rederived in Edinburgh and phenotypic analysis of Mp/Mp homozygotes showed runting, malformed pinnae with microphthalmia but not anophthalmia. The apparent hind-limb oligodactyly was due to osseous syndactyly. Mp heterozygotes had milder microphthalmia and pinnae deformities, but lacked the syndactyly. In both heterozygotes and homozygotes the eye malformations were fully penetrant, pan-ocular and characterised by failure of both the ciliary apparatus and vitreous body to form and abnormal retinal lamination. Genome-wide microsatellite marker analysis showed linkage of the Mp phenotype to chromosome 18. Fbn2 mapped within the linkage interval and was a good candidate for Mp based on the finding of hind limb osseous syndactyly in Fbn2-null mice. However, Fbn2-null mice have no eye phenotype. 3’-RACE identified that Mp was as a 660 kb inversion affecting the 3’-regions of Fbn2 and the adjacent gene Isoc1. This created two aberrant reciprocal fusion transcripts: Fbn2 exons 1-63 are fused to Isoc1 exon 5; and Isoc1 exons 1-4 are fused to Fbn2 exons 64-65. This predicts nonsense-mediated decay of the Isoc1 Mp transcript and production of a truncated Fbn2 Mp protein. Ocular development was analysed in homozygote and wild type embryos to define the basis of the “worse than null phenotype” seen in Mp mice. RNA in situ hybridisations (ISH) failed to detect expression of Isoc1 in the embryonic eye. In contrast, normal expression of Fbn2 in the ciliary body and retina was consistent with the Mp phenotype. A combination of EM and immunocytochemistry showed that truncated Fbn2 (Fbn2Mp) was retained within the ER. Fbn2Mp co-localised with markers of ER stress: Grp78 expression and UPR-specific Xbp1 splicing. Signalling by Wnt2b is thought to be critical for ciliary development and Lef1, a Wnt-responsive transcription factor, showed increased and ectopic ocular expression in the region affected by ER stress. Sox2 is a direct transcriptional target of Lef1 and we observed apparent ectopic expression of Sox2 in the ciliary body. Throughout the developing retina in mutant embryos we also observed individual cells that were ectopically expressing the transcription factor Chx10 and other cells expressing the apoptotic marker Activated- Caspase-3. The apoptotic marker did not specifically co-localise with Fbn2Mp. Taken together, these findings suggest that the ocular malformations in Mp are a direct result of the ER stress induced by Fbn2Mp in a specific group of cells in the early ciliary body. The ER stress presumably halts post-translational modification of a developmentally critical signaling molecule, possibly Wnt2b, which happens to be expressed in the same cells. We have termed the resulting pathological mechanism a synodiporic effect (synodiporia = the ones walking the street together or fellow travellers). Such effects may have significant implications for human genetic disease analysis, and may provide an explanation for other “worse than null” mutations.

During optic cup morphogenesis, the neuroectodermal layers of the optic vesicle (OV) invaginate ventrally, and fuse at the choroid fissure (CF) along the proximo-distal axis such that the retina and retinal pigment epithelium (RPE) are confined within the cup. Failure of CF closure results in colobomas, which are characterized by the persistence of a cleft or hole at the back of the eye. While CF closure is a critical aspect of ocular development, the molecular and cellular mechanisms underlying this process are poorly understood. My research examined CF closure and colobomas using zebrafish as a model system. In the first study, I determined that early cell fate changes within the eye field could cause colobomas using the zebrafish mutant blowout. Colobomas in blowout resulted from defects in optic stalk morphogenesis whereby the optic stalk extended into the retina and impeded the edges of the CF from meeting and fusing. Positional cloning of blowout identified a nonsense mutation in patched1, a negative regulator of the Hedgehog pathway. Up-regulation of Hedgehog pathway activity causes disruption in the patterning of the OV into proximal and distal territories, revealing that cell fate determination, mediated by Hedgehog signaling, is intimately involved in regulating CF closure. In the second study, I examined Bcl6 function and regulation during zebrafish eye development. bcl6 encodes a transcriptional repressor expressed in the ventral retina during zebrafish eye development. Loss of Bcl6 function leads to colobomas along with up-regulation of p53, a previously known Bcl6 target, and an increase in the number of apoptotic cells in the retina, demonstrating that Bcl6 plays a critical role in preventing apoptosis in the retina during early eye development. I also showed that Vax1 and Vax2 act upstream of bcl6 in the ventral retina. Furthermore, I identified functional interactions between Bcl6, Bcor and Hdac1 during eye development, demonstrating that Bcl6 functions along with Bcor and Hdac1 to mediate cell survival by regulating p53 expression. Together my studies expand the gene regulatory network involved in cell fate determination and cell survival during CF closure and ventral retina formation, and provide mechanistic insight into coloboma formation. / text

Submitted by (lucia.rodrigues@ufrpe.br) on 2016-05-25T12:57:30Z No. of bitstreams: 1 Fabiana Felix de Oliveira.pdf: 1456917 bytes, checksum: 0e4831795041d99c98c8196d60199a9b (MD5) / Made available in DSpace on 2016-05-25T12:57:30Z (GMT). No. of bitstreams: 1 Fabiana Felix de Oliveira.pdf: 1456917 bytes, checksum: 0e4831795041d99c98c8196d60199a9b (MD5) Previous issue date: 2012-05-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The present study it aimed at to describe the ocular development, with emphasis in the retina of the Betta splendens fish, an ornamental fish that presents an extra-embryonic development, favoring research that involves one better agreement of its embryology and studies on degenerations and regenerations, ontogeny and differentiation occured in the eye, this research was carried through of the embryonic period up to ten days post-eclosão. The complete development of the eye when it has the eclosion of the animal. For the study of the stages of development the animals had been anesthetized in water with Eugenol 50ppm, were fixed in a solution of 4% paraformaldehyde in phosphate buffer, dehydrated and infiltrated with Paraplast Plus. After that, confectioned sections of 1-5μm of thickness and stained with HE, PAS and staining TUNEL. As result was possible to observe that the eyes had appeared of evaginações of diencéfalo, forming the optic cup. Neuroectoderm, the mesoderm and the ectoderm of the surface are involved in the formation of the eye. The staining TUNEL identified to process of apoptotic cells in the retina and lens, proving that it has a reduction of cells during the development of the animal. In the adult fish, through the chemistry it was possible to observe the morphology and the cellular arrangement of the photoreceptors. / O presente estudo visou descrever o desenvolvimento ocular, com ênfase na retina do Betta splendens, um peixe ornamental que apresenta um desenvolvimento extra-embrionário, o que favorece pesquisas que envolvam um melhor entendimento da sua embriologia e estudos relacionados ao conhecimento de degenerações e regenerações ocorridas no olho, à pesquisa foi realizada do período embrionário até dez dias pós eclosão. O desenvolvimento do olho já esta completo quando o animal eclode por ser um órgão de extrema importância para locomoção, reprodução, alimentação e defesa deste em relação ao predador. Para o estudo das etapas de desenvolvimento os animais foram anestesiados com Eugenol, sendo utilizado 50ppm em água, perfundidos com Paraformaldeido a 4% em Tampão Fosfato, desidratados e embebidos em Paraplast Plus. Foram confeccionados cortes histológicos com 5 e 1μm de espessura e corados com HE, PAS e submetidos ao método TUNEL. Como resultado foi possível observar que os olhos surgiram de evaginações do diencéfalo, formando o cálice óptico, o neuroectoderma, o mesoderma e o ectoderma da superfície estão envolvidos na formação do olho. O método TUNEL identificou processo de apoptose na retina e cristalino, comprovando que há uma redução de células durante o desenvolvimento do animal. No peixe adulto, através da histoquímica foi possível observar a morfologia e o arranjo celular dos fotorreceptores.