Publicação
Molecular mechanisms triggering differentiation of the embryonic endodermal stem cells
| Resumo: | Mouse small intestine is a highly organized tissue with a three dimensional structure with finger-like protrusions – villi – surrounded at the base my multiple invaginations – crypts of Lieberkühn – making it the body largest interface. Due to the constant exposure to harsh conditions, the small intestine has a high cell renewal rate fuelled by stem cells present at the bottom of the crypts. In embryos, the structure that will give rise to the small intestine is formed around e8.5 to e9.0 and progressively acquires the characteristics of the small intestine with villus emerging at e15. At this time, cell proliferation is restricted to the intervillus region and will continue to be located there until the crypts develop in the postnatal period. This work aimed to 1) validate the transcription profile of the ISCs previously obtained by performing in-situ hybridization analysis on paraffin sections of mouse embryos (e12.5, e13.5, e14.5 and e15.5) and adult small intestine and 2) understand in more detail the mechanisms involved in gene regulation by focusing in DNA methylation performing MBD-seq analysis. Bisulfite sequencing protocol was optimized for further DNA methylation analysis. ISH analysis of Hes1, Notch2, Ascl2, Muc4, Dusp1, Cps1, Kcne3, Igfbp5 and Shh was indicative that the genetic program leading to the adult small intestine change, as target genes were detected to be expressed at different developmental stages. MBD-seq data allowed to detect also changes in DNA methylation in the genes Shh, Grb10, Foxa1, Id2, Ndufaf3, Nrp, Fz2, Meis1, Mdk, Efbn2, Lgr5, Vdr, Olfm4, Elf3, Ephb3 and Oct4. For the majority of the genes, an active transcription was associated with unmethylation of the DNA and methylation increased as the genes were inactive. In respect to the genes where this relation was not possible to establish, transcription regulation as result of other epigenetic markers should be investigated. |
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| Autores principais: | Guerreiro, Eduarda Mazagão |
| Assunto: | Ciências biomédicas Intestino Células estaminais ADN |
| Ano: | 2013 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Algarve |
| Idioma: | inglês |
| Origem: | Sapientia - Universidade do Algarve |
| Resumo: | Mouse small intestine is a highly organized tissue with a three dimensional structure with finger-like protrusions – villi – surrounded at the base my multiple invaginations – crypts of Lieberkühn – making it the body largest interface. Due to the constant exposure to harsh conditions, the small intestine has a high cell renewal rate fuelled by stem cells present at the bottom of the crypts. In embryos, the structure that will give rise to the small intestine is formed around e8.5 to e9.0 and progressively acquires the characteristics of the small intestine with villus emerging at e15. At this time, cell proliferation is restricted to the intervillus region and will continue to be located there until the crypts develop in the postnatal period. This work aimed to 1) validate the transcription profile of the ISCs previously obtained by performing in-situ hybridization analysis on paraffin sections of mouse embryos (e12.5, e13.5, e14.5 and e15.5) and adult small intestine and 2) understand in more detail the mechanisms involved in gene regulation by focusing in DNA methylation performing MBD-seq analysis. Bisulfite sequencing protocol was optimized for further DNA methylation analysis. ISH analysis of Hes1, Notch2, Ascl2, Muc4, Dusp1, Cps1, Kcne3, Igfbp5 and Shh was indicative that the genetic program leading to the adult small intestine change, as target genes were detected to be expressed at different developmental stages. MBD-seq data allowed to detect also changes in DNA methylation in the genes Shh, Grb10, Foxa1, Id2, Ndufaf3, Nrp, Fz2, Meis1, Mdk, Efbn2, Lgr5, Vdr, Olfm4, Elf3, Ephb3 and Oct4. For the majority of the genes, an active transcription was associated with unmethylation of the DNA and methylation increased as the genes were inactive. In respect to the genes where this relation was not possible to establish, transcription regulation as result of other epigenetic markers should be investigated. |
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