The extraembryonic endoderm of mammals is essential for nutritive support of the foetus and patterning of the early embryo. Visceral and parietal endoderm are major subtypes of this lineage with the former exhibiting most, if not all, of the embryonic patterning properties. Extraembryonic endoderm (XEN) cell lines derived from the primitive endoderm of mouse blastocysts represent a cell culture model of this lineage, but are biased towards parietal endoderm in culture and in chimaeras. Here, I further characterise XEN cells and show that these cell lines exhibit high levels of heterogeneity. In an effort for XEN cells to adopt visceral endoderm character different aspects of the in vivo environment were mimicked. I found that BMP4 and laminin promote a mesenchymal-to-epithelial transition of XEN cells with upregulation of epithelial markers and downregulation of mesenchymal markers. Gene expression analysis showed the differentiated XEN cells most resembled extraembryonic visceral endoderm. Correspondingly, inhibition of Erk and BMP signalling drives XEN cells toward parietal endoderm fate. Finally, I show that BMP4 treatment of freshly isolated parietal endoderm from Reichert’s membrane promotes its visceral endoderm differentiation. This suggests that parietal endoderm is still developmentally plastic and can be transdifferentiated to a visceral endoderm in response to BMP. Generation of visceral endoderm from XEN cells uncovers the true potential of these blastocyst-derived cells and is a significant step towards modelling early developmental events ex vivo.

Made available in DSpace on 2016-08-15T20:31:06Z (GMT). No. of bitstreams: 1 AndreMV_DISSERT.pdf: 26149415 bytes, checksum: 8052cd9f22e05a0bada5a1c6b968c995 (MD5) Previous issue date: 2011-10-12 / The inversion period of the yolk sac as the dynamic which results from this process was studied in 30 female of prea on days 6, 10, 11 to 15, 20, 25, and 30 of gestation. Six groups of animals (one male for five females) were formed and kept in boxes of 5m2. Vaginal cytology was performed daily for detection of copulation. Pregnant females were separated of the other individuals of the group. They were euthanized using a specific anesthetic protocol for collection of the gestational sacs. Then, the material was processed for histology, immunohistochemistry, scanning electron microscopy, and transmission electron microscopy. At days 11 to 13 of gestation was observed the development of the parietal and visceral endoderm delimiting the yolk sac cavity. The parietal endoderm was coating the surface of the fetal side of the chorioallantoic placenta and surrounding the space bounded by the capsular decidua. These endoderm layers showed a prismatic shape and were separated of the trophoblast by a developed Reichert s membrane. The visceral endoderm contained vitelline vessels and villi in some areas. On the 13th day it was possible to establish as a basic condition before the inversion of teh yolk sac, the permanence of at least one of the three elements that characterized the bilaminar omphalopleure. On the 14th day of gestation the complete inversion of the yolk sac, which was characterized by the degeneration of both parietal endoderm and mural trophoblast, coupled with the gradual disappearance of the Reichert s membrane was observed. As a consequence of this, the visceral endoderm constitutes the interface with the uterine epithelium. After the inversion of the yolk sac, the parietal endoderm remained intact was the one which rested on the surface of the chorioallantoic placenta, which showed high columnar cells, characteristic of the pseudostratified epithelium. The visceral endoderm showed numerous apical villi mainly in regions close to the chorioallantoic placenta. The intense vascularization was confirmed by the immunohistochemistry reaction for vimentin associated with the scanning electron microscopy. During the continuous development of the embryo and chorioallantoic placenta it was also observed the development of an important area of apposition of both visceral and parietal endoderm whose proliferative capacity of these cells was demonstrated by immunohistochemistry using PCNA antibody. Therefore, we concluded the inversion of the yolk sac represents an anatomical arrangement in favor of embryonic development as well as an evolutionary characteristic in this rodent specie. / O período de inversão do saco vitelino bem como a dinâmica resultante deste processo foi estudado em 30 fêmeas de preás nos dias 6, 10, 11 a 15, 20, 25 e 30 de gestação. Foram formados seis grupos de animais numa relação de um macho para cinco fêmeas, mantidos em boxes de 5m2. Após formação dos grupos, diariamente eram realizados exames de citologia vaginal, para verificação de cópula, separando-se dos grupos as fêmeas que eram cobertas. A partir da ocorrência da cópula programaram-se as coletas dos sacos gestacionais mediante sacrifício das fêmeas gestantes através de protocolo anestésico específico. O material, então, era processado segundo técnicas para histologia convencional, imunohistoquímica, microscopia eletrônica de varredura e de transmissão. Nos dias de 11 a 13 de gestação observou-se o desenvolvimento dos endodermas parietal e visceral delimitando a cavidade do saco vitelino. O endoderma parietal foi evidenciado revestindo a superfície fetal da placenta corioalantoide bem como contornando o espaço delimitado pela decídua capsular. Estes endodermas apresentaram formato prismático e encontraram-se separados do trofoblasto por uma desenvolvida membrana de Reichert. Já o endoderma visceral continha vasos vitelínicos e possuía vilosidades apenas em determinadas áreas. No décimo terceiro dia, foi possível estabelecer como condição primordial antes da inversão, a permanência de pelo menos um dentre os três elementos que caracterizavam a onfalopleura bilaminar. No décimo quarto dia de gestação verificou-se a inversão do saco vitelino, caracterizada pela degeneração do endoderma parietal e trofoblasto mural, associado ao desaparecimento gradual da membrana de Reichert. Como consequência deste fenômeno, o endoderma visceral passou a constituir interface com o epitélio uterino. Após a inversão, o endoderma parietal que permaneceu íntegro foi aquele que se apoiava na superfície da placenta principal, apresentando células em formato colunar alto e característica de epitélio pseudoestratificado. O endoderma visceral apresentou numerosas vilosidades apicais principalmente em regiões próximas a placenta principal. A intensa vascularização desse endoderma foi evidenciada pela reação imunohistoquímica para vimentina e microscopia eletrônica de varredura. Com o contínuo desenvolvimento do embrião e placenta principal, observou-se o surgimento de importante área de aposição entre os endodermas visceral e parietal cuja capacidade proliferativa destas células foi demonstrada pela reação imunohistoquímica para o PCNA. Por conseguinte, concluiu-se que a inversão do saco vitelino representou uma disposição anatômica favorável ao desenvolvimento embrionário além de uma característica evolutiva nesta espécie de roedor.