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Stromal Support of Erythropoiesis During DevelopmentSimon Cridland Unknown Date (has links)
Adult haematopoiesis occurs in the context of a supportive stromal cell niche. The bone marrow, spleen and thymus all contain specific, but relatively poorly defined, stromal cells, which are important for maintenance of quiescence and directed differentiation. Even less is known about the haematopoietic niche during haematopoietic development. The formation of red blood cells (erythropoiesis) occurs during haematopoiesis, and is also controlled by a variety of stromal cells. This thesis examined the visceral endoderm, a group of cells that surrounds the developing epiblast and is required for primitive erythropoiesis (early blood production). We attempted to determine which factors in the visceral endoderm were responsible for induction of primitive erythropoiesis, and whether they would be useful as blood induction factors in embryonic stem cell differentiation. Thus, I attempted to immortalise the visceral endoderm using an immortalising agent (SV40Tag), driven off of a previously identified visceral endoderm gene, Indian hedgehog. We modified a bacterial artificial chromosome so that SV40Tag was driven off of the Indian hedgehog gene. The modified bacterial artificial chromosome was used in both pronuclear injections of mouse blastocysts and the electroporation of embryonic stem cells. After neither attempt produced a visceral endoderm cell line, we examined a visceral endoderm-like cell line, END2, for the presence of the blood inducing factors. We demonstrated the ability of END2 conditioned media to apparently increase expression of blood transcripts in differentiating embryonic cells indicating the presence of blood inducing factors. Expression profiles of END2 cells were compared to a previously completed embryonic stem cell differentiation profile to identify enriched genes. Two genes, angiopoietin-like 7 and Bc064033, were tested for an ability to induce blood in differentiating embryonic stem cells. When neither protein was capable of inducing blood, the END2 cells were examined for the presence of other known blood inducing factors and similarity to in vivo visceral endoderm. The END2 cells were found to produce bone morphogenetic protein 4, a potent inducer of blood in embryonic stem cell differentiation, which complicated the search for additional factors. Examination of END2 cells also indicated a lack of visceral endoderm markers such as alpha fetoprotein, indicating that the END2 cells may not be as visceral endoderm-like as expected from the current literature. The previously identified Indian hedgehog gene was also examined for its blood induction abilities in vivo. Indian hedgehog knockout mice were examined for the effect gene removal had on both primitive and definitive erythropoiesis. Levels of primitive erythrocytes were unaffected in the Indian hedgehog knockout mice, but levels of definitive erythrocytes were found to be significantly decreased. Further examination of Indian hedgehog knockout fetal livers also showed that they had decreased numbers of haematopoietic stem cells. The haematopoietic stem cells were fully capable when cultured and generated appropriate numbers of progenitor cells, indicating a non-intrinsic cause for this defect. Levels of hedgehog target genes that are usually highest in the stromal compartment were also found to be most significantly decreased in Indian hedgehog knockout fetal livers. Another hedgehog gene, desert hedgehog, was also shown to be expressed in the fetal liver and may act with Indian hedgehog to regulate stromal production in the fetal liver.
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Differentiation of extraembryonic endoderm stem cell lines and parietal endoderm into visceral endoderm : the art of XEN cellsPaca, Agnieszka Maria January 2012 (has links)
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.
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Dinâmica da inversão do saco vitelino em preás Galea spixii Wagler, 1831 / Dynamic of the yolk sac inversion in Preas Galea spixii Wagler, 1831Vale, André Menezes do 12 October 2011 (has links)
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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.
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