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.
| Identifer | oai:union.ndltd.org:ADTP/279302 |
| Creators | Simon Cridland |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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