Bovine Pre-gastrulation Development and the role Of Rauber's Layer
van Leeuwen, J. (2016). Bovine Pre-gastrulation Development and the role Of Rauber’s Layer (Thesis, Doctor of Philosophy (PhD)). University of Waikato, Hamilton, New Zealand. Retrieved from http://hdl.handle.net/10289/10385
Permanent Research Commons link: http://hdl.handle.net/10289/10385
Although conception rates in cattle are estimated to be 90%, little over half of each insemination results in the birth of offspring. The majority of total combined foetal and embryonic loss is sustained between the bovine blastocyst hatching at Day 8 and the onset of gastrulation after Day 14. Post hatching, the bovine embryo develops a morphological architecture which is very different to the mouse, with a flat epiblast and the loss of the polar trophoblast, known as Rauber’s layer. In mice, the polar trophoblast is essential for the induction of gastrulation and the formation of the placenta. The pre-gastrulation layout of cattle embryos is shared by a diverse phylogeny of placental forming mammals, from rabbits to pigs, and it is therefore considered the more typical mode of mammalian embryonic development. Yet, compared to mouse embryonic development, relatively little is known about the gene expression and signalling networks required for successful gastrulation in these species, particularly cattle which are also economically important. This thesis studied the morphological and gene expression events during Days 11-15 in cattle which coincides with the period of greatest embryonic loss. A detailed morphological staging system is presented that is supported with spatio-temporal gene expression patterns of genes known to play fundamental roles in mouse pre-gastrulation development. Novel morphological structures and processes were identified such as the formation of the cattle anterior visceral hypoblast (equivalent to the mouse AVE) and cavitation of the epiblast in preparation for epithelialisation of this tissue. Both of these processes are considered pre-requisites for the induction of gastrulation in mammals. The spatio-temporal gene expression patterns prior to gastrulation showed overall high concordance with mouse development however, processes that lead to asymmetry in the mouse involving extra-embryonic tissues have diverged and this can be related back to the different embryonic layouts and polar trophoblast fates between cattle and mice. Functional studies were carried out in cattle embryos to investigate the relative roles of the three first cell lineages (trophoblast, epiblast and hypoblast) in contributing to embryonic loss and to investigate the possible role of Rauber’s layer in pre-gastrulation development. Over-expression of the weak pro-apoptotic factor BAD resulted in the selective loss of the epiblast between Days 7 and 13/14 of development. The trophoblast was not measurably impaired and although the hypoblast was not lost, the gene expression profile of this tissue was dramatically altered. These results indicate a gradient of sensitivity of epiblast>hypoblast>trophoblast tissue and suggest the epiblast contributes more greatly overall to embryonic loss. This result provides insights into the phenomenom of “phantom pregnancies”, which also are due to loss of the epiblast/embryo proper and are thought to occur in 12-22% of cattle pregnancies. The loss of Rauber’s layer is thought to occur by apoptosis and the results shown in this thesis support this idea. Over-expression of anti-apoptotic BCL2 resulted in a delayed loss of Rauber’s layer. BCL2 embryos also had an increased epiblast length and one embryo showed ectopic mesoderm formation. These results are consistent with the hypothesis that a maintained Rauber’s layer could lead to an increase in NODAL signalling. The findings in this thesis provide a foundational step in the establishment of the bovine embryo as a model for mammalian development. The results also demonstrate functional perturbations of the cattle embryo are experimentally feasible, and how these have revealed insights into cattle embryonic loss and the role of Rauber’s layer in development.
University of Waikato
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