Establishment and use of a revised embryo staging, novel in vitro assays for pluripotency, potency and specification for investigating early mammalian ectoderm germ layer development in wildtype and gene knockout mouse embryos

Abstract

The ectoderm, together with the other two embryonic germ layers (mesoderm and endoderm, collectively known as mesendoderm), are the progenitors of all tissues of the foetus/new-born. Ectoderm is the least understood mammalian germ layer. Its development begins within that part of the epiblast epithelium (progenitor of foetus/new-born) whose cells: (a) are fated to form ectodermal derivatives, mainly neural and surface ectoderm tissues, and (b) remain within the epiblast, as they do not exit it through the primitive streak during gastrulation to form mesendoderm. This project focused on investigating early mammalian ectoderm development using the mouse as a model by studying the development of anterior epiblast, which is fated to mainly form brain and head surface ectoderm, the earliest-formed ectodermal derivatives. The differentiation of epiblast cells into their differentiated cell types, such as ectodermal derivatives, is preceded by important spatio-temporally regulated developmental events. Specifically, epiblast cells, which are initially pluripotent (they are capable of differentiating to all the cell types derived from all three embryonic germ layers) and have a certain specification state (the cell type they are programmed to differentiate), gradually: (a) restrict their potency (reduce the collection of cell types they are capable of differentiating to), and therefore stop being pluripotent, and (b) have to change their specification if it is not according to what they differentiate into during normal development. However, these developmental events during early mouse ectoderm development are poorly understood and were the main interest of this project. Investigating them, not only requires the use of assays for pluripotency, potency and specification, but also knowing when these events occur. The latter requires employment of embryo staging: the subdivision of development into a specific temporal sequence of embryos with different structural features called stages, which unlike embryonic age (time elapsed since fertilization) are an accurate index of the degree of development reached. The study of early ectoderm development, therefore, could benefit from a more refined embryo staging of the period during which it occurs. The six major findings of this study are the following. First, using novel combinations of external embryo features and gene expression validation, a revised embryo staging for the period from just before gastrulation until the late headfold stage was established. It resulted in subdividing this period into fifteen stages, as opposed to the existing nine ones. This new staging includes the hitherto unidentified stage of gastrulation initiation and, unlike existing staging, subdivides the pre-headfold period into stages without being depended on the timing of allantoic bud presence/size as a diagnostic feature since the latter is not applicable across all mouse strains. v Second, new in vitro pluripotency and potency assays for mouse postimplantation tissues that are simpler and faster than existing ones, were established using explant culture and gene expression validation. Both assays identify whether the tested postimplantation tissue is pluripotent, but only the potency assay identifies the potency of non-pluripotent tissues. Third, the first specification assay for mouse embryo tissues was established by culturing explants under completely defined culture conditions that are considered neutral, so as to allow the manifestation of the programmed differentiation of the tested tissue. Fourth, combining our revised staging, our novel pluripotency/potency assays and marker gene expression, we show for the first time that during anterior epiblast development the following take place. (i) The earliest loss of pluripotency occurs at pre-headfold-2 (PH2) stage, the earliest stage when primitive streak reaches its full length. This is the earliest stage when anterior-proximal epiblast restricts its potency to only ectodermal fates (neural and surface ectoderm fates) and this bipotent ectoderm state is be marked by co-expression of the pluripotency-related markers Fgf5 (low levels) and Oct4 and the early surface ectoderm marker Dlx5, at a time before expression of the earliest neural genes Six3 and Hesx1. In contrast, the anterior-distal epiblast at this stage is still pluripotent and expresses Fgf5 (high levels), Oct4, but not Dlx5. (ii) At pre-headfold-3 (PH3) stage, the previously pluripotent anterior-distal epiblast also restricts its potency to ectodermal fates and this coincides with low level expression of Fgf5 in this part of anterior epiblast, suggesting that low expression of Fgf5 marks anterior epiblast with bipotent ectodermal potency. The earliest anterior neural markers Six3/Hesx1 start being expressed in anterior-proximal epiblast at this stage, suggesting neural plate formation. (iii) At pre-headfold-4 (PH4) stage, there is further restriction of potency of the entire anterior epiblast fated to become brain (most proximal edge of anterior epiblast is fated to form surface ectoderm) to only neural fates. This coincides with undetectable Fgf5 expression in anterior epiblast, suggesting that the earliest loss of Fgf5 expression marks neurally committed anterior epiblast. Fifth, using our specification assay, we show that just prior to gastrulation (exact stage needs further clarification), the pluripotent anterior-proximal and anterior-distal epiblast regions (previouslyshown to be fated towards mainly non-neural ectoderm and neural derivatives, respectively), are specified differently: the former is specified mainly towards surface ectoderm fates and the latter towards neural fates. Lastly, we used our pluripotency assay on the anterior epiblast of Ets2 gene knockout embryos, an in vivo system for studying influences from early trophoblast (extraembryonic progenitor tissue of the placenta) on embryo development. This is because unpublished data from our lab showed that early ectoderm development requires vi signals from the trophoblast, since the anterior epiblast of these mutants fails to express neural genes (indicating failure of neural induction) and coexpresses Fgf5, Oct4 and Dlx5. Our pluripotency assay revealed that anterior epiblast of these mutants in not pluripotent. Taken together with our finding that coexpression of Fgf5, Oct4 and Dlx5 marks the transient, ectodermally bipotent anterior-proximal epiblast state, it is suggested that anterior epiblast of these mutants may be ‘stuck’ or confined at this state and that the role of trophoblast signalling might be to promote exit from this state, so as to allow further ectodermal development. It is concluded that the revised staging and the new pluripotency/potency/specification assays developed here are useful tools for mouse developmental biologists in general, and the results of their application to early ectoderm development have contributed to the understanding of this important developmental process.