On ultrastructure and gene expression during epiblast morphogenesis and axial differentiation in the prestreak embryonic disc of the pig

The epiblast of the amniote embryo is of paramount importance during early development as it gives rise to all tissues of the embryonic proper. In most mammals, the epiblast emerges by segregation of the inner cell mass into (a) flat one-layered hypoblast also known as visceral endoderm and (b) epiblast precursors which subsequently undergo epithelialization and create the embryonic disc. In a next step the anterior-posterior axis emerges by directed migration of hypoblast cells towards the future anterior pole. These cells display a cuboidal morphology and, molecularly, are involved in suppression of the posterior fate in the overlying epiblast cells. In the pig embryonic disc, the flat epithelial epiblast emerges after anterior-posterior axis formation and during the disintegration of the polar trophoblast. Both epiblast epithelialization and polar trophoblast loss are still ill-understood despite being crucial processes for ensuring tissue integrity during gastrulation and will, therefore, be subject of this thesis. High resolution light and transmission electron microscopy, three-dimension (3D) reconstruction and in situ hybridization of the organizer gene goosecoid (gsc) as an early marker for the formation and polarity of the anterior-posterior axis were applied to 8- to 10-days-old pig embryos. Because of the morphological complexity of the early embryonic disc during the epiblast shape transition I refined – as the first step of my thesis – the staging system of the two existing pregastrulation stages into four stages (1-, 1+, 2- and 2+): At stage 1- the epiblast is partially polarized and soon segregates into (1) a dorsal part which forms a closely associated non-polarized mass of cells and (2) ventral epiblast cells which form special immature desmosomal contacts located near the center of the embryonic disc and bordered by a dense felt of microfilaments parallel to the desmosomal plaque. Ventral and dorsal epiblast cells form rosette-like arrangements at their apical narrow ends by interdigitation. The cytoplasm of the intact polar trophoblast is rich with grey lipid droplets, primary lysosomes and different types of heterolysosomes and (auto)phagosomes and three types of mitochondria. Trophoblast cells closely adhere to each other by many lateral desmosomes and long bundles of intermediate filaments interwoven into the cytoplasm and cortical bundles of microfilaments oriented parallel to the apical surface. At stage 1+ the epiblast forms an extracellular cavity which separates horizontally dorsal epiblast cells from ventral epiblast cells. In addition, long polar trophoblast protrusions towards the epiblast separate the two epiblast populations vertically. The polar trophoblast shows increase sign of disintegration with cytoplasmic condensations, apical blebs, large vacuoles, residual bodies, and numerous lysosomes. Dorsal epiblast cells are only closely attached to the remnants of the polar trophoblast by desmosomes while ventral epiblast cells show epithelial characteristics and are closely attached to each other by special triplet desmosomes. At stage 2- only remnants of polar trophoblast are found and lie mostly posteriorly and show apical blebs and long microvilli. The ventral epiblast forms a concave epithelium under the remnants of polar trophoblast and shares adhesion contacts with mural trophoblast and with each other by what is known as zipper-like desmosomes, the mature serial form of the special desmosome. The expression of the homeobox transcription factor goosecoid (gsc) is found early at stage 0 of 8 dpc embryos in the flat hypoblast of the ICM and in the extraembryonic trophoblast. Later it is confined to the extraembryonic and embryonic parts of the anterior hypoblast. The expression pattern develops from an anterior crescent shape into half-ring shape. Intriguingly, two opposite domains of gsc are found at pregastrulation stage 2: (1) in the anterior hypoblast, which shifts both more centrally and more anteriorly towards the extraembryonic tissue and (2) in posterior epiblast cells where the primitive streak will eventually form. At stage 4+ the node (the mammalian equivalent to Spemann’s organizer) is strongly gsc positive. The results of the study are discussed in the context of epiblast development and early axis formation, cavity formation by the rosette-like cellular rearrangement as seen in pre-amniotic cavity formation in the mouse and human, and I speculate that the separation of the apical membrane of epiblast cells is not only caused by charge repulsion mediated through actomyosin contraction but also involves the role of filopodia in cell mobility and migration. The polar trophoblast disintegration shares overlapping characteristics of two modes of programmed cell death, autophagy and apoptosis. The dorsal, non- epithelialized mass of cells shares the fate of the attached polar trophoblast. Both cell types show signs of apoptosis and they may be eventually phagocytosed by the neighboring ventral epiblast cells as the latter contain up to stage 2- few but large phagocytosis vacuoles. In sum, I propose that the epiblast does not undergo an unfolding process but that its cells (1) rearrange their position with the help of highly flexible immature special desmosome during epithelialization, and (2) form a concave epithelium fusing with mural trophoblast anteriorly or posteriorly, while the central mass of dorsal epiblast is lost with the disappearing polar trophoblast. Furthermore, I propose that the pattern of the gsc expression in the pregastrulation stages reflects two different roles of the gene: (1) in the anterior hypoblast, gsc may repress the activity of the BMP4 at stages 0, 1- and 1+ in the epiblast; (2) in the epiblast gsc may be involved in activation of the fate and features of the prospective prechordal mesoderm.2021-09-2