We describe the internal organization of murine embryoid bodies (EBs) in terms of the
structures and cell types formed as Oct4 expression becomes progressively lost. This is
done by making the EBs from iPS cells carrying an inducible and permanent Oct4
reporter (Oct4-MerCreMer;mTmG). When these EBs are treated with tamoxifen, the
Oct4 expressing cells switch from a red to a green fluorescence color, and this is
maintained thereafter by their progeny. We show that there is no specific pattern in
which Oct4 is downregulated, rather it appears to be spatially random. The earliest cells
to lose Oct4 expression are internal and stain positive for -fetoprotein (AFP) indicating
that they are visceral endoderm. However, GATA4, characteristic of primitive
endoderm, was found in Oct4-expressing cells at this stage. This indicates that the first
formed visceral endoderm does not arise from primitive endoderm, a difference from
normal embryonic development. Contrary to previous reports, our EBs did not form a
layer of primitive endoderm, or visceral endoderm, around the outside. Markers of the
early body axis, BRACHYURY (T) and FOXA2, behaved somewhat differently from
each other. BRA, which marks the early mesoderm, node and notochord, arises in Oct4
expressing cells on days 3-4. FOXA2, which marks the floor plate of the neural tube and
definitive endoderm, as well as the node and notochord, arises at the same time but
mostly in cells that have already lost Oct4 expression. Although there is usually a
concentration of T or FOXA2 cells in one region of the EB, the morphology is not
predictable and there are also scattered cells expressing these markers. Several clumps of cardiomyocytes are visible by day 7 of EB development, and we show that the cells
forming these clumps lose Oct4 expression between days 3 and 5. Overall, our results
indicate that EBs recapitulate normal development quite well in terms of the tempo of
events and the appearance of specific markers, but they do not resemble embryos in terms
of their morphology and structure, which is contrary to the previous reports.
University of Minnesota M.S. thesis. April 2012. Major: Stem cell biology. Advisor: James Dutton. 1 computer file (PDF); vi, 52 pages, appendix p. 52.
Sajini, Abdulrahim Abdulrahman M.
Loss of Oct4 expression during the development of murine embryoid bodies.
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