The extraembryonic endoderm (XEN) defines the yolk sac, a set of membranes that
provide essential support for mammalian embryos. Recently, the committed XENprecursor
was identified in the embryonic Inner Cell Mass (ICM) as a group of cells that
intermingles with the closely related, anatomically indistinguishable epiblast (EPI)-
precursor that gives rise to the fetus. In vitro, the EPI-precursor is represented by the
well-known embryonic stem (ES) cell lines, but cell lines representing the XENprecursor
are not known. Furthermore, since the XEN-precursor cells were discovered
only very recently, the unexpected fact that they express the key pluripotency marker
Oct4 has not been explored. Recently, however, our laboratory has isolated rat XEN cell
lines that express Oct4, leading to the following two questions: (i) Do these new XEN
cell lines represent XEN-precursor cells? (ii) Is their Oct4 expression regulated similarly
as previously known from ES cells? These two questions are addressed here by lineage
marker and reporter gene analyses. Whole culture analyses showed that rat XEN cell lines expressed markers of all
XEN stages including XEN-precursor, primitive endoderm (PrE) and/or visceral
endoderm (VE), and parietal endoderm (PE) but trophoectoderm and EPI-precursor
markers were missing. In line with this, immunocytochemistry demonstrated
heterogeneity and directly visualized the XEN-precursor, PrE/VE, and PE
subpopulations. Low-density plating and time-dependent immunocytochemistry on
resulting colonies strongly suggested that XEN-precursor cells generate the other XEN
stages. Moreover, by analyzing single-cell derived clones, it was shown that culture
heterogeneity results from the self-renewal and differentiation of a single cell. Reporter
gene analyses using the 5’ regulatory region of the mouse Oct4 gene revealed that a
DNA fragment containing the previously described distal enhancer drove reporter gene
expression only in ES cells whereas inclusion of an upstream fragment led to high
expression in both mouse ES and rat XEN cells.
In conclusion, our rat XEN cell lines contain XEN-precursor cells that differentiate
extensively, providing for the first time an in vitro model that mimics the natural process
of early XEN differentiation. In addition, they regulate Oct4 gene transcription
differently than ES cells suggesting heterogeneous Oct4 regulation within the
mammalian ICM.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/ETD-TAMU-2657 |
| Date | 15 May 2009 |
| Creators | Debeb, Bisrat Godefay |
| Contributors | Binas, Bert |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Dissertation, text |
| Format | electronic, application/pdf, born digital |
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