Whence pluripotency?
Interaction between Cdx2 and Oct3/4 sorts out the early embryo
Differentiation, the process by which cells assume sets of properties
and specialized functions, is one of the most fundamental mechanisms of
development. In the embryo, cells progress from a less differentiated
to a more specifically differentiated state, a maturation that generally
results in the loss of the differentiated cell's ability to take on other
roles. This trade-off between potential and lineage commitment begins
very early in the life of the mammalian embryo, as shown by the loss of
totipotency (the autonomous ability of a cell to develop into an organism
entire) of cells after only a few rounds of division. This first differentiation
event results in the segregation of the early, ball-shaped embryo (called
a blastocyst) into two distinct tissue types: an outer layer of trophectoderm,
which goes on to form placenta, and the inner cell mass (ICM), a population
of cells that both serves as the wellspring for every one of the cells
in the embryo proper and contributes additional extraembryonic tissue
as well. Despite the primary importance of this pivotal event, its molecular
underpinnings have remained a tantalizing puzzle to researchers for nearly
two decades.
A significant portion of that puzzle has now been fit into place by
researchers from the Laboratory for Pluripotent Cell Studies (Hitoshi
Niwa; Team Leader), who described the interaction between a pair of factors,
Oct3/4 and Cdx2, that sets up the trophectoderm/ICM divide. Working with
colleagues from Kobe University, the Japan Science and Technology Agency,
and Mount Sinai Hospital (Canada), the study proposed a new model for
the genetic basis of the earliest instance of cellular differentiation
in the December 2005 issue of the journal Cell.
In
previous work with embryonic stem (ES) cells, which share many of the
properties of cells from the blastocyst interior, Niwa discovered that
he could induce the ES cells to differentiate into trophectoderm (something
they do not normally do) by repressing the function of the transcription
factor, Oct3/4. Other work has further recently identified a second molecule,
Cdx2, as intimately involved in trophectoderm formation in the mouse blastocyst.
With that finding as a cue, the Niwa team tested the effects of overexpressing
Cdx2 in ES cells in vitro and found that this also could induce trophoblast
stem (TS) cells under certain culture conditions. On injection back into
a blastocyst, Cdx2-induced TS cells that had been labeled with a fluorescent
transgene to allow them to be visualized were seen to give rise to placental
cell lineages in the chimeric embryos, as evidenced by the green glow
from the placenta when exposed to an excitation wavelength of light.
Prompted by their discovery to investigate the possibility of a relationship
between Oct3/4 and Cdx2 (specifically, that they might act in a mutually
inhibitory fashion), the team transfected ES cells with both molecules
and observed the effects on genes normally activated by endogenous Oct3/4.
They found that Cdx2 represses Oct3/4's transcriptional activating effects
and, interestingly, that this repression depended on Oct3/4; in the absence
of Oct3/4, no Cdx2-mediated inhibitory effect was seen on its downstream
target genes. Intriguingly, Oct3/4 seems to exert a suppressive influence
on Cdx2 as well. Cdx2 is thought to positively regulate its own expression
in ES cells, but when these same cells are cultured with an Oct3/4 expression
vector, this autoregulation is significantly damped.
This set of findings hinted that Cdx2 and Oct3/4 are locked in a mutually
inhibitory relationship. Curious about the mechanics of this interaction,
the Niwa team first labeled the two molecules to study their localization
within cells and found that when both were expressed together, they relocated
to inactive regions of the nucleus. Next, they performed immunoprecipitation
analysis (a means of determining whether two molecules bind to each other)
and found that Oct3/4 and Cdx2 do indeed co-precipitate, indicating a
direct interaction on the molecular level.
The story took on a new twist when Niwa and colleagues looked at ES
cells lacking both Cdx2 and Oct3/4 function. Although Cdx2 was thought
to be an inducer of trophectoderm, they discovered that, when Oct3/4 function
is inhibited, even ES cells with homozygous deletions of Cdx2 can give
rise to trophectoderm. The unexpected dispensability of Cdx2 in trophectodermal
differentiation led them to inquire just what does Cdx2 actually do. Creating
a line of cells in which Cdx2 expression could be controlled conditionally,
they compared TS cells derived solely as a result of the down regulation
of Oct3/4 with those actively induced by Cdx2 expression, and found that
the TS cells lacking Cdx2 were deficient in their ability to self-renew,
which is one of the hallmark properties of all stem cells. As it turns
out, a second factor, Eomesodermin (Eomeso), is capable of inducing trophectodermal
differentiation even in the absence of Cdx2; Eomeso, however, could not
entirely compensate for Cdx2 function, and had no repressive effect on
Oct3/4.
The Niwa team next observed how cells immunostained for Cdx2 and Oct3/4
behave in morula and blastocyst-stage embryos. In the early morula, both
factors were detected in all cells' nuclei, but by the later 10-16 cell
stage Cdx2 could only be found in some of the outermost cells (Oct3/4
expression remained pervasive). By the blastocyst stage, the segregation
was complete with Cdx2 limited to the outer layer and Oct3/4 restricted
to the interior. These findings present the strong possibility that the
loss of Cdx2 expression in the inner cells of the morula may be the trigger
for the territorial sorting of the Cdx2-expressing trophectoderm from
the Oct3/4-expressing inner cell mass. Niwa suggests that this process
may be the result of the mutual repression of the two molecules, but whether
the dynamics of this sorting out are simply stochastic, or actively determined
by cell polarity or size is yet to be determined. Other questions, such
as whether this reciprocal inhibition is both necessary and sufficient
to drive Cdx2 out to the morula periphery and the mechanism by which Oct3/4
maintains pluripotency in ES cells, also await answers. Answers that no
doubt will stand on the solid foundations laid by this landmark series
of experiments.
