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among all vertebrates. This was confirmed when Chordin and Noggin were identi-
fied as the endogenous, neural inducing, and dorsalizing signals expressed in the
amphibian Organizer, teleost shield, and amniote node (Smith and Harland 1992 ;
Sasai et al. 1994 ; Connolly et al. 1997 ; Fisher et al. 1997 ; Schulte-Merker et al.
1997 ; Streit et al. 1998 ; Furthauer et al. 1999 ; Bachiller et al. 2000 ).
7.5.3.2 Koller’s Sickle/PMZ
It has been more challenging to identify the equivalent of the Nieuwkoop center in
amniotes than in other phyla, mostly due to the more complicated anatomy of the
blastoderm. Attention has focused on three extraembryonic tissues: the hypoblast,
Koller’s sickle and the posterior marginal zone (PMZ). The location of the hypo-
blast underneath the epiblast is reminiscent of the location of the vegetal blasto-
meres under the marginal zone of amphibians. Waddington was the first to test the
capacity of the hypoblast to induce mesoderm, working in chicken and duck
embryos (Waddington 1932 , 1937 ). He separated the hypoblast from the epiblast,
rotated the it 90° or 180° along the anteroposterior axis, recombined the pieces and
grew them in culture. In all cases, the primitive streak developed abnormally, and
sometimes secondary primitive streak and body axis formed with its polarity coor-
dinated with that of the rotated endoderm. Although this result was suggestive, the
lack of adequate lineage-tracing techniques meant that Waddington could not defin-
itively conclude that the formation of a new node and streak was due to an inductive
event. More recent experiments used lineage labels to unambiguously demonstrate
that the node is induced, but have variously identified Koller’s sickle, the PMZ, and
the hypoblast as the inductive tissue (Azar and Eyal-Giladi 1979 ; Eyal-Giladi et al.
1992 ; Callebaut and Van Nueten 1994 ; Khaner 1998 ; Callebaut et al. 2003 ). For
instance, a secondary body axis forms when a square piece of tissue from the PMZ
including Koller’s sickle, is grafted orthogonally to the endogenous PMZ (Fig. 7.8a)
(Khaner and Eyal-Giladi 1989 ; Eyal-Giladi et al. 1992 ). Subsequent experiments
found that Koller’s sickle alone could not induce a primitive streak (Khaner 1998 ).
Furthermore, detailed fate mapping showed that cells in Koller’s sickle contribute to
the axial mesoderm (Bachvarova et al. 1998 ). These results eliminated the sickle
from consideration as the Nieuwkoop Center. PMZ dissected from a chicken or
duck embryo in the absence of Koller’s sickle can induce a primitive streak and
body axis when grafted to an epiblast (Fig. 7.8a) (Bachvarova et al. 1998 ; Khaner
1998 ). Importantly, labeled cells in the graft did not contribute to the new body axis,
indicating that the avian PMZ expresses signals that induce formation of the primi-
tive streak near the marginal zone, which is comprised of future mesoderm and
endodermal cells (Fig. 7.8b, large arrows). Similar grafting experiments have not
been performed in mammals, but genetic studies in the mouse demonstrated that
signals from the extraembryonic visceral endoderm (VE) are required to induce the
node and primitive streak (Varlet et al. 1997 ). Thus, the avian PMZ and the mam-
malian VE are functionally equivalent and meet the criteria of the Nieuwkoop cen-
ter. As the primitive streak extends and the node migrates toward the anterior,
signals from the posterior streak induce surrounding epiblast cells to form lateral
W. Tseng et al.