245streak (Brennan et al. 2001 ; Ben-Haim et al. 2006 ), indicating that Wnt regulation of
Nodal remains conserved, but is deployed modularly in development.
Interestingly, although Wnt may not activate early Nodal expression in the
blastocyst, data suggest that Tcf3 repression of Nodal is a conserved feature of ver-
tebrate development. Deletion of mouse Tcf3 (Merrill et al. 2004 ) results in the
formation of secondary axes (Hensen’s nodes and notochord) and in upregulation of
Nodal target genes (e.g., Foxa2; Hoodless et al. 2001 ; Yamamoto et al. 2001 ) in the
pregastrula embryo. And, Nodal is upregulated in Tcf3 null embryonic stem (ES)
cells (Cole et al. 2008 ). In fish and frogs, early-localized Wnt signals provide an
initial signal to relieve repression by Tcf3 (Kim et al. 2000 ; Houston et al. 2002 ;
Dorsky et al. 2003 ). Mammals must have evolved different mechanisms to over-
come this repression in the absence of pervasive early Wnt activity. Recent data
suggest that Nodal initiation and potentiation may be controlled by a distinct
enhancer bound by pluripotency transcription factors Oct4 and Klf4, in addition to
ongoing Smad2-dependent signaling (Papanayotou et al. 2014 ). Interestingly, Tcf3
null ES cells also exhibit derepression of other genes, in addition to Nodal, that are
co-regulated by pro-pluripotency factors (Cole et al. 2008 ), suggesting that in nor-
mal ES cells, these proteins may help overcome Tcf3-mediated repression in the
absence of strong activation of Wnt/beta-catenin signaling.
Is there a mammalian Nieuwkoop center? In line with a lack of asymmetry in the
mammalian egg, clonal analyses and transplant experiments performed in the pre-
gastrula mouse embryo have not identified a region with the requisite Nieuwkoop
properties; indeed transplanted epiblast cells typically change fate according to their
new position (Lawson et al. 1991 ; Lawson and Pedersen 1992 ; Parameswaran and
Tam 1995 ). Also, there is a high degree of cell mixing in the epiblast during normal
development (Gardner and Cockroft 1998 ), which would be inconsistent with a lin-
eage-restricted Nieuwkoop center. However, there is overlap of Nodal and Wnt3 in
the posterior proximal epiblast and subsequently throughout the primitive streak
(Tam et al. 2006 ) and high levels of Nodal signaling are required for organizer for-
mation (Vincent et al. 2003 ). The early proximal epiblast may have some hallmarks
of a Nieuwkoop center, although the region of highest and most complete axis-
inducing activity is the anterior primitive streak itself, prior to Hensen’s node forma-
tion (Kinder et al. 2001 ), similar to the case in chicken. Interestingly, these cells do
not significantly contribute to the organizer/Hensen’s node or notochord, instead
making contributions to the anterior mesendoderm (Kinder et al. 2001 ). Thus, orga-
nizer induction in mammals may still require non-cell-autonomous induction by Wnt
and Nodal signals, with the signals being generated from the nascent mesendoderm,
as opposed to strictly extra-mesodermal Nieuwkoop center- like mechanisms.
6.4 The Organizer and Dorsoventral Patterning
Axis formation and patterning are largely determined by a discrete zone of midline
mesoderm in the prospective dorsal region of the gastrula. The collective cell sig-
naling and morphogenetic properties of these cells and their descendants during
6 Vertebrate Axial Patterning: From Egg to Asymmetry