233signaling are also critical for the Wnt/calcium pathway, suggesting that these path-
ways overlap considerably (Fig. 6.7). In line with this idea, overexpression of Dvl
can initiate calcium flux and activate Camk2 and Prkca in fish and frog embryos
(Sheldahl et al. 2003 ). Similarly, overexpression of Prickle1 indirectly regulates
calcium dynamics (Veeman et al. 2003b). Also, recruitment of Dvl to the membrane
during PCP signaling requires a calcium-regulated PKC isoform, Prkcd (Kinoshita
et al. 2003 ). Wnt/PCP and Wnt/Calcium are likely to be tightly integrated, owing to
shared components and shared roles in regulating morphogenesis during gastrula-
tion and beta-catenin antagonism.
Evidence suggests that Wnt/Calcium signaling is essential for inhibiting beta-
catenin activation during axis formation. Loss of maternal Wnt5b in zebrafish elimi-
nates calcium flux in the blastula and triggers ectopic beta-catenin activity, resulting
in dorsalized embryos (Westfall et al. 2003 ). This effect was partially rescued by
Camk2, suggesting that calcium-mediated activation of this pathway is sufficient to
suppress beta-catenin activity. Wnt/Calcium is also implicated in activating Nemo-
like kinase (Nlk) (Ishitani et al. 1999 , 2003 ; Meneghini et al. 1999 ) and Nfatc
nuclear translocation (Saneyoshi et al. 2002 ) to antagonize beta-catenin activity.
6.3.1.5 Wnt Secretion and Extracellular Regulation
Wnts are secreted and are modified by glycosylation (Brown et al. 1987 ; Papkoff
et al. 1987 ) and lipidation (Willert et al. 2003 ). Efficient secretion of Wnts requires
glycosylation and palmitoleoylation, the latter of which is mediated by the Porcupine
(Porcn) family of acyl transferases (van den Heuvel et al. 1993 ; Kadowaki et al.
1996 ; Hofmann 2000 ; Tanaka et al. 2000 ). Tyrosine sulfation has also been observed
and may be necessary for activity in some cases (Cha et al. 2009 ). Wnt secretion
also requires trafficking of Wnt from the Golgi apparatus to the plasma membrane
by the Wntless Wnt ligand secretion mediator (Wls; alias Evi/Gpr177/Wingful) as
well as efficient recycling of Wls through the endosome-retromer system
(Bartscherer et al. 2006 ; Coudreuse 2006 ). Interestingly, Wls is a direct Wnt/beta-
catenin target gene in mouse and is required for extracellular Wnt signaling during
mouse axis formation (Fu et al. 2009 ), indicating that Wnt activity potentiates its
own signaling. Additional evidence suggests Wnt proteins may also be packaged
into lipoprotein particles and/or exosome vesicles (Panáková et al. 2005 ; Gross
et al. 2012 ). Wnts can act as both long-and short-range signaling molecules in the
extracellular space, acting as developmental morphogens (Zecca et al. 1996 ). Wnt
signaling gradients can also interact with those of a Wnt antagonist, Dkk1, to estab-
lish hair follicle spacing through a Turing-like reaction-diffusion mechanism (Sick
et al. 2006 ), illustrating one of the complex ways this pathway can used to establish
tissue patterns in development.
Wnt signaling can be tightly regulated in the extracellular space by a host of dif-
ferent Wnt antagonists. Many of these proteins belong to large protein families and
have redundant and tissue-specific functions throughout development (Cruciat and
Niehrs 2013 ). The main secreted Wnt antagonists involved in axial patterning are the
6 Vertebrate Axial Patterning: From Egg to Asymmetry