352
and ndr2/cyc in the margin depends upon the function of the EGF-CFC/Cripto/FRL
co-receptor, One-Eyed Pinhead (Oep), and is expanded in embryos lacking the
secreted Nodal antagonist, Antivin/Lefty (Gritsman et al. 1999 ; Meno et al. 1999 ;
Chen and Schier 2002 ; Feldman et al. 2002 ). Overexpression of Antivin/Lefty
inhibits expression of ndr1/sqt and ndr2/cyc (Thisse and Thisse 1999 ). Expression
of a ndr1/sqt transgene depends on a conserved Activin/Nodal response element in
the ndr1/sqt first intron (Fan et al. 2007 ). Thus, as in other vertebrates, expression
of the zebrafish nodal-related genes depends upon an autoregulatory feedback loop
(Fig. 7.9b). Nodal signals expressed in the extraembryonic YSL are required to
induce expression of both ndr1/sqt and ndr2/cyc in the margin (Fan et al. 2007 ).
Nodal expression in the YSL is independent of Nodal signals, however, since the
YSL does not express the EGF-CFC co-receptor, Oep, or have activated Smad2
(Fan et al. 2007 ; Harvey and Smith 2009 ). Expression of ndr1/sqt and ndr2/cyc in
the YSL is controlled in part by the Mix/Bix transcription factor Mxtx2, although
evidence suggests that a T-box containing transcription factor may also be involved
(Fig. 7.9b) (Hong et al. 2011 ; Xu et al. 2014a). sqt/ndr1, zDVR-1 and activin are all
expressed maternally in zebrafish, and drug inhibition studies indicate that embryos
develop normally when these maternal signals are blocked before the mid-blastula
transition (Hagos et al. 2007 ). Elimination of extraembryonic ndr1/sqt and ndr2/cyc
in maternal sqt/ndr1 mutants phenocopies ndr1/cyc-/-; ndr2/sqt-/- double mutants
(Hong et al. 2011 ). This indicates that maternal Activin-like signals and zygotic
Nodal signals are required to boost expression of nodal-related genes in the embryo,
acting in parallel to transcription factors in the YSL (Fig. 7.9b).
7.7.3 Conclusion
Taken together, these experiments show that the regulatory logic of nodal-related
gene expression is remarkably consistent. In all vertebrates, expression of Nodal
signals is maintained by a positive feedback loop, and their expression is induced by
other Activin-like signals acting in parallel to transcription factors. This feedback
loop acts through an Activin/Nodal Response Elements in the first introns of nodal
genes in mice, frogs, and fish. In both Xenopus and zebrafish, the Activin-like sig-
nals are provided maternally. In frogs, the maternally provided Activin and Vg1 act
in parallel to the T-box transcription factor, VegT transcription factor to induce xnr
expression (Fig. 7.9a). In zebrafish, maternal Ndr1/Sqt acts in parallel with the
zygotically expressed Mix/Bix transcription factor, Mxtx2, which is controlled by
the maternal T-box gene, Eomesa (Fig. 7.9b) (Bruce et al. 2005 ). The transcription
factors that control Nodal expression in mice and chicken have not been identified.
Another common feature of Nodal regulation is that signals from extraembry-
onic tissues induce Nodal expression in mice, chicken, and fish. In the mouse, nodal
expression is induced by signals from the extraembryonic ectoderm (Exe) (Brennan
et al. 2001 ). Nodal expression is partially dependent on the Vg1 orthologues, gdf1
and gdf3, but neither of these genes can be detected in the ExE at this stage
W. Tseng et al.