Our studies have established a role for Nanog-like during zebrafish YSL development. We propose a model in which Nanog-like regulates endoderm formation through an Mxtx2-Nodal pathway. During the early blastula stage (2.5 hpf), marginal blastomere cells collapse to form the YSL precursor. In the YSL precursor, Nanog-like directly activates Mxtx2, which in turn specifies the YSL lineage by directly activating the expression of YSL genes. During the late blastula stage, the YSL functions as a signaling center producing Nodal molecules for ventrolateral endoderm induction. The dorsal endoderm is induced by stabilized β-catenin-activated Nodal independent of Nanog-like ().
The pluripotency network described in embryonic stem cells has not been studied in the developmental ICM or in the epiblast. Zebrafish produce a large number of embryos, providing us with an opportunity to access the in vivo network. One important element in succeeding in a ChIP-Seq experiment is having a reliable antibody for the transcription factor of interest. To overcome the lack of antibodies in zebrafish studies, we developed a technique allowing us to perform ChIP-Seq analysis utilizing embryos expressing Myc tagged transcription factors. Using this technique, we examined the Nanog-like and Mxtx2 gene regulatory network in blastula stage zebrafish embryos. We found that Nanog-like binds to known pluripotency genes like pou5f1, sox2, and nanog-like. We also found that Nanog-like bound to genes involved in extraembryonic lineage differentiation, like gata3 and krt4 for EVL differentiation, and mxtx2 and slc26a1 for YSL differentiation, mesoderm specification like ntl and tbx3, cell movement like wnt11 and cxcr4b, and signaling genes like ndr1, bmp2b, fgf8a and wnt8a. The binding profile suggests that Nanog-like may play a versatile role involving many developmental processes. Another intriguing finding is that Nanog-like and Mxtx2 bound to nanog-like and mxtx2 loci, suggesting potential cross-regulation and auto-regulation loops between these two genes. This observation prompted us to test if Nanog-like could rescue mxtx2 morphants. By overexpressing Nanog-like, we were not able to rescue the YSL and endoderm defects in mxtx2 morphants (data not shown). This is consistent with our findings that Mxtx2 functions downstream of Nanog-like.
Mxtx2 is critical for YSL induction. The expression of mxtx2 is highly regulated, suggested by the fact that both the high stage Nanog-like and Mxtx2 binding sites at the mxtx2 locus are the strongest across the entire genome. Nanog-like is ubiquitously expressed in all blastomere cells and its overexpression does not induce ectopic mxtx2 expression, suggesting that it is not responsible for the spatial expression of mxtx2. The spatial restriction of mxtx2 expression to the YSL may be a result of the formation of the YSL structure through collapsing of marginal cells into the yolk. One possibility is that one signaling pathway is activated in the YSL by this event, which in turn activates the expression of mxtx2 in the YSL.
The YSL is unique to teleosts, and has been considered an equivalent of the mouse primitive endoderm. Interestingly, Nanog is required for primitive endoderm formation, but with a distinct mechanism. Recent studies found that Nanog regulates primitive endoderm formation through a non-cell autonomous mechanism (Frankenberg et al., 2011
; Messerschmidt and Kemler, 2010
). Our study suggests that in zebrafish Nanog-like regulates YSL formation through a cell-autonomous mechanism. While the YSL and primitive endoderm share similar gene expression and both function as the signaling center that patterns the head mesoderm and endoderm, the regulation mechanism involving their formation is different. The FGF signal that is required for mouse primitive endoderm induction seems not to be involved in YSL formation (Chazaud et al., 2006
; Yamanaka et al., 2010
). Moreover, Gata4 and Gata6, key transcription factors in regulating primitive endoderm formation, are dispensable for YSL formation (Holtzinger and Evans, 2007
; Koutsourakis et al., 1999
; Peterkin et al., 2007
Compelling evidence suggests that pluripotency factors have distinct roles in mammals and teleosts. In zebrafish, the acquisition of pluripotency by deep cells (the mouse epiblast equivalent) is independent of Nanog-like, as nanog-like
-deficient deep cells can readily differentiate into the three germ layers ( and data not shown). It should be noted that maternally deposited Nanog-like protein cannot be eliminated by the morpholino knockdown approach. A similar observation was made in the maternal-zygotic (MZ) pou5f1
mutant. Pou5f1 is required for the formation of pluripotent ICM in the mouse blastocyst (Nichols et al., 1998
), but the zebrafish MZ pou5f1
mutant, lacking both maternal and zygotic expression, was still capable of differentiating into three germ layers (Lunde et al., 2004
; Reim et al., 2004
). Despite Schuff et al
recently showed that zebrafish Nanog-like prevents murine embryoid body (EB) differentiation (Schuff et al., 2011
), our data reveals that overexpression of zebrafish Nanog-like does not rescue LIF dependence in murine ES cells (Figure S1D, S1E, and S1F
). Certain structural domains may be responsible, as zebrafish pou5f1
does not rescue mouse Pou5f1
mutant ES cells (Morrison and Brickman, 2006
). The conservation of Nanog-like and Pou5f1 is supported by the fact that both zebrafish mutants can be rescued by their mammalian counterparts ()(Onichtchouk et al., 2010
). However, Nanog-like’s role in YSL differentiation and Pou5f1’s role in endoderm differentiation do not seem to be conserved in mammals ()(Lunde et al., 2004
; Reim et al., 2004
). We speculate that the ancestors of pluripotency regulators adopted distinct functions during teleost evolution.
Our work reveals a role for Nanog-like in regulating the formation of the extra-embryonic lineage, which later secretes Nodal signals to break down the pluripotency of the deep cell. Our studies suggest that the pluripotency network may have distinct roles on germ layer formation in vivo.