Nodal signals mediate interactions between the extra-embryonic and embryonic tissues in zebrafish

Abstract

In many vertebrates, extra-embryonic tissues are important signaling centers that induce and pattern the germ layers. In teleosts, the mechanism by which the extra-embryonic yolk syncytial layer (YSL) patterns the embryo is not understood. Although the Nodal-related protein Squint is expressed in the YSL, its role in this tissue is not known. We generated a series of stable transgenic lines with GFP under the control of squint genomic sequences. In all species, nodal-related genes induce their own expression through a positive feedback loop. We show that two tissue specific enhancers in the zebrafish squint gene mediate the response to Nodal signals. Expression in the blastomeres depends upon a conserved Nodal response element (NRE) in the squint first intron, while expression in the extra-embryonic enveloping layer (EVL) is mediated by an element upstream of the transcription start site. Targeted depletion experiments demonstrate that the zebrafish Nodal-related proteins Squint and Cyclops are required in the YSL for endoderm and head mesoderm formation. Thus, Nodal signals mediate interactions between embryonic and extra-embryonic tissues in zebrafish that maintain nodal-related gene expression in the margin. Our results demonstrate a high degree of functional conservation between the extra-embryonic tissues of mouse and zebrafish.

The evolutionary origin of nodal-related genes in teleosts

“…The level and distribution of fluorescence is comparable to that observed in embryos injected with a construct containing 9.9 kb of zebrafish sqt genomic sequences driving GFP (88%, N=36; Fig. 3a; Fan et al 2007). This demonstrates that the zebrafish transcription factors that control sqt expression can recognize enhancers in the Tetraodon gene, despite the great evolutionary distance.…”

Transcriptional profiling of endogenous germ layer precursor cells identifies dusp4 as an essential gene in zebrafish endoderm specification

“…Transgenic zebrafish lines with mesoderm-, endoderm-, or ectoderm-restricted fluorescent protein expression have been reported (11,12), but at the late-blastula stage, none of these are bright enough for FACS-based purification (J.L.B. and B.F., unpublished observations).…”

Biphasic wnt8a expression is achieved through interactions of multiple regulatory inputs

“…The YSL is an extra-embryonic tissue that is important for both mesoderm induction and gastrulation (Carvalho and Heisenberg, 2010), and several studies have highlighted the role of YSL factors in mediating the patterning activities of this tissue (Ober and Schulte-Merker, 1999;Fan et al, 2007;Hong et al, 2011). The YSL enhancer we identified functions independently of the margin enhancer, raising the question of what role Wnt8a plays in the YSL.…”

“…Cis-regulatory analysis of nodal related 1 (ndr1, aka squint) showed its expression to be regulated by two enhancers: an intronic enhancer comprising a Nodal response element (NRE) that drives expression in marginal blastomeres, and an upstream enhancer that drives expression in the extra-embryonic enveloping layer and YSL (Fan et al, 2007). The NRE contains two putative FoxH1-binding sites that are required for the Nodal response and expression in marginal blastomeres (Osada et al, 2000;Fan et al, 2007). FoxH1 is a key effector of Nodal signaling and mediates most early Nodal signaling in zebrafish (Osada et al, 2000;Pogoda et al, 2000).…”

“…The FoxH1 consensus binding site is highly conserved across taxa, and several studies have shown that FoxH1 consensus binding sites in diverse genes mediate a Nodal response (e.g., Labbe et al, 1998;Zhou et al, 1998). Interestingly, ndr1 expression driven by the Nodal-responsive intron enhancer initiates shortly after the midblastula transition and declines in the margin by 6 hpf (Fan et al, 2007). Thus, ndr1 expression in marginal blastomeres is governed by a transient autoregulatory mechanism that is active during Nodal-dependent phase I wnt8a expression.…”