Abstract
The vertebrate organizer and notochord have conserved, essential functions for embryonic development and patterning. The restricted expression of developmental regulators in these tissues is directed by specific cis-regulatory modules (CRMs) whose sequence conservation varies considerably. Some CRMs have been conserved throughout vertebrates and likely represent ancestral regulatory networks, while others have diverged beyond recognition but still function over a wide evolutionary range. Here we identify and characterize a mammalian-specific CRM required for node and notochord specific (NNC) expression of NOTO, a transcription factor essential for node morphogenesis, nodal cilia movement and establishment of laterality in mouse. A 523 bp enhancer region (NOCE) upstream the Noto promoter was necessary and sufficient for NNC expression from the endogenous Noto locus. Three subregions in NOCE together mediated full activity in vivo. Binding sites for known transcription factors in NOCE were functional in vitro but dispensable for NOCE activity in vivo. A FOXA2 site in combination with a novel motif was necessary for NOCE activity in vivo. Strikingly, syntenic regions in non-mammalian vertebrates showed no recognizable sequence similarities. In contrast to its activity in mouse NOCE did not drive NNC expression in transgenic fish. NOCE represents a novel, mammal-specific CRM required for the highly restricted Noto expression in the node and nascent notochord and thus regulates normal node development and function.
Highlights
The organizer of vertebrate embryos is essential for early embryonic patterning
To delineate the region(s) directing the highly restricted node and notochord specific (NNC) expression of Noto we analyzed the expression of reporter transgenes in chimeric embryos obtained with ES cells carrying reporter constructs in their genome
To minimize effects of copy number and integration sites on reporter gene expression we introduced by homologous recombination single-copy reporter constructs in the same orientation upstream of the Hprt gene (Figure 1; see Mat and Methods for details)
Summary
The organizer of vertebrate embryos is essential for early embryonic patterning (reviewed in [1]). The zebrafish Not gene floating head (flh) and its mouse orthologue Noto are pivotal for normal development and function of the organizer and notochord in both species [9,12]. Noto is essential for morphogenesis of the node, which constitutes the mouse late organizer (reviewed in [5]), for posterior localization of cilia on node cells and nodal ciliogenesis, and for the establishment of laterality [13,16]. Flh function in zebrafish embryos is required for the formation of normal Kupffer’s vesicle [14], a transient structure that functions in the establishment of left-right asymmetry equivalent to the mammalian posterior notochord [17,18,19,20]
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