Determination of left/right asymmetric expression of nodal by a left side-specific enhancer with sequence similarity to a lefty-2 enhancer

Abstract

The nodal gene is expressed on the left side of developing mouse embryos and is implicated in left/right (L-R) axis formation. The transcriptional regulatory regions of nodal have now been investigated by transgenic analysis. A node-specific enhancer was detected in the upstream region (−9.5 to −8.7 kb) of the gene. Intron 1 was also shown to contain a left side-specific enhancer (ASE) that was able to direct transgene expression in the lateral plate mesoderm and prospective floor plate on the left side. A 3.5-kb region of nodal that contained ASE responded to mutations in iv, inv, and lefty-1, all genes that act upstream of nodal. The same 3.5-kb region also directed expression in the epiblast and visceral endoderm at earlier stages of development. Characterization of deletion constructs delineated ASE to a 340-bp region that was both essential and sufficient for asymmetric expression of nodal. Several sequence motifs were found to be conserved between the nodal ASE and the lefty-2 ASE, some of which appeared to be essential for nodal ASE activity. These results suggest that similar transcriptional mechanisms underlie the asymmetric expression of nodal and of lefty-2 as well as the earlier expression of nodal in the epiblast and endoderm.[Key Words: Left/right asymmetry; transcriptional regulation; nodal; lefty]Received April 17, 1999; revised version accepted May 10, 1999. Vertebrates exhibit numerous left/right (L-R) asymmetries such as the positioning of the heart and spleen on the left side of the body. Important insights into the molecular mechanisms that underlie L-R axis formation have been obtained only recently (Fujinaga 1997;King and Brown 1997;Levin and Mercola 1998;Beddington and Robertson 1999). The entire process by which L-R asymmetry is achieved can be divided into three phases: (1) initial determination of L-R polarity; (2) establishment of such polarity by the asymmetric expression of signaling molecules; and (3) L-R asymmetric morphogenesis induced by these signaling molecules.The mechanism of initial determination of L-R polarity remains unknown, although several models have been proposed (Brown and Wolpert 1990;Brown et al. 1991;Yost 1991). Several mutations that affect L-R patterning have been identified in mice (Hummel and Chapman 1959;Yokoyama et al. 1993;Heymer et al. 1997;Chen et al. 1998;Melloy et al. 1998;Nonaka et al. 1998) and in human (Gebbia et al. 1997). Two of the more extensively studied of these mutations are iv and inv, the corresponding genes of which encode axonemal dynein and a protein with ankyrin repeats, respectively (Supp et al. 1997;Mochizuki et al. 1998;Morgan et al. 1998). However, the precise functions of the proteins encoded by most of these affected genes (including Lrd, Inv, KIF3A, and HFH4) remain unclear. The initial determination of L-R polarity in mice probably occurs in or near the node, with recent observations suggesting an important role for ciliary movement in the node (Nonaka et al. 1998). In Xenopus, maternal Vg1 is implicated in the initiation of...

TGF-beta Family Signaling in Embryonic Stem Cells

“…Lefty locus contains two genes with the same transcriptional orientation in human, mice, and zebrafish. Human Lefty1 is identical to mice LeftyB and Lefty2 is identical to human LeftyA (10)(11)(12)(13). LeftyB has 96% sequence identity with LeftyA and these two proteins differ only in 16 amino acids.…”

NOTCH activation interferes with cell fate specification in the gastrulating mouse embryo

“…In agreement with its major role during early embryonic patterning, Nodal expression is extremely dynamic and tightly regulated by positive and negative inputs integrated on enhancer sequences. In the post-implantation epiblast at E5.5, Nodal expression is controlled by pluripotency factors, and notably by OCT4 and by an auto-regulatory loop involving NODAL signalling (Adachi et al, 1999;Norris et al, 2002;Norris and Robertson, 1999). In addition, WNT3/β-catenin signalling triggers Nodal upregulation in the E6.5 posterior epiblast and primitive streak (Ben-Haim et al, 2006;Vincent et al, 2003), and NOTCH/RBPJ signalling controls its expression in the node from E7.5 (Krebs et al, 2003;Raya et al, 2003).…”

“…In addition, WNT3/β-catenin signalling triggers Nodal upregulation in the E6.5 posterior epiblast and primitive streak (Ben-Haim et al, 2006;Vincent et al, 2003), and NOTCH/RBPJ signalling controls its expression in the node from E7.5 (Krebs et al, 2003;Raya et al, 2003). The ectopic anterior expression of Nodal observed in E7.5 N1ICD epi embryos might result from a direct action of N1ICD/ RBPJ on the Nodal node-specific enhancer (NDE) (Adachi et al, 1999;Krebs et al, 2003;Norris and Robertson, 1999; Raya et al, 2003). This direct regulation of Nodal by NOTCH signalling is also at play in pathological situations, as the NDE enhancer is involved in Nodal upregulation in response to elevated NOTCH signalling in melanoma and breast cancer cell lines (Hardy et al, 2010;Quail et al, 2011).…”

“…However, we could not detect such an effect in N1ICD epi embryos. Alternatively, NOTCH activation could interfere with the auto-regulatory loop that controls Nodal expression in the early epiblast (Adachi et al, 1999;Norris et al, 2002;Norris and Robertson, 1999). NICD has been shown to antagonise TGFβ signalling in various cell types via direct binding to SMAD3 or its co-activators (Carlson et al, 2008;Masuda et al, 2005;Sun et al, 2005).…”

“…S2). The maintenance of Nodal expression in the epiblast, the primitive streak and the VE depends both on an autoregulatory loop involving NODAL signalling through its co-receptor CRIPTO (TDGF1), on the transcription factor OCT4, and on the WNT3/β-catenin pathway (Adachi et al, 1999;Ben-Haim et al, 2006;Granier et al, 2011; 2002; Norris and Robertson, 1999;Papanayotou et al, 2014). Oct4, Cripto and Wnt3 were all normally expressed in E6.5 N1ICD epi embryos (n=3, n=5 and n=3, respectively; Fig.…”