Distinct transcriptional regulation and phylogenetic divergence of human LEFTY genes Kenta Yashiro,1,2 Yukio Saijoh,1 Rui Sakuma,1 Masatomo Tada,3 Naohiro Tomita,3 Kenji Amano,4 Youichi Matsuda,4 Morito Monden,3 Shintaro Okada2 and Hiroshi Hamada1,* 1
Division of Molecular Biology, Institute for Molecular and Cellular Biology, Osaka University, and CREST, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan 2 Deparment of Paediatrics and 3Department of Surgery II, Osaka University Medical School, 1-3 Yamada-oka, Suita, Osaka 5650871, Japan 4 Laboratory of Animal Genetics, School of Agricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8601, Japan
Abstract Background: Mouse lefty1 and lefty2 genes are expressed on the left side of developing embryos and are required for left-right determination. Here we have studied expression and transcriptional regulatory mechanisms of human LEFTY genes. Results: The human LEFTY locus comprises two functional genes (LEFTY1 and LEFTY2) and a putative pseudogene. LEFTY1 is expressed in colon crypts. However, whereas LEFTY1 mRNA is present in basal cells of the crypts, LEFTY1 protein is localized in the apical region, suggesting that this secreted protein undergoes long-range transport. Human LEFTY2 possesses a left side-speci®c enhancer (ASE) like mouse lefty2; however, the
Introduction Determination of the left-right (L-R) axis in vertebrates is mediated by a series of regulatory genes that act during early stages of embryogenesis and is thought to consist of three distinct steps: (1) an initial event that establishes L-R asymmetry (2) L-R asymmetric expression of signalling molecules, and (3) L-R asymmetric morphogenesis induced by these signalling molecules (for reviews, King & Brown 1997; Levin & Mercola 1998; Varlet & Robertson 1997). The mechanism by which L-R symmetry is initially broken remains unknown, although several models have been proposed (Brown & Wolpert 1990; Brown et al. Communicated by: Shinichi Aizawa * Correspondence: E-mail:
[email protected] q Blackwell Science Limited
LEFTY2 ASE shows markedly higher activity in the ¯oor plate than does the lefty2 ASE. In contrast to mouse lefty1, which is expressed predominantly in the ¯oor plate under the control of a right sidespeci®c silencer, human LEFTY1 is expressed mainly in left lateral plate mesoderm under the control of an ASE-like left side-speci®c enhancer. The presence of FAST-binding sites in the LEFTY1 enhancer (and their absence in lefty1) contributes to the difference. Conclusion: These observations suggest that humans and mice have acquired distinct strategies during evolution for determining the asymmetric expression of LEFTY and lefty genes.
1991; Yost 1991). Several mouse mutants, including iv and inv animals (Hummel & Chapman 1959; Yokoyama et al. 1993), appear defective in the initial determination of L-R asymmetry. Although the iv and inv genes have been molecularly cloned (Suppl. et al. 1997; Mochizuki et al. 1998; Morgan et al. 1998), the precise functions of the encoded proteins remain unclear. Recent evidence suggests that leftward nodal ¯ow of extraembryonic ¯uid, generated by monocilia on the node pit cells, may result in a graded distribution of an unkown morphogen (Nonaka et al. 1998). Such nodal ¯ow is impaired in several situs-defective mutants, including both iv and inv mice (Okada et al. 1999). Asymmetrically expressed signalling molecules in mammals include three transforming growth factor b (TGFb)-related proteins (Nodal, Lefty1, and Lefty2), all of which are expressed in the left half of developing Genes to Cells (2000) 5, 343±357
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mouse embryos (Collignon et al. 1996; Lowe et al. 1996; Meno et al. 1996, 1997). Nodal is expressed in the lateral plate mesoderm (LPM) on the left side (Levin et al. 1995; Collignon et al. 1996; Lowe et al. 1996), and most likely acts as a `leftness' determinant (Levin et al. 1997; Sampath et al. 1997; Meno et al. 1998). The Lefty proteins are atypical members of the TGFb superfamily, in that they lack a cysteine residue required for dimer formation. The mouse lefty1 gene is expressed in the prospective ¯oor plate (PFP) on the left side, and genetic evidence suggests that Lefty1 restricts the expression of nodal and lefty2 to the left side, perhaps by acting as a midline barrier (Meno et al. 1998). The lefty2 gene is expressed predominantly in left LPM, and the recent characterization of lefty2 mutant mice has indicated that Lefty2 antagonizes Nodal signalling and restricts the range and duration of Nodal activity, perhaps by competing for a common receptor (Meno et al. 1999). Thus, Lefty2 probably inhibits the action of Nodal in left LPM and thereby precisely controls the duration and sites of activity of the leftness-determining factor. Situs-speci®c morphogenesis in response to L-R asymmetric signals is mediated by Pitx2, a bicoid-type homeobox-containing transcription factor (Logan et al. 1998; Meno et al. 1998; Piedra et al. 1998; Ryan et al. 1998; Yoshioka et al. 1998). Several genes that function in the pathway of L-R determination play roles that are evolutionarily conserved among vertebrates. In particular, nodal (Levin et al. 1995; Lowe et al. 1996; Sampath et al. 1997), lefty (Bisgrove et al. 1999; Thisse & Thisse 1999), and Pitx2 (Logan et al. 1998; Piedra et al. 1998; Ryan et al. 1998) genes show similar expression patterns and appear to perform similar functions in chick, Xenopus and zebra®sh. However, several differences among vertebrate systems have complicated our understanding of L-R axis determination. Thus, many genes that are expressed asymmetrically in one species show a different pattern of expression in other species. Such genes include those that encode Activin bB, Activin receptor IIA, Sonic hedgehog (Shh), ®broblast growth factor 8 (FGF8), and Nkx3.2 (Levin et al. 1995; Boettger et al. 1999; Meyers & Martin 1999; Rodoriguez-Esteban et al. 1999). Furthermore, some genes appear to play distinct (even opposite) roles in different vertebrates. Thus, Shh acts as a leftness determinant in the chick (Levin et al. 1995), whereas it is required for maintenance of the midline barrier in the mouse (Izraeli et al. 1999; Meyers & Martin 1999). In addition, FGF8 functions as a leftness determinant in the mouse (Meyers & Martin 1999) but it appears to act as a rightness determinant in the chick (Boettger et al. 1999). These discrepancies may re¯ect 344
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the morphological diversity apparent among vertebrates. Thus, although the positions of visceral organs are conserved in general, vertebrates exhibit various positional differences, such as the direction of aortic arch looping. Such differences are even apparent among mammals; for example, the lobation of the lung and the position of the azygous vein differ between human and mouse. To increase our understanding of the molecular basis of L-R morphological diversity, we have now examined the expression and transcriptional regulation of human LEFTY genes. Our data reveal marked differences between human LEFTY and mouse lefty genes in terms of both their expression domains and transcriptional regulation.
Results Chromosomal organization of the human LEFTY locus: a cluster of two functional genes and a putative pseudogene Screening of human genomic libraries with a mouse lefty cDNA probe yielded several overlapping clones (®ve BAC and ®ve phage clones) (Fig. 1A and 1B). Exons were mapped by hybridization with mouse lefty cDNA and then sequenced. Alignment of the clones indicated the presence of three genes that spanned a region of
