Role of asymmetric signals in left–right patterning in the mouse

INTRODUCTION

Generation of morphological asymmetries is fundamental for embryonic patterning. Studies of left–right (L-R) asymmetry in vertebrates have provided insight into how symmetry is initially broken and how asymmetric structures are eventually generated [Brown and Wolpert, 1990; Brown et al., 1991; Yost, 1991; Fujinaga, 1997]. In particular, much progress has been made in recent years in characterization of the molecular mechanisms by which L-R asymmetry is established in vertebrates [Harvey, 1998; Levin and Mercola, 1998; Beddington and Robertson, 1999; Capdevila et al., 2000]. This entire process can be divided into four phases: (1) initial determination of L-R polarity in or near the node, (2) transfer of asymmetric signals from the node to the lateral plate mesoderm (LPM), (3) asymmetric expression of signaling molecules such as Nodal and Lefty in the left LPM, and (4) L-R asymmetric morphogenesis induced by these signaling molecules. Although differences in the expression patterns (and functions) of certain regulatory genes are apparent among vertebrates, the central mechanism for determination of L-R asymmetry has been widely conserved. Thus, nodal, lefty, and Pitx2 exhibit similar expression patterns among vertebrates and appear to play conserved roles in this process. Here we will describe the role of nodal and lefty genes and their transcriptional regulation in the mouse.

GENERATION OF LEFTY MUTANT MICE

Genomic lefty1 clones were isolated from a genomic library constructed from E14 ES cells. A targeting vector was constructed by subcloning the 5′-flanking region (the Sal I-Sac I 3-kb fragment), a STneo cassette from pSTneo ( the Sac I-Xba I fragment) and the 3′-flanking region (the Xba I-Sal I 9-kb fragment) in Bluescript. Gene targeting was performed as described previously [Sawai et al., 1991]. The targeting vector was linearized with Not I before electroporation into E14 ES cells. Among 134 G418-resistant ES clones examined, 3 (2.2%) were found to have undergone homologous recombination, as demonstrated by Southern blot analysis with probes specific for the 5′-flanking region, the 3′-flanking region and the neo gene. Two targeted ES cell lines were separately injected into blastocysts obtained from the mating of C57BL/6Cr mice with (C57BL/6Cr x C3H) F1 mice, resulting in the birth of chimeric animals. Male chimeras derived from each ES cell line were bred with C57BL/6C females, yielding heterozygous F1 offspring. The F1 heterozygotes were mated with each other, producing lefty1^−/− homozygotes. The lefty1^−/− homozygotes derived from each of the two targeted ES cell lines showed indistinguishable phenotypes. All analyses were performed on a mixed C57BL/129 background unless otherwise indicated.

TRANSIENT TRANSGENIC ASSAY

Transgenic mice were generated by pronuclear injection of lacZ fragments (4 ng/ml) into fertilized eggs obtained from intercrosses between (C57BL/6 X C3H)F1 mice [Hogan et al., 1994; Sasaki and Hogan, 1996]. The injected embryos were transferred into pseudopregnant recipients, and allowed to develop in utero until E8.2. Because of the narrow window of lefty expression, it was necessary to recover the embryos at a stage between three and eight somite pairs, for which the recipient mice were anesthetized with pentobarbital sodium (Nembutal), and the stage of embryos was estimated by the size of the decidua. The E8.2 embryos were examined for the presence of the transgene [by the polymerase chain reaction] and for lacZ expression (by X-Gal staining). The activity of b-galactosidase in dissected embryos was detected by a standard protocol. For each construct, more than 120 embryos were microinjected and transferred, and 10 or more transgene-positive embryos were examined by X-Gal staining. The amount of β-galactosidase activity was estimated from the extent of X-Gal staining after incubating in the staining buffer for 2, 8, and 24 hr. Primers used to amplify the lacZ sequence were 5′-TTGCCGTCTGAATTTGACCTG-3′ and 5′ TCTGCTTCAATCAGCGTGCC-3′ [Sasaki and Hogan, 1996].

ROLE OF NODAL AND LEFTY IN L-R DETERMINATION

Asymmetric signaling molecules in mammals include three transforming growth factor β (TGFβ)-related factors—Nodal, Lefty1, and Lefty2—all of which are expressed on the left half of developing mouse embryos [Collignon et al., 1996; Lowe et al., 1996; Meno et al., 1996, 1997]. Nodal is a typical member of the TGFβ superfamily that seems to transduce signals through its receptor (not established yet but most likely ActRIs and ActRIIs), an EGF-CFC protein (as a cofactor), and transcription factors Smad2, Smad4, and FAST2 [Schier and Shen, 1999; Saijoh et al., 2000]. Lefty1 and Lefty2 comprise a unique subgroup of the TGFβ superfamily that act as antagonists against Nodal, being able to bind to receptors but unable to transduce signals.

Nodal is initially expressed throughout the epiblast and primitive endoderm at pregastrulation stages. At the early somite stage, it is expressed in the lateral plate and in the node. Although its expression in the lateral plate is exclusively on the left side, the expression in the node shows subtle asymmetry (expression on the left side of the node is stronger and wider than that on the right side). The expression in left LPM is highly transient, and is no longer detectable by the eight-somite stage. The L-R asymmetric expression pattern of nodal is conserved among vertebrates [Levin et al., 1995; Collignon et al., 1996; Lowe et al., 1996]. Several lines of evidence suggest that Nodal is a determinant for “leftness” [Levin et al., 1997]. Perhaps the role of Nodal is to instruct mesoderm cells in the left LPM (mainly splanchnopleure and somatopleure) by inducing the expression of genes such as Pitx2, and to adopt left-side morphology at later stages.

Lefty1 and Lefty2 are atypical TGFβ-related factors lacking the cysteine residue required for dimer formation. Although both genes are expressed in a L-R asymmetric manner on embryonic day 8.0 (E8.0), their expression patterns differ: lefty1 is expressed predominantly in the left half of the prospective floor plate (PFP), whereas lefty2 is strongly expressed on the left side of the LPM. To examine the role of lefty1 in L-R asymmetry, we generated mutant mice deficient in lefty1 and analyzed their phenotype. The lefty1^−/− mice showed various L-R positional defects, the most common of which was thoracic left isomerism associated with malpositioning of the cardiac outflow tracts, the inferior vena cava, and the azygos vein. In the absence of Lefty1, nodal and lefty2 were bilaterally expressed in the LPM. Therefore, the role of Lefty1 is to serve as a midline barrier to prevent the diffusion of a molecule that regulates expression of nodal and lefty2. However, the exact action of Lefty1 remains to be clarified.

Lefty2 is also expressed in the left LPM. Lefty2 expression begins in newly formed mesoderm at the early primitive streak stage. This expression domain disappears by the end of the neural fold stage, but a new expression domain appears in the left LPM at the 2/3 somite stage. The left-sided expression of lefty2, like that of nodal, is very transient. Recent analysis of lefty2 mutant mice indicates that Lefty2 antagonizes Nodal signaling and restricts the range and duration of Nodal activity during gastrulation, perhaps by competing for a common receptor [Meno et al., 1999]. Direct evidence for the role of Lefty2 in the L-R axis formation is not available because lefty2 null mutants die before L-R defects can be judged [Meno et al., 1999]. However, it is conceivable that the role of Lefty2 in the left LPM also the duration and site of the action of Nodal, a leftness-inducing signal. Lefty1 is not a determinant for “leftness,” but rather is a regulator that restricts the expression of nodal and lefty2 to the left side, possibly by acting as a midline barrier [Meno et al., 1998].

TRANSCRIPTIONAL REGULATION OF LEFTY GENES

We examined the transcriptional activity of upstream regions of lefty1 and lefty2 [Saijoh et al., 1999]. For lefty1, the 9.5-kb upstream region containing the TATA box was linked to lacZ, yielding L1-9.5. For lefty2, the 5.5-kb upstream region including the TATA box was fused to lacZ, yielding L2-5.5. L1-9.5 and L2-5.5 were injected separately into the pronuclei of fertilized embryos at the one-cell stage. The embryos were allowed to develop in utero until embryonic day 8.2 (E8.2), when they were recovered and the expression of the lacZ transgene was examined by staining with X-Gal (5-bromo-4-chloro-3-indoyl-b-D-galactoside). L1-9.5 and L2-5.5 were able to recapitulate the expression patterns of lefty1 and lefty2, respectively. These results also indicate that lefty1 and lefty2 are regulated independently, even though they are tightly linked on the same chromosome.

To locate cis-elements responsible for the asymmetric expression of lefty2, we tested two sets of deletion mutants from L2-5.5. The results indicated the presence of a left side–specific enhancer referred to as ASE (asymmetric enhancer), in a 380-bp region between −4.1 and −3.7 kb. Internal deletion of the 380-bp region containing ASE from L2-5.5 abolished asymmetric expression. The ASE-lef2p construct, in which the 380-bp ASE fragment is linked to the minimal promoter region of lefty2 (from −300 to +90 bp), yielded left-sided X-Gal staining in left LPM and PFP. Finally, constructs in which the 380-bp fragment was linked to the hsp68 promoter instead of the lefty2 promoter gave rise to X-Gal staining in left LPM and left PFP. These results demonstrate that ASE is essential and sufficient for the asymmetric expression of lefty2.

To localize the transcriptional regulatory elements of lefty1, we tested various restriction fragments derived from the 9.5-kb upstream region for the ability to confer asymmetric expression. Two enhancers were found in the upstream region, but they were bilateral enhancers. L-R specificity was determined not by these enhancers but by a silencer (right side–specific silencer) located in the proximal promoter region that can repress transcription on the right side when combined with the bilateral enhancers.

POSITIVE AND NEGATIVE REGULATORY LOOPS BETWEEN lefty AND nodal

Both nodal and lefty2 are coexpressed in the left LPM at the early somite stage. Then, what would the regulatory relationship between the two genes? Are they regulated independently or similarly, or is one of them regulating the other? Studies on transcriptional regulatory mechanisms of nodal and lefty2 has demonstrated the presence of complex yet beautiful regulatory loops between the two genes. The L-R asymmetric expression of nodal and lefty2 is controlled by a left side–specific enhancer, ASE [Adachi et al., 1999; Norris et al., 1999; Saijoh et al., 1999; Osada et al., 2000]. The transcription factor FAST2, which can mediate signaling by TGFβ and activin [Whitman, 1998], has been identified as a protein that binds to a conserved sequence (AATCCACAT) in the ASE elements of mouse lefty2 and nodal [Saijoh et al., 2000]. These FAST2 binding sites were shown to be both essential and sufficient for L-R asymmetric gene expression in mouse embryos. The Fast2 gene is bilaterally expressed in mouse embryos at a time when nodal and lefty2 are expressed on the left side. TGFβ and activin can activate the ASE activity in a FAST2-dependent manner, whereas Nodal can do so in the presence of an EGF-CFC protein. These results suggest that the asymmetric expression of lefty2 and nodal in mouse embryos is mediated by FAST2 and is induced by a left side–specific TGFβ-related factor, which is most likely Nodal itself.

On the basis of these observations, we propose the following scenario for the asymmetric expression of nodal and lefty2: The left-sided expression of nodal is initially induced by an unknown factor (step 1). The Nodal protein thereby produced autoregulates the nodal gene and activates lefty2 expression in the left LPM (step 2). Components of the Nodal signaling pathway, including an EGF-CFC protein (most likely Cryptic), FAST2, and Smad2-Smad4 would therefore all be required for the maintenance of nodal expression and for induction of lefty2 expression. Thus, Lefty2 produced as a result of Nodal signaling likely antagonizes Nodal action (step 3), thereby rapidly terminating the left-sided expression of nodal and lefty2. This feedback mechanism would restrict the range and duration of Nodal signaling in developing embryos.

The relationship between Nodal and Lefty2 resembles that of two hypothetical molecules that comprise “reaction-diffusion mechanism” proposed by Turing [1952]. The nodal-lefty2 connection may be the first example of the reaction-diffusion mechanisms that exist in developing organisms. The regulatory loops between nodal and lefty may be involved not only in embryonic patterning such as mesoderm induction and L-R (and possibly A-P) determination, but also in generating periodic patterns during organogenesis.