Department of Molecular and Cellular Biology, Harvard University, The Biological Laboratories, Cambridge, MA 02138, USA
* Present address: Medical Research Council Mammalian Genetics Unit, Harwell OX11 0RD, UK
Author for correspondence (e-mail: ejrobert{at}fas.harvard.edu)
Accepted 12 April 2002
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SUMMARY |
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Key words: Mouse, Nodal, Axis specification, Foxh1
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INTRODUCTION |
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Nodal signals via the Alk4 or Alk7 type I receptor in association with either the ActRIIA or ActRIIB type II receptor (reviewed by Whitman, 2001). The activated receptor complex phosphorylates the intracellular molecules Smad2 and Smad3, which in turn associate with Smad4 (Massague et al., 2000
) and translocate to the nucleus. Cooperatively with other DNA-binding proteins, Smad proteins regulate transcription of target genes (reviewed by Wotton and Massague, 2001
). Studies of activin responsive promoters in Xenopus have identified two Smad2/4-interacting DNA partners. The forkhead domain protein Foxh1 (formerly known as FAST1/2) binds as part of a complex to the activin responsive element of the Mix2 gene (Chen et al., 1996
; Chen et al., 1997
). Similarly members of the Mix family of homeodomain proteins, in association with Smad2/4, activate the Gsc promoter (Germain et al., 2000
). Loss of Foxh1 function in mouse results in a range of developmental defects broadly consistent with a role in mediating Nodal signals (Hoodless et al., 2001
; Yamamoto et al., 2001
). Foxh1 is not required for mesoderm formation, but mutant embryos show AP axis patterning defects, as well as abnormal development of the node and definitive endoderm, both of which are derivatives of the anterior primitive streak. Similarly, zebrafish schmalspur (Foxh1 homolog) loss-of-function mutant embryos lack axial mesendoderm (Boggetti et al., 2000
; Pogoda et al., 2000
; Sirotkin et al., 2000
). These defects are considerably less severe than those resulting from loss of Nodal function (Zhou et al., 1993
; Conlon et al., 1994
), suggesting that Nodal activates target genes required for initial axis specification and mesoderm formation via Foxh1-independent pathways.
Nodal expression is tightly regulated in a highly dynamic fashion at discrete tissue sites during early mouse development (Collignon et al., 1996; Varlet et al., 1997
). Nodal mRNA is first detected throughout the epiblast and overlying visceral endoderm (VE). As development proceeds, Nodal is confined to the prospective posterior epiblast, marking the site of primitive streak formation. Nodal is lost from the VE after the onset of gastrulation, and becomes progressively down-regulated in the primitive streak. In the node, Nodal transcripts appear in a discrete population of cells located on the edges of the ventrally located notochordal plate, and become more strongly expressed on the left. At later stages Nodal is induced in a broad stripe of left lateral plate mesoderm (LPM). Mutations that disturb asymmetric Nodal expression inevitably cause defective LR axis patterning (Collignon et al., 1996
; Lowe et al., 1996
). Similarly, Nodal mis-expression in chick, fish or Xenopus embryos reverses the orientation of the LR body axis (reviewed by Capdevila et al., 2000
).
Transgenic approaches have been used to map cis-regulatory elements responsible for spatially and temporally restricted Nodal expression patterns (Adachi et al., 1999; Norris and Robertson, 1999
; Brennan et al., 2001
) (D. P. N. and E. J. R., unpublished). An intronic enhancer, termed the ASE, controls early expression in the epiblast and VE, and also at later stages on the left side of the embryo. The minimal ASE enhancer region contains two Foxh1-binding sites (Saijoh et al., 2000
). Similar Foxh1-dependent regulatory elements control asymmetric Lefty2 (Leftb Mouse Genome Informatics) and Pitx2 expression (Saijoh et al., 1999
; Shiratori et al., 2001
). Moreover, Foxh1-dependent cis-acting regulatory elements have been mapped within the Nodal locus of all vertebrates examined (reviewed by Whitman, 2001
). We have used gene targeting techniques to delete the ASE and test its role in axial patterning. The developmental abnormalities seen in trans-heterozygous embryos carrying the ASE deletion and a null allele (Nodal
600/), and homozygous mutant embryos (Nodal
600/
600) were compared. In Nodal
600/ embryos, Nodal is expressed at low levels in the early epiblast and becomes confined to proximal epiblast cells; however, expression is undetectable in the VE. Reduced Nodal expression levels are sufficient for formation of the anterior visceral endoderm (AVE) and mesoderm induction. Many of these embryos, however, incorrectly position the AP axis, leading to abnormal gastrulation and cell movements. Moreover, Nodal
600/ embryos lack anterior definitive endoderm (ADE) and display rostral CNS patterning defects. By contrast, in homozygous Nodal
600/
600 embryos, Nodal is more efficiently expressed in the epiblast but absent from the VE. Unexpectedly, these embryos develop normal AP pattern. Later, at early somite stages, Nodal expression in the LPM is significantly reduced, leading to failure to activate Lefty2, delayed activation of Pitx2 and defective LR axis patterning. Overall, we conclude the Foxh1-dependent autoregulatory enhancer maintains and amplifies Nodal signals that selectively activate target genes responsible for patterning the embryonic body plan.
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MATERIALS AND METHODS |
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Mouse breeding and genotyping
NodalLacZ, Nodal100.lacZ, Nodal
600.LacZ and transgenic lines expressing lacZ were PCR genotyped for the presence of the lacZ gene.
600 mice were genotyped by PCR using
600-5 (5' GCT AGT GGC GCG ATC GGA ATG GA 3') and
600-6 (5' AAG GGA AGT GAA CTG GAA AGG TAT GT 3'): a 350 bp fragment shows presence of the deletion; a 950 bp fragment shows the wild-type allele. Foxh1 mutant mice were a kind gift from Jeff Wrana and Pamela Hoodless, and were genotyped according to published protocols (Hoodless et al., 2001
). All strains used in this analysis were maintained by breeding to ICR mice (Taconic).
WISH and histology
Whole-mount in situ hybridization was performed according to standard procedures. Probes for the following genes were used in this study: cardiac actin (Roebroek et al., 1998
), Bmp4 (Winnier et al., 1995
), cripto (Ding et al., 1998
), Eomes (Russ et al., 2000
), Foxa2 (previously known as Hnf3ß) (Sasaki and Hogan, 1996
), Gsc (Blum et al., 1992
), Hex (Thomas et al., 1998
), Lefty (Ebaf Mouse Genome Informatics) (Meno et al., 1996
), Lhx1 (Barnes et al., 1994
), Nodal (Conlon et al., 1994
), Otx2 (Ang et al., 1994
), Pitx2 (Ryan et al., 1998
), Shh (Echelard et al., 1993
), T (Herrmann, 1991
) and Wnt3 (Liu et al., 1999
). For histology embryos were fixed in 4% paraformaldehyde (PFA), dehydrated through an ethanol series and embedded in wax before sectioning. Hematoxylin and Eosin staining and X-gal staining were performed according to standard protocols. India ink was injected into the left ventricle of embryos at 14.5 dpc and 18.5 dpc, and allowed to fill the ventricle. Whether or not the ink moved directly into the right ventricle was scored visually.
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RESULTS |
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Failure to specify anterior definitive endoderm in Nodal600/ embryos
While only 40% of Nodal600/ embryos exhibit overt morphological defects at early gastrulation stages, by 7.5 dpc, all Nodal
600/ embryos are distinctly abnormal (Table 1). As mentioned above, approximately 40% exhibit a visible constriction at the embryonic/extra-embryonic boundary at 6.5 dpc, which varies in severity ranging from a slightly noticeable narrowing to almost complete separation of these regions (Fig. 4A,B; summarized in Table 1). Slightly later at 8.5 dpc, this class of most severely disturbed embryos develop external to the visceral yolk sac (VYS) (Fig. 4E). In some cases, the anterior regions are partially externalized, while the primitive streak is retained within the VYS (Fig. 4D). Histological analysis demonstrates the presence of neural tissue (Fig. 4D,E), but midline tissues are largely absent resulting in fused somites (data not shown). This is likely to be due to defective gastrulation movements that secondarily lead to abnormal node morphogenesis and failure to form a notochord. Cells expressing the myocardial marker
cardiac actin are juxtaposed to the VYS in severely affected embryos (data not shown), suggesting that cardiac precursors are specified normally.
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AP and LR patterning in Nodal600/
600 embryos
As shown in Fig. 5, in contrast to Nodal600/ embryos, we found that Nodal
600/
600 embryos efficiently undergo AVE rotation, display a correctly positioned primitive streak, and gastrulate normally. In comparison with Nodal
600/ embryos, Nodal expression within the epiblast is elevated roughly twofold in Nodal
600/
600 homozygous mutants. This increased dose is sufficient to rescue ADE formation and axis patterning defects. Nodal mRNA is expressed at significantly reduced levels in the epiblast, and is undetectable in the VE (Fig. 5A-D). Thus, Nodal expression within the VE is not essential for either specification or positioning of the AVE. Rather, these processes depend on the strength of Nodal signals within the epiblast. At 7.5 and 8.5 dpc, expression within the node closely resembles that in wild type (Fig. 5E,F), except that asymmetry of expression is lost. Later, at early somite stages, Nodal
600/
600 embryos display only weak Nodal expression in LPM (Fig. 5J-L), although invariably on the left side of the axis.
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To analyze embryonic turning and establishment of organ situs, Nodal600/
600 embryos were allowed to develop beyond 8.5 dpc. At early somite stages, none of a large panel of mutant embryos showed morphological defects (Table 1). However, by 9.0 dpc, heart looping is visibly abnormal in Nodal
600/
600 embryos, with the heart positioned more ventrally than in wild type (Fig. 7A,B). The heart tubes appear to fold normally, suggesting they have acquired regional patterning. Moreover, heart contractions and blood circulation develop normally (data not shown). By 11.5 dpc, mutant hearts display an ambiguously positioned apex (Fig. 7C). In addition, the lungs of mutant embryos display lobation defects (Fig. 7C; Table 1). Differences in branching morphogenesis normally leads to the formation of four lobes on the right and a single lobe on the left side of the thoracic cavity. By contrast, Nodal
600/
600 embryos develop partial right isomerisms (Fig. 7C).
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DISCUSSION |
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Nodal expression in visceral endoderm is not required for anterior patterning
Embryos that lack the ASE fail to express Nodal mRNA in the VE. Thus, activation of the Nodal locus in the VE is strictly controlled by the Smad2/Foxh1-dependent pathway. However, markers of the AVE are activated normally in Nodal600/ embryos. Thus, Nodal signals within the VE are nonessential for early anterior patterning. Consistent with this, embryos deficient in Foxh1, and therefore lacking Nodal activity in the VE, also establish a normal AVE (Hoodless et al., 2001
; Yamamoto et al., 2001
). An interesting feature of Nodal
600/ embryos is that they develop a severe physical constriction at the extra-embryonic/embryonic boundary that subsequently leads to externalization and growth of the embryo outside of the yolk sac. Similar defects have been described for Foxa2 and Lhx1 mutants (Ang and Rossant, 1994
; Shawlot and Behringer, 1995
). In the case of Foxa2, this probably reflects a requirement for Foxa2 within the VE, as the defect is not seen in chimeric embryos in which the VE is wild type (Dufort et al., 1998
). Loss of Foxh1 also results in VE constriction, and this defect is also alleviated in mutant embryos in which the VE is wild type (Yamamoto et al., 2001
). By contrast, Nodal
600/
600 embryos that lack Nodal activity in the VE develop normally. The morphological defects in Nodal
600/ embryos are therefore most likely due to decreased Nodal signaling from the epiblast that in turn attenuates expression of downstream target genes acting in the VE. In keeping with this idea, we find that expression of genes such as Gsc and Lefty1, which represent downstream targets of the Nodal pathway (Labbe et al., 1998
; Yamamoto et al., 2001
), are induced poorly, if at all, in Nodal
600/ embryos. We conclude that the strength of Nodal signaling from the epiblast is crucial in controlling activation of discrete Nodal targets in the VE that in turn are responsible for normal growth and morphogenesis of the VE.
Nodal signals control formation of the definitive gut endoderm
Interestingly Foxh1/ (Hoodless et al., 2001; Yamamoto et al., 2001
) and Nodal
600/ embryos both develop anterior CNS truncations, a phenotype similar to that which develops in chimeric embryos lacking Nodal function in the VE (Varlet et al., 1997
). Here, by contrast, we observe normal development of Nodal
600/
600 embryos, arguing that Nodal is not required in the VE. Similarly Foxh1, while essential for Nodal activation in the VE, is not required for formation of the AVE (Hoodless et al., 2001
; Yamamoto et al., 2001
). The current findings suggest an alternative explanation for the development of these anterior defects, namely that lowering the dose of Nodal signaling in the epiblast, either genetically or via cell mixing in chimeras, leads to a failure to correctly specify definitive endoderm tissue. The definitive endoderm (DE) initially appears as a discrete cell layer at the most distal end of the primitive streak (Lawson et al., 1986
; Lawson and Pedersen, 1987
; Kinder et al., 2001
), displacing primitive visceral endoderm into the extra-embryonic regions (Lawson and Pedersen, 1987
; Tam and Beddington, 1992
; Thomas and Beddington, 1996
). Recent evidence has shown that the signals provided by the anterior gut endoderm are essential for reinforcing and correctly elaborating pattern in the developing neural plate after displacement of the AVE. Thus, embryos lacking the transcription factor Hex fail to form anterior definitive endoderm and develop anterior CNS truncations (Martinez Barbera et al., 2000
). Similarly, the Wnt inhibitor Dkk-1 acting in the anterior mesendoderm (Mukhopadhyay et al., 2001
), and the pro-protein convertase SPC4 expressed in the foregut (Constam and Robertson, 2000
) are both required for patterning of the rostral CNS. Our current findings indicate in mouse, as for Xenopus (Agius et al., 2000
), that Nodal signaling is essential for specification of the definitive endoderm lineage.
Neither Foxh1 (Hoodless et al., 2001) nor Smad2 (Tremblay et al., 2000
) -deficient ES cells are competent to form definitive endoderm, suggesting Foxh1/Smad2 complexes govern endodermal fate in the anterior streak. Chimeras composed of Nodal-deficient VE, with Nodal expression only partially restored in the epiblast, phenocopy Nodal
600/ embryos (Varlet et al., 1997
). Thus, decreasing Smad2 signals in the anterior streak is sufficient to disrupt formation of DE, in turn leading to cardiac and anterior CNS defects. By contrast, in Nodal
600/
600 embryos, increased levels of Nodal activity allow normal specification of the definitive endoderm lineage. Collectively, these findings establish a dose-dependent requirement for the Nodal signaling pathway in eliciting endodermal fates in the mouse.
Attenuated Nodal signals in the absence of the feedback loop cause relatively mild late onset LR patterning defects
Conserved Nodal activities are known to be essential for specification of the LR body axis (reviewed by Capdevila et al., 2000). Asymmetric activation of Nodal expression in the left lateral plate mesoderm at early somite stages in turn activates the downstream targets Lefty2 and Pitx2 (reviewed by Capdevila et al., 2000
) and enhances Nodal transcription via the Foxh1-dependent autoregulatory enhancer. Deletion of the Nodal ASE enhancer severely compromises this pathway, and only low levels of asymmetric Nodal expression are transiently detected in Nodal
600/
600 embryos.
Asymmetric Lefty2 transcription is also mediated by an enhancer containing two Foxh1-binding motifs (Saijoh et al., 1999; Saijoh et al., 2000
). Deletion of these sequences in the mouse germline results in failure to activate asymmetric Lefty2 expression and causes ectopic Nodal expression leading to situs defects (Meno et al., 2001
). Nodal
600/
600 embryos either fail or only weakly activate Lefty2 expression. Lefty2 target gene expression thus requires continuous Nodal signaling. In addition, we found that Lefty1, which is normally expressed in the midline prospective floorplate tissue, is only rarely induced in Nodal
600/
600embryos. These results implicate Lefty1 as a downstream target. However, as yet, Nodal responsive cis-acting elements such as Foxh1-binding sites have not been identified at the Lefty1 locus (Saijoh et al., 1999
).
By contrast, decreased Nodal activity in Nodal600/
600 embryos is sufficient to activate robust Pitx2 expression. Recent experiments have described a Pitx2 transcriptional enhancer containing three Foxh1-binding motifs. In contrast to Nodal, Pitx2 transcription is maintained via the activity of an Nkx2.1-dependent enhancer (Shiratori et al., 2001
). The increased number or distinct configuration of the Foxh1-binding sites may allow Pitx2 occupancy by activated Smad2/3/4 complexes transiently formed in Nodal
600/
600 embryos. Nonetheless delayed activation of Pitx2 transcription in the LPM results from the markedly reduced Nodal expression, that correlates with the development of right pulmonary isomerisms and heart defects in Nodal
600/
600 embryos. As Pitx2-deficient embryos show similar thoracic defects (Gage et al., 1999
; Kitamura et al., 1999
; Lin et al., 1999
; Lu et al., 1999
), these abnormalities probably reflect altered Pitx2 expression patterns.
Cryptic, a Nodal co-factor (Yan et al., 1999), and Gdf1, a TGFß family member (Rankin et al., 2000
), are both essential for asymmetric activation of Nodal, Lefty2 and Pitx2. Loss-of-function mutants display right pulmonary isomerism and randomization of cardiac and abdominal situs, associated with randomization in the direction of embryonic turning and heart looping. Surprisingly, we found that dramatically decreasing Nodal activity in the lateral plate by elimination of the positive feedback loop had a relatively minor impact on LR patterning. Thus, the direction of embryonic turning or initial cardiac looping is unaffected in Nodal
600/
600 embryos. This cannot be due to asymmetric activation of Pitx2 in these embryos, as loss-of-function of Pitx2 does not affect the direction of turning or prevent initial establishment of abdominal situs (Gage et al., 1999
; Kitamura et al., 1999
; Lin et al., 1999
; Lu et al., 1999
). Embryonic turning and abdominal situs may be therefore be controlled by distinct, Nodal-independent, molecular pathways. However, in chick (Logan et al., 1998
; Piedra et al., 1998
; Ryan et al., 1998
) and Xenopus (Sampath et al., 1997
; Ryan et al., 1998
; Campione et al., 1999
), ectopic Nodal expression can reverse body situs, suggesting that as yet undescribed Nodal targets may be activated to control this process. In this case, based on the phenotype of Nodal
600/
600 embryos, only low levels of asymmetric Nodal activity are predicted to be required to activate these pathways.
It has been suggested that LR asymmetry is initiated in the mouse by a net leftward flow of extracellular fluid generated by the cilia located on the ventral surface of the node (reviewed by Capdevila et al., 2000). This asymmetric morphogen gradient is thought to act at a distance to induce asymmetric Nodal expression in the left lateral plate. Mutations affecting the formation or motility of the cilia disturb establishment of the asymmetric Nodal expression domain. Nodal ligand produced at the edge of the notochordal plate potentially represents this morphogen and acts to induce its own transcription in the LPM. Consistent with this, recent work in zebrafish shows that the Nodal homolog squint can act at a distance (Chen and Schier, 2001
). Similarly removal of the asymmetric expression domain of the Nodal antagonist Lefty2, results in bilateral Nodal expression, consistent with long range diffusion and autoactivation by secreted Nodal ligand (Meno et al., 2001
).
However, the present findings challenge this simple model and allow us to draw two new conclusions about Nodal activities involved in establishing the LR axis. First, we demonstrate that asymmetric Nodal expression in the developing node is not essential to induce asymmetric Nodal transcription in the lateral plate, as Nodal600/
600 embryos express wild-type levels of Nodal transcripts in the node in a symmetric fashion, but Nodal is correctly induced exclusively on the left side of the axis. Thus, the functional significance of Nodal asymmetry in the mouse node remains unclear. Second, deletion of the autoregulatory enhancer fails to eliminate Nodal expression in the left LPM. Thus, tissue-specific activation of the locus occurs selectively on the left via an ASE- and probably Foxh1-independent mechanism(s). While key molecules controlling LR patterning have been identified in recent years, clearly additional work will be required to dissect the components of the functional pathways they control, and how they interact with each other to regulate this process.
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ACKNOWLEDGMENTS |
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