1 Howard Hughes Medical Institute and Department of Cell Biology, Vanderbilt University Medical Center, 1161 21st Ave. South, Nashville, TN 37232-2175
2 Department of Cell and Developmental Biology, The University of North Carolina, Chapel Hill, NC 27599-7178, USA
3 Bowles Center for Alcohol Studies, The University of North Carolina, Chapel Hill, NC 27599-7178, USA
* Present address: Dana-Farber Cancer Institute, Department of Pediatric Oncology, Harvard Medical School, 44 Binney Street, Boston, MA 02115, USA
Author for correspondence (e-mail: brigid.hogan{at}mcmail.vanderbilt.edu)
Accepted 16 July 2002
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SUMMARY |
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Key words: Bone morphogenetic protein 4, Mouse, Embryo, LR asymmetry, Node
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INTRODUCTION |
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The initiation of LR asymmetry in the vertebrate embryo is thought to involve the unidirectional flow of molecules across the body axis to establish asymmetric gene expression in the node (Wright, 2001; Hamada et al., 2002
). In the chick embryo, ion flow in the blastoderm at the streak stage may induce Shh on the left side of Hensens node and this, in turn, activates the left-sided program in the embryo (Pagan-Westphal and Tabin, 1998
; Levin and Mercola 1999
). In the mouse, monocilia in the ventral node cells rotate unidirectionally and may generate a left-specific flow of morphogens within the node to establish the program for LR asymmetry (Wright, 2001
; Hamada et al., 2002
). In both cases, the node, influenced by its surrounding environment, serves as a center for establishing LR asymmetry.
The mechanisms for reinforcing the LR molecular cascade within and from the node are different in the chick and the mouse (Wright, 2001; Hamada et al., 2002
). In the chick embryo, it is thought that Shh induces Nodal to reinforce the left-side pathway while on the right Bmp4 induces Fgf8 that, in turn, locally represses Shh (Levin et al., 1995
; Monsoro-Burq and le Douarin, 2001
). The left-side signaling cascade in the node is subsequently extended to the lateral plate mesoderm (LPM), possibly by the diffusion of Nodal from the node. Significantly, caronte, a secreted Bmp antagonist induced by Shh, is expressed in the left paraxial mesoderm and left LPM and inhibits the activity of Bmp2, Bmp4 and Bmp7 in the LPM that would otherwise repress Nodal. Caronte thereby permits Nodal expression in the left LPM (Rodriguez Esteban et al., 1999
; Yokouchi et al., 1999
). Nodal in turn induces a transcription factor, Pitx2, in the left LPM to complete the establishment of the molecular left-side patterning (Logan et al., 1998
).
In the mouse, as described earlier, the first asymmetric expression of Nodal is thought to be driven by asymmetric physical flow leading to higher levels of transcripts on the left side of the node compared with the right (Collignon et al., 1996). However, unlike in the chick, Shh and Fgf8 are not asymmetrically expressed in the mouse node, at least at the RNA level: Shh is transcribed throughout the node (Collignon et al., 1996
), while Fgf8 is exclusively expressed in the primitive streak but not in the node (Crossley and Martin, 1995
). Genetic studies have provided evidence that these genes have functions distinct from those proposed in the chick. Specifically, Shh is required to maintain the midline and so prevent left-side identity invading into the right side (Meyers and Martin, 1999
), while Fgf8 positively facilitates left-side patterning (Meyers and Martin, 1999
). How molecular asymmetries at the node are transferred to the left LPM in the mouse embryo remains obscure because, among other things, no caronte homolog has been identified. However, cryptic, which encodes a membrane-bound protein of the EGF-CFC family that is a co-factor of Nodal signaling, is a known player in LR patterning in the mouse. It is expressed in the node, midline and bilaterally in the LPM; analysis of cryptic-null embryos suggests that the protein is required in the left LPM to enable the expression of Nodal and other left-side determinants (Shen et al., 1997
; Yan et al., 1999
; Shen and Schier, 2000
; Yeo and Whitman, 2001
). Once Nodal is induced in the LPM, the molecular left-side signaling pathway seems to be conserved between most vertebrates (Wright, 2001
; Hamada et al., 2002
). Nodal induces Lefty2 (Leftb Mouse Genome Informatics), which acts as a feedback repressor of Nodal expression (Meno et al., 1999
; Whitman, 2001
), and Pitx2, which maintains the left-sided environment (Meno et al., 2001
; Shiratori et al., 2001
).
In the early mouse embryo, Bmp4 is expressed in a dynamic pattern, first in the extra-embryonic ectoderm (ExE) and then in epiblast-derived tissues, including extra-embryonic mesoderm (ExM), posterior primitive streak and bilaterally in the LPM. Moreover, genetic loss of function and chimeric embryo analyses have demonstrated distinct functions for Bmp4 in these different tissues (Winnier et al., 1995; Lawson et al., 1999
; Fujiwara et al., 2001
). Bmp4 expressed in the ExE is required for patterning the epiblast along the proximodistal axis that is later transformed during gastrulation into the anteroposterior axis. Consequently, epiblast cells closest to the source of Bmp4 in the ExE give rise to the most posterior cell types, the allantois, ExM, and primordial germ cells (PGCs) (Lawson et al., 1999
). Bmp4 expressed in the ExM regulates the subsequent migration and survival of PGCs, and the development of the allantois and its blood vessels (Fujiwara et al., 2001
). We employ a variety of strategies to further investigate the function of Bmp4 in the early patterning of the mouse embryo. Our data indicate that Bmp4 expression in the ExE is essential for the normal morphological development of the node and primitive streak. At the same time, Bmp4 made in epiblast-derived tissues is required for the propagation of the left-side molecular cascade. Taken together, our results highlight the dynamic interplay that must occur between different tissues, signaling pathways and anatomical structures for establishing and maintaining LR axial patterning.
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MATERIALS AND METHODS |
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Whole-mount in situ hybridization and immunostaining
Whole-mount in situ hybridization was performed essentially as previously described (Hogan et al., 1994). For double whole-mount in situ hybridization, RNA probes for Nodal and Foxf1 (Foxf1a Mouse Genome Informatics) were labeled with digoxigenin and fluorescein, respectively. BM-purple and BCIP were used for the color reactions. BCIP Reaction buffer is NTM [100 mM Tris-HCl (pH 9.5), 100 mM MgCl2, 150 mM NaCl]. For sectioning after in situ hybridization, embryos were fixed in 4% paraformaldehyde in PBS, dehydrated into 100% isopropanol, washed with 1:1 isopropanol/paraffin wax, embedded in wax and sectioned at 7 µm. Whole-mount immunostaining with anti VCAM1 antibody (Pharmingen) was performed as previously described (Fujiwara et al., 2001
).
Scanning electron microscopy
Dissected embryos were immediately washed three times in Sorensons phosphate buffer (Sulik et al., 1994). After fixation with 2.5% glutaraldehyde in Sorensons phosphate buffer at 4°C for 48 hours, embryos were rinsed in Sorensons phosphate buffer, then post-fixed in 2% osmium tetroxide for 2 hours. After dehydration in a graded series of ethanols, the specimens were critical-point dried, mounted on metal stubs and sputter-coated with gold palladium. Electron microscopy was performed on a JOEL microscope.
Whole embryo culture
Headfold-stage wild-type embryos were isolated and Reicherts membrane removed mechanically. Three to four embryos were cultured with rotation in a silicon-coated vial in 500 µl culture medium with/without 1 µg/ml noggin for 20 hours at 37°C, 5% CO2/95% air. The culture medium was Dulbeccos Modified Eagles Medium (DMEM) supplemented with 50% rat serum (Harlan Bioproducts), 2 mM L-Glutamine (GIBCO) and 50 µg streptomycin/penicillin (GIBCO). Recombinant mouse noggin (R&D Systems) was dissolved at 100 µg/ml in PBS containing 0.1% bovine serum albumin (BSA) (BSA/PBS). The BSA/PBS solution was used as a negative control. After culture, the embryos were washed in BSA/PBS three times and fixed with 4% PFA in PBS for 75 minutes at 4°C, and subsequently analyzed by whole-mount in situ hybridization.
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RESULTS |
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It was not possible in the above study to compare the detailed morphology (Sulik et al., 1994) of ventral node cells of mutant and wild-type embryos because the cell boundaries and architecture of the monocilia were disrupted by the in situ hybridization protocol. We therefore analyzed by SEM the gross morphology of five additional, untreated, Bmp4tm1 mutants from the headfold to the six-somite stage. Wild-type embryos (n=5) showed a well-formed primitive streak and a concave and tear-drop shaped node with monocilia in almost every cell. The extended notochord had well demarcated borders with the endoderm (Fig. 1B, parts a,b). By contrast, a ventrally projected bulge was present in the posterior region of all Bmp4tm1 mutants examined (n=5). The shape of the node was also abnormal and was either flat or slightly convex (Fig. 1B, parts, e,f). Most cells had monocilia, but one embryo at the headfold stage showed large and irregularly shaped unciliated cells, resembling endodermal cells, scattered within the node (Fig. 2B, part i). The notochord was formed in all mutants examined, although the boundary of the notochord and the adjacent endoderm was less regular than in the wild-type embryos (Fig. 1B, part e).
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Reduced Lefty2 and cryptic expression in Bmp4-null mutants
To further examine the L-sided molecular cascade in Bmp4tm1 mutants, we analyzed expression of Lefty2 and cryptic. Lefty2 is normally transiently expressed in the left LPM between the two- and five-somite stage (Fig. 1C, part a) (Meno et al., 1996), while cryptic is expressed bilaterally in the LPM, node, and the midline from the headfold to the six- to eight-somite stage (Fig. 1C, parts d,g) (Shen et al., 1997
). In about 65% (n=6/9) of Bmp4tm1 mutants at the 3-4 somite stage, Lefty2 expression was not detected (Fig. 1C, part b). The remainder (n=3/9) had relatively normal left-side expression (Fig. 1C, part c). In the case of cryptic, three out of three Bmp4 mutants at the two- to four-somite stage and four out of four at the five- to six-somite stage had no expression of the gene in the LPM, node and midline (Fig. 2C, parts e,f). However, in all earlier (headfold to one somite-stage) mutants examined (n=4/4), cryptic was expressed bilaterally in the LPM, but was greatly reduced in the node (Fig. 2C, part h). This suggests that Bmp4 signaling is required for the maintenance of cryptic expression in the LPM.
The absence of Lefty2 and cryptic transcripts in the LPM again raised the issue of whether this tissue fails to develop or degenerates in Bmp4tm1 mutants. To address this possibility, we made use of the fact that in Bmp4lacZ/+ embryos the expression of Bmp4 in the LPM is marked by lacZ activity (Fig. 1C, parts i,j) (Lawson et al., 1999). Staining for ß-galactosidase revealed abundant Bmp4lacZ-positive cells with a normal morphology in the LPM of Bmp4lacZ/lacz null mutants at the four- to six-somite stage (Fig. 1C, parts l,m). Moreover, analysis of Foxf1 expression as described above (Fig. 1A, parts d-f) also confirmed that the LPM is formed in all Bmp4tm1 mutants examined at the two- to six-somite stage (n=8/8) (Fig. 1C, parts k,n).
Abnormal expression of Fgfs and Dte but maintenance of midline tissues in Bmp4-null mutants
Fgf8 has been implicated as a positive facilitator of left-sided patterning in the mouse embryo, and as a right-sided determinant in the chick (Meyers and Martin, 1999; Monsoro-Burq and le Douarin, 2001
). In the mouse, Fgf8 is symmetrically expressed in the primitive streak, and expression of Fgf4 and Nodal is dependent on the gene dose of Fgf8 (Crossley and Martin, 1995
; Meyers and Martin, 1999
; Sun et al., 1999
). We therefore examined the expression of both Fgf genes in one- to six-somite-stage Bmp4tm1 mutants. In five- to seven-somite stage wild-type embryos, Fgf8 expression was detected in the primitive streak (Fig. 2A). By contrast, Fgf8 expression was significantly reduced in around 80% (n=5/6) of five- to six-somite stage Bmp4tm1 mutants (Fig. 2B,C). One six-somite stage Bmp4tm1 mutant showed Fgf8 expression in the primitive streak at a level comparable with the wild type (data not shown). As for Fgf4, about 70% (n=3/4) of one- to two-somite stage Bmp4tm1 mutants showed reduced levels in the primitive streak compared with wild-type embryos (Fig. 2D,E), while one mutant showed comparatively normal expression (Fig. 2F).
Because of the importance of the midline in maintaining LR asymmetry, we confirmed that trunk midline tissues still develop robustly despite the posterior abnormalities in Bmp4tm1 mutants. In the mouse, Shh is normally symmetrically expressed in the node and midline tissues such as the notochord and the prospective floor plate in the headfold to 6-somite stage embryos (Collignon et al., 1996). In 10/11 Bmp4tm1 mutants examined at the pre- to 6-somite stage, expression of Shh was observed in the node and midline tissues at a level comparable with that seen in wild-type embryos (Fig. 2G-J). One headfold-stage Bmp4tm1 mutant showed scattered Shh expression only in the node, possibly reflecting a delay of midline development (data not shown).
Members of the cerberus/Dan-related gene family encode proteins that are thought to directly bind to Nodal and Bmps and to inhibit signaling through their receptors (Pearce et al., 1999; Massague and Chen, 2000
). One gene, Dante (Dte), is expressed specifically in the periphery of the node in the mirror image of Nodal, with somewhat higher expression on the right than the left at the four- to six-somite stage (Fig. 2K) (Pearce et al., 1999
). In 80% (n=4/5) of Bmp4tm1 mutants examined, Dte expression in the node periphery was reduced and patchy but still relatively higher on the right (Fig. 2L), while one mutant was normal (Fig. 2M). Taken together, our results with Nodal, cryptic and Dte suggest that the normal development of the node requires Bmp4 expression in the embryo.
Mesocardia in Bmp4 null mutants
Homozygous null Bmp4tm1 mutants do not survive to the stage when there is clear asymmetry of organs such as the lung and gut, and the only anatomical indicator of early LR patterning that can be scored is heart looping. In about half of the Bmp4tm1 mutant embryos examined at the nine- to ten-somite stage (n=4/7) the heart tube lay centrally in the midline, and showed no evidence of heart looping, either to the left or right (mesocardia) (Fig. 3A-F). This was seen both in the intact embryo and after sectioning. The remainder of the Bmp4tm1 mutants (n=3/7) had normal rightward looping, as in the wild-type embryo at the same somite stage.
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In spite of the restoration in node morphology, a reduction in Nodal expression was still observed in tetraploid chimeras, at both the late streak (n=2/2) (compare Fig. 5A,D) and the headfold stage (n=2/2) (compare Fig. 5B,E). Later somite-stage tetraploid chimeras (n=2/2) also showed significant reduction of Nodal expression in both the node and left LPM (compare Fig. 5C with 5F). These results suggest that Bmp4 expression in epiblast-derived cells is required for Nodal expression in the node and left LPM, independently of node morphogenesis.
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Effect of exogenous noggin on LR patterning
Previous studies in the chick embryo suggested that Bmp activity suppresses Nodal expression in the left LPM, because caronte inhibits Bmp2, Bmp4 and Bmp7 and permits Nodal expression in this tissue (Rodriguez Esteban et al., 1999; Yokouchi et al., 1999
). In the mouse embryo, Bmp2 is expressed bilaterally in the LPM but not in the midline (Fig. 6A) but no specific expression of Bmp6 or Bmp7 is observed in the LPM, although Bmp7 expression is seen in the node and midline (data not shown). We therefore addressed the role of Bmps produced in the LPM by culturing embryos in the presence of noggin, an inhibitor of Bmps normally expressed in the node and notochord at the headfold to four-somite stage (Fig. 6B) (Massague and Chen, 2000
). This was carried out at the headfold stage when embryos have already established Nodal expression in the node. Of the nine control embryos cultured to the two- top five-somite stage, seven showed left-sided and two showed bilateral Nodal expression in the LPM (Fig. 6C and data not shown). By contrast, none of the embryos cultured with noggin to the same stage (n=17) showed any Nodal expression in the LPM (Fig. 6D). Unlike in tetraploid chimeras, Nodal expression in the periphery of the node was still observed in all noggin-treated embryos (Fig. 6D). Moreover, the addition of noggin did not affect cryptic expression in the LPM and midline (Fig. 6E; n=16/16), a result that indirectly demonstrates that the LPM did develop in cultured embryos. Proper midline development was confirmed by Shh expression in embryos cultured with exogenous noggin (Fig. 6F; n=7/7).
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DISCUSSION |
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Morphogenesis of the node and primitive streak requires Bmp4 made in the extra-embryonic ectoderm (ExE)
Comparison of the phenotype of Bmp4-null mutants and tetraploid chimeras suggests that Bmp4 signaling from the ExE is a key factor in regulating the development of the posterior mesoderm and the node in the headfold to 6-somite stage mouse embryo (Fig. 1A, parts e,f,h,i; Fig. 2B, parts e,f). This conclusion (summarized in model form in Fig. 7A) is based on the hypothesis that Bmp4 in the ExE patterns the proximodistal axis of the epiblast that is converted, by gastrulation, into the dorsal/anteroposterior/ventral axis of the embryo (Beddington and Robertson, 1999). Fate-mapping studies and previous analysis of Bmp4-null embryos, suggest that the epiblast cells closest to the source of Bmp4 are specified as precursors of the posterior ExM, allantois and PGCs, while somewhat more distal cells are specified as precursors of the posterior embryonic mesoderm (Lawson et al., 1991
; Lawson et al., 1999
). Accordingly, in the absence of ExE Bmp4 the epiblast is dorsalized, fewer posterior-proximal epiblast cells are specified as prospective ExM, and the development of posterior mesoderm-derived tissues is disrupted. Two kinds of data obtained here support this model. First, posterior proximal epiblast and cells in the posterior primitive streak normally express Fgf8, and Fgf8 regulates Fgf4 expression in the primitive streak (Crossley and Martin, 1995
; Sun et al., 1999
). Both Fgf8 and Fgf4 are strongly downregulated in Bmp4-null embryos, while Evx1-positive embryonic mesodermal cells accumulate in the posterior region (Fig. 2B, part k; Fig. 3B,C,E). Second, the posterior phenotype of Bmp4-null embryos analyzed by SEM strongly resembles that of Fgf8/ mutants, with an accumulation of cells in the primitive streak region filling the amniotic cavity and creating a characteristic bulge [compare Fig. 2B, parts g,h with Fig. 1C,D by Sun et al. (Sun et al., 1999
)]. This posterior streak defect and the expression of Fgf8 are rescued in tetraploid chimeras, in which ExE Bmp4 is restored.
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One important character of the mouse node is the formation and function of monocilia. Although these cilia are present in almost all ventral cells in Bmp4-null mutants and tetraploid chimeras analyzed, whether leftward nodal flow is present remains to be determined.
Bmp4 made in epiblast-derived tissues is required for molecular LR patterning
A key finding of this study is that in both Bmp4-null mutants and tetraploid chimeras, there is reduction of Nodal expression in the node and left LPM (Fig. 1A, parts b,c; Fig. 5D-F). This suggests that Bmp4 made in epiblast-derived tissues is an upstream regulator of Nodal signaling and the establishment or maintenance of the left-side molecular pathway.
One unanswered question is whether defects in the molecular LR pathway in Bmp4 mutants are translated into defect in anatomical structures. Homozygous Bmp4 mutants die before asymmetries in gut derivatives develop, and the only anatomical read-out that can be assayed is heart looping. Hypomorphic Nodal mutants have randomized heart looping, with 50% to the right and 50% to the left (Lowe et al., 2001
). By contrast, about half of the Bmp4-null mutants examined here have mesocardia, in which the heart does not loop at all. We have observed that Bmp4 is strongly expressed, symmetrically, in the outflow tract of the eight- to ten-somite stage embryo (data not shown). This raises the possibility that abnormalities in the LR patterning of the heart are compounded by a second Bmp4 function intrinsic to cardiac morphogenesis. Early cardiac specific inactivation of Bmp4 will be required to test this hypothesis.
The cardiac and LR molecular marker expression defects caused by loss of Bmp4 function are observed in 50-75% of Bmp4-null mutants, but in all tetraploid chimeras. This discrepancy is possibly due to the different genetic backgrounds on which the null mutation is presented. Tetraploid chimeras are completely ES cell-derived (Nagy et al., 1993), so that our chimeras reflect the phenotype of Bmp4-null mutants on the 129/SvEvTaqfBR inbred background. As previously shown, homozygous Bmp4 mutants on an inbred background have more severe defects than on an outbred background (Winnier et al., 1995
; Lawson et al., 1999
), although the genetic basis for this effect is not yet known.
Role of epiblast-derived Bmp4 in regulating Nodal expression in the node
Several epiblast-derived cell populations express Bmp4, including the ExM, the most posterior mesoderm of the primitive streak, and, later, the LPM (Lawson et al., 1999). Based on timing of expression, it is likely that Bmp4 expressed in the ExM and/or the most posterior primitive streak affects the initiation of Nodal expression in the node, while consolidation and maintenance of this Nodal expression is regulated by Bmp4 expressed in the posterior LPM from the late neural plate stage. Previous genetic loss of function studies using hypomorphic Fgf8 mutants proposed that Fgf8 produced in the primitive streak positively regulates Nodal expression in the node (Meyers and Martin, 1999
). Fgf8 expression in our tetraploid chimeras was moderately reduced, but still present (Fig. 5H). However, whether the level of Fgf8 in these chimeras reaches the threshold necessary to support Nodal expression in the node still remains unclear. An attractive alternative possibility is that molecules other than Fgf8 are required in this signaling pathway. Examining the expression of other signaling molecules in Bmp4/ mutants might shed light on the answer to this question in the future.
Role of epiblast-derived Bmp4 in regulating Nodal expression in the lateral plate mesoderm
Left side determinant genes, including Nodal and Lefty2, are first expressed in the left LPM of the embryo at the two- to six-somite stage (Collignon et al., 1996; Meno et al., 1996
). By contrast, expression of Bmp4 in the ExM, posterior primitive streak and LPM starts earlier, at least from the headfold stage. This suggests that Bmp4 positively regulates downstream genes that establish and maintain LR asymmetry, including expression of Nodal, Lefty2 and cryptic. Our genetic loss-of-function studies alone do not address the specific role of LPM-produced Bmp4 in regulating Nodal signaling in the LPM. We therefore took an alternative approach and inhibited the activity of Bmps, including Bmp4, in the LPM by applying noggin in culture to embryos that already express Nodal in the node. This resulted in the absence of Nodal expression in the LPM, while transcription continued in the node. This finding in the mouse is inconsistent with the model proposed in the chick embryo in which the function of Bmps (Bmp2, Bmp4, Bmp7) is to suppress Nodal signaling in the LPM. An important feature of the chick model is the presence of caronte in the left LPM that inhibits Bmp signaling and consequently permits Nodal expression in the left LPM (Rodriguez Esteban et al., 1999
; Yokouchi et al., 1999
). In the mouse, no caronte homolog has been identified, and the Bmp antagonists, noggin and chordin, are not expressed in the LPM (Davidson and Tam, 2000
) (Fig. 6A). Therefore, it can be concluded that mouse and chick embryos use different molecular mechanisms in the LPM to establish and maintain LR asymmetry.
Cryptic is a positive co-factor of Nodal signaling (Shen and Schier, 2000; Yeo and Whitman, 2001
), and is expressed bilaterally in the LPM, node, and midline axis, from the headfold to six- to eight-somite stage (Shen et al., 1997
). Bilateral cryptic expression in the LPM is seen in Bmp4-null mutants at the headfold to one somite stage (Fig. 2C, part g), but is undetected in later two- to six-somite-stage mutants (Fig. 2C, parts e,f). This suggests that embryo-derived Bmp4 functions as a maintenance factor for cryptic expression in the LPM. It is of interest that, unlike Bmp4-null mutants and tetraploid chimeras, the headfold-stage embryos cultured in vitro with added noggin did express cryptic in the LPM and midline (Fig. 6E). A significant difference between the mutants and the wild-type embryos cultured with noggin (n=17) is that in the latter, Nodal expression is still observed in the node (Fig. 6D). This argues that cryptic expression in the LPM and/or midline is initiated and can be maintained by Nodal expressed in the node (Fig. 7C). In the future, sorting out the precise contribution of the different tissues expressing Bmp4 will be best achieved by inactivation of the gene by conditional gene targeting strategies.
Downstream Bmp4 signaling in LR axis formation
Bmps signal through transmembrane receptors and downstream components such as Smad1, Smad5, Smad8 and Smad4 (Massague and Chen, 2000). Recently, it was reported that about half of the Smad5-null mutants at the one- to five-somite stage have bilateral Nodal expression, while the remainder show no expression in the LPM. Moreover, in Smad5-null mutants, Nodal expression in the node is normal, with enrichment in the left periphery (Chang et al., 2000
). However, Lefty1, which functions as a midline barrier, is absent in all Smad5-null mutants examined, suggesting that the bilateral LPM Nodal expression is a result of a midline defect (Chang et al., 2000
). By contrast, we did not observe any Bmp4-null mutants with bilateral Nodal expression in the LPM, and the loss of left LPM Nodal expression in Bmp4-null mutants and tetraploid chimeras was, in all cases, accompanied by strongly reduced Nodal expression in the node (mutants, n=14/14; chimeras, n=6/6). The discrepancy in LR patterning between the Smad5- and Bmp4-null phenotypes may therefore be due to differences in the level of Nodal production in the node, which is required to activate Nodal signaling in the left LPM.
Summary: a model for the establishment of LR asymmetry in the mouse embryo
Our genetic loss of Bmp4 function analyses provide new insight into the signaling cues that initiate Nodal expression in the node and left LPM of the mouse embryo. We propose that Bmp4 functions at two stages. As summarized in Fig. 7, Bmp4 made in the ExM and/or posterior primitive streak promotes the initial expression of Nodal in the node, possibly by a mechanism independent of Fgf8 (Fig. 7B). This effect is apparently independent of an earlier role for Bmp4 in proximodistal patterning of the epiblast and the morphogenesis of the node (Fig. 7A). Second, we propose that Bmps, including Bmp2 and Bmp4, function later in the LPM to positively regulate the expression of Nodal. Cryptic might ensure the competence of the LPM to respond to the signal that activates the left-side signaling cascade (Fig. 7C). The essential feature of this proposal is that Bmp4 is a left-side-signaling facilitator during mammalian embryonic development.
Note added in proof
Since the submission of this manuscript, two papers have appeared demonstrating that BMP2 is a positive regulator of Nodal signaling in the chick embryo (Schlange et al., 2002; Piedra and Ros, 2002
).
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ACKNOWLEDGMENTS |
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REFERENCES |
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Beddington, R. S. and Robertson, E. J. (1999). Axis development and early asymmetry in mammals. Cell 96, 195-209.[Medline]
Chang, H., Zwijsen, A., Vogel, H., Huylebroeck, D. and Matzuk, M. M. (2000). Smad5 is essential for left-right asymmetry in mice. Dev. Biol. 219, 71-78.[CrossRef][Medline]
Collignon, J., Varlet, I. and Robertson, E. J. (1996). Relationship between asymmetric nodal expression and the direction of embryonic turning. Nature 381, 155-158.[CrossRef][Medline]
Crossley, P. H. and Martin, G. R. (1995). The mouse Fgf8 gene encodes a family of polypeptides and is expressed in regions that direct outgrowth and patterning in the developing embryo. Development 121, 439-451.
Davidson, B. P. and Tam, P. P. (2000). The node of the mouse embryo. Curr. Biol. 10, 617-619.
Dufort, D., Schwartz, L., Harpal, K. and Rossant, J. (1998). The transcription factor HNF3beta is required in visceral endoderm for normal primitive streak morphogenesis. Development 125, 3015-3025.
Dush, M. K. and Martin, G. R. (1992). Analysis of mouse Evx genes: Evx-1 displays graded expression in the primitive streak. Dev. Biol. 151, 273-287.[Medline]
Fujiwara, T., Dunn, N. R. and Hogan, B. L. (2001). Bone morphogenetic protein 4 in the extraembryonic mesoderm is required for allantois development and the localization and survival of primordial germ cells in the mouse. Proc. Natl. Acad. Sci. USA 98, 13739-13744.
Hamada, H., Meno, C., Watanabe, D. and Saijoh, Y. (2002). Establishment of vertebrate left-right asymmetry. Nat. Rev. Genet. 3, 103-113.[CrossRef][Medline]
Hogan, B., Beddington, R., Constantini, F. and Lacy, E. (1994). Manipulating the Mouse Embryo. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Kinder, S. J., Tsang, T. E., Wakamiya, M., Sasaki, H., Behringer, R. R., Nagy, A. and Tam, P. P. (2001). The organizer of the mouse gastrula is composed of a dynamic population of progenitor cells for the axial mesoderm. Development 128, 3623-3634.
Lawson, K. A., Meneses, J. J. and Pedersen, R. A. (1991). Clonal analysis of epiblast fate during germ layer formation in the mouse embryo. Development 113, 891-911.[Abstract]
Lawson, K. A., Dunn, N. R., Roelen, B. A., Zeinstra, L. M., Davis, A. M., Wright, C. V., Korving, J. P. and Hogan, B. L. (1999). Bmp4 is required for the generation of primordial germ cells in the mouse embryo. Genes Dev. 13, 424-436.
Levin, M. and Mercola, M. (1999). Gap junction-mediated transfer of left-right patterning signals in the early chick blastoderm is upstream of Shh asymmetry in the node. Development 126, 4703-4714.
Levin, M., Johnson, R. L., Stern, C. D., Kuehn, M. and Tabin, C. (1995). A molecular pathway determining left-right asymmetry in chick embryogenesis. Cell 82, 803-814.[Medline]
Logan, M., Pagan-Westphal, S. M., Smith, D. M., Paganessi, L. and Tabin, C. J. (1998). The transcription factor Pitx2 mediates situs-specific morphogenesis in response to left-right asymmetric signals. Cell 94, 307-317.[Medline]
Lowe, L. A., Yamada, S. and Kuehn, M. R. (2001). Genetic dissection of nodal function in patterning the mouse embryo. Development 128, 1831-1843.
Mahlapuu, M., Ormestad, M., Enerback, S. and Carlsson, P. (2001). The forkhead transcription factor Foxf1 is required for differentiation of extraembryonic and lateral plate mesoderm. Development 128, 155-166.
Massague, J. and Chen, Y. G. (2000). Controlling TGF-beta signaling. Genes Dev. 14, 627-644.
Meno, C., Takeuchi, J., Sakuma, R., Koshiba-Takeuchi, K., Ohishi, S., Saijoh, Y., Miyazaki, J., ten Dijke, P., Ogura, T. and Hamada, H. (2001). Diffusion of Nodal signaling activity in the absence of the feedback inhibitor Lefty2. Dev. Cell 1, 127-138.[CrossRef][Medline]
Meno, C., Saijoh, Y., Fujii, H., Ikeda, M., Yokoyama, T., Yokoyama, M., Toyoda, Y. and Hamada, H. (1996). Left-right asymmetric expression of the TGF beta-family member lefty in mouse embryos. Nature 381, 151-155.[CrossRef][Medline]
Meno, C., Gritsman, K., Ohishi, S., Ohfuji, Y., Heckscher, E., Mochida, K., Shimono, A., Kondoh, H., Talbot, W. S., Robertson, E. J., Schier, A. F. and Hamada, H. (1999). Mouse Lefty2 and zebrafish antivin are feedback inhibitors of nodal signaling during vertebrate gastrulation. Mol. Cell 4, 287-298.[Medline]
Meyers, E. N. and Martin, G. R. (1999). Differences in left-right axis pathways in mouse and chick: functions of FGF8 and SHH. Science 285, 403-406.
Monsoro-Burq, A. and le Douarin, N. M. (2001). BMP4 plays a key role in left-right patterning in chick embryos by maintaining Sonic Hedgehog asymmetry. Mol. Cell 7, 789-799.[CrossRef][Medline]
Monsoro-Burq, A. and le Douarin, N. M. (2000). Left-right asymmetry in BMP4 signalling pathway during chick gastrulation. Mech. Dev. 97, 105-108.[CrossRef][Medline]
Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W. and Roder, J. C. (1993). Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc. Natl. Acad. Sci. USA 90, 8424-8428.
Pagan-Westphal, S. M. and Tabin, C. J. (1998). The transfer of left-right positional information during chick embryogenesis. Cell 93, 25-35.[Medline]
Pearce, J. J., Penny, G. and Rossant, J. (1999). A mouse cerberus/Dan-related gene family. Dev. Biol. 209, 98-110.[CrossRef][Medline]
Piedra, M. E. and Ros, M. A. (2002). BMP signaling positively regulates Nodal expression during left right specification in the chick embryo. Development 129, 3431-3440.
Rodriguez Esteban, C., Capdevila, J., Economides, A. N., Pascual, J., Ortiz, A. and Izpisua Belmonte, J. C. (1999). The novel Cer-like protein Caronte mediates the establishment of embryonic left-right asymmetry. Nature 401, 243-251.[CrossRef][Medline]
Schlange, T., Arnold, H. H. and Brand, T. (2002). BMP2 is a positive regulator of Nodal signaling during left-right axis formation in the chicken embryo. Development 129, 3421-3429
Shen, M. M. and Schier, A. F. (2000). The EGF-CFC gene family in vertebrate development. Trends Genet. 16, 303-309.[CrossRef][Medline]
Shen, M. M., Wang, H. and Leder, P. (1997). A differential display strategy identifies Cryptic, a novel EGF-related gene expressed in the axial and lateral mesoderm during mouse gastrulation. Development 124, 429-442.
Shiratori, H., Sakuma, R., Watanabe, M., Hashiguchi, H., Mochida, K., Sakai, Y., Nishino, J., Saijoh, Y., Whitman, M. and Hamada, H. (2001). Two-step regulation of left-right asymmetric expression of Pitx2: initiation by nodal signaling and maintenance by Nkx2. Mol. Cell 7, 137-149.[Medline]
Sulik, K. K., Dehart, D. B., Inagaki, T., Carson, J. L., Vrablic, T., Gesteland, K. and Schoenwolf, G. C. (1994). Morphogenesis of the murine node and notochordal plate. Dev. Dyn. 201, 260-278.[Medline]
Sun, X., Meyers, E. N., Lewandoski, M. and Martin, G. R. (1999). Targeted disruption of Fgf8 causes failure of cell migration in the gastrulating mouse embryo. Genes Dev. 13, 1834-1846.
Whitman, M. (2001). Nodal signaling in early vertebrate embryos: themes and variations. Dev. Cell 1, 605-617.[Medline]
Winnier, G., Blessing, M., Labosky, P. A. and Hogan, B. L. (1995). Bone morphogenetic protein-4 is required for mesoderm formation and patterning in the mouse. Genes Dev. 9, 2105-2116.[Abstract]
Wright, C. V. (2001). Mechanisms of left-right asymmetry: whats right and whats left? Dev. Cell 1, 179-186.[Medline]
Yan, Y. T., Gritsman, K., Ding, J., Burdine, R. D., Corrales, J. D., Price, S. M., Talbot, W. S., Schier, A. F. and Shen, M. M. (1999). Conserved requirement for EGF-CFC genes in vertebrate left-right axis formation. Genes Dev. 13, 2527-2537.
Yeo, C. and Whitman, M. (2001). Nodal signals to Smads through Cripto-dependent and Cripto-independent mechanisms. Mol. Cell 7, 949-957.[CrossRef][Medline]
Yokouchi, Y., Vogan, K. J., Pearse, R. V., 2nd and Tabin, C. J. (1999). Antagonistic signaling by Caronte, a novel Cerberus-related gene, establishes left-right asymmetric gene expression. Cell 98, 573-583.[Medline]