View metadata, citation and similar papers at core.ac.uk brought to you by CORE
provided by Elsevier - Publisher Connector
Developmental Biology 276 (2004) 185–193 www.elsevier.com/locate/ydbio
BMP signaling through ACVRI is required for left–right patterning in the early mouse embryo
Satoshi Kishigamia,1, Shun-Ichi Yoshikawab,c, Trisha Castranioa, Kenji Okazakib, Yasuhide Furutac, Yuji Mishinaa,*
aMolecular Developmental Biology Group, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, United States bDepartment of Molecular Biology, Biomolecular-Engineering Research Institute, Suita, Osaka 565-0874, Japan cUniversity of Texas, M.D. Anderson Cancer Center, Houston, TX 77030, United States
Received for publication 14 September 2003, revised 7 July 2004, accepted 20 August 2004 Available online 18 September 2004
Abstract
Vertebrate organisms are characterized by dorsal–ventral and left–right asymmetry. The process that establishes left–right asymmetry during vertebrate development involves bone morphogenetic protein (BMP)-dependent signaling, but the molecular details of this signaling pathway remain poorly defined. This study tests the role of the BMP type I receptor ACVRI in establishing left–right asymmetry in chimeric mouse embryos. Mouse embryonic stem (ES) cells with a homozygous deletion at Acvr1 were used to generate chimeric embryos. Chimeric embryos were rescued from the gastrulation defect of Acvr1 null embryos but exhibited abnormal heart looping and embryonic turning. High mutant contribution chimeras expressed left-side markers such as nodal bilaterally in the lateral plate mesoderm (LPM), indicating that loss of ACVRI signaling leads to left isomerism. Expression of lefty1 was absent in the midline of chimeric embryos, but shh, a midline marker, was expressed normally, suggesting that, despite formation of midline, its barrier function was abolished. High-contribution chimeras also lacked asymmetric expression of nodal in the node. These data suggest that ACVRI signaling negatively regulates left-side determinants such as nodal and positively regulates lefty1. These functions maintain the midline, restrict expression of left-side markers, and are required for left–right pattern formation during embryogenesis in the mouse. D 2004 Elsevier Inc. All rights reserved.
Keywords: BMP signaling; Midline; Perinode; Left–right asymmetry
Introduction process that is fairly well conserved in amphibians, birds, and mammals, involves several members of the trans- During normal vertebrate development, the asymmetry of forming growth factor-h (TGF-h) protein superfamily the left–right (L–R) axis plays an important role in (Capdevila et al., 2000; Wright, 2001). Studies in chicken positioning and morphogenesis of internal organs. The and Xenopus suggest that bone morphogenetic proteins molecular mechanism to determine L–R asymmetry, a (BMPs), which are members of TGF-h superfamily, play a critical role in L–R patterning. In Xenopus, one of BMP type I receptors, ACVRI/ALK2, transduces right-sidedness * Corresponding author. Molecular Developmental Biology Group, in a manner that is antagonistic to Vg1-dependent signaling Laboratory of Reproductive and Developmental Toxicology, National (Ramsdell and Yost, 1999). Little is currently known about Institute of Environmental Health Sciences, 111 Alexander Drive, Research the role of BMP signaling in establishment of L–R Triangle Park, NC 27709. Fax: +1 919 541 3800. E-mail address: [email protected] (Y. Mishina). asymmetry in mammals (Hamada et al., 2002). 1 Present address: RIKEN Center for Developmental Biology, 2-2-3 Nodal is a TGF-h superfamily protein that is expressed Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan. bilaterally but asymmetrically in the node in early somite
0012-1606/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2004.08.042 186 S. Kishigami et al. / Developmental Biology 276 (2004) 185–193 stage mouse embryos, with notably higher expression on the Materials and methods left than on the right side. In contrast, nodal expression is restricted on the left side in the lateral plate mesoderm Chimera production and analysis (LPM) (Brennan et al., 2001; Collignon et al., 1996). Brennan et al. (2002) showed that node-specific inactivation Generation of Acvr1 mutant ES cells was described of nodal prevents L–R axis asymmetry from being previously (Mishina et al., 1999). In this study, Acvr1 established in the node. Smad2 is a downstream target of mutant ES cells (A2-98) were injected into wild-type CD-1 Nodal, and the importance of the Nodal/Smad2 pathway for blastocysts (Charles River, Wilmington, MA). Results left-side expression was shown by the right isomerism of obtained with A2-98 cells were confirmed by performing mice deficient in Smad2 and the type I receptor Alk4 the same experiment with two other lines of Acvr1 mutant (Nomura and Li, 1998). Lefty1 and Lefty2, distant members ES cells A2-166 and A2-134 (data not shown). Control of the BMP subfamily, are also important for L–R chimeric embryos were generated using Acvr1 heterozygous patterning. lefty1 is expressed in the prospective floor plate ES cell line (A2-65) in the same host blastocysts. All ES (PFP) and is thought to form a bmidline barrierQ that lines except A2-134 carry Rosa26, which carries a lacZ prevents left-side signals from acting on right-side targets transgene. After injection, blastocysts were transferred into (Meno et al., 1998). Like nodal, lefty2 is expressed in the uteri of pseudopregnant foster mothers for reimplantation. left LPM as well as the midline, with a slightly larger Embryos were dissected, fixed, and stained for h-galacto- expression domain than nodal (Meno et al., 1998). sidase activity with X-gal or Salmon-gal (Biosynth) as Biochemical and genetic studies suggest that Lefty proteins described (Mishina et al., 1999). and Nodal bind competitively and act antagonistically on the same receptor complex (Sakuma et al., 2002), suggest- Double staining for b-galactosidase and whole-mount in ing that Lefty1 and Lefty2 facilitate finely tuned control of situ hybridization Nodal during L–R patterning. In early stage chick embryos, BMP signaling suppresses Double staining was carried out using Salmon-gal expression of nodal. Thus, BMP signaling is thought to staining (magenta) for h-galactosidase and alkaline phos- initiate a right-sided molecular cascade that establishes phatase (purple) for whole-mount in situ hybridization with right-side identity in the LPM (Rodriguez-Esteban et al., digoxigenin-labeled RNA probes (Mishina et al., 1999). 1999; Yokouchi et al., 1999). This theory is consistent with Briefly, embryos were stained for h-galactosidase for 30 the fact that constitutive activation of Acvr1 in frog embryos min, fixed in 4% paraformaldehyde in PBS, and then used causes right isomerism (Ramsdell and Yost, 1999). Smad5 is for whole-mount in situ hybridization. Digoxigenin-labeled an intracellular transducer for BMPs, and smad5-deficient probes for whole-mount in situ hybridization are described mice express nodal and lefty2 bilaterally in the LPM and do in the following references: nodal (Lowe et al., 1996), lefty- not express lefty1 in the midline (Chang et al., 2000). 1 (Meno et al., 1998), lefty-2 (Meno et al., 1998), Shh However, a role for BMP signaling in left-side identity has (McMahon et al., 1998), Pitx-2 (Yoshioka et al., 1998). not been ruled out. For example, BMP signaling can induce nodal expression in the LPM in chick embryos (Piedra and Histological analysis and immunohistochemistry Ros, 2002; Schlange et al., 2002), and tetraploid mouse embryo chimeras lacking Bmp4 do not express left-side Whole-mount immunohistochemistry was carried out markers in the LPM (Fujiwara et al., 2002). These findings with monoclonal antibody 2A7 against ACVRI (Yoshikawa suggest that BMPs may also stimulate a left-sided molecular et al., 2000). Stained embryos were fixed in Bouin fixative, cascade involved in L–R patterning. These discrepancies dehydrated, and embedded in paraffin. Serial sections were could indicate that different BMPs have spatially and/or cut at 7 Am and counterstained with eosin. temporally different functions mediated by different recep- tors and transducers, and the combined action of several BMP-dependent signaling pathways may collaborate during Results L–R pattern formation. The goal of this study was to determine the molecular ACVRI is highly expressed in the node and midline, and details of BMP signaling and define the roles of distinct weakly expressed in LPM BMP receptors during L–R axis determination in mouse embryos. In particular, L–R pattern formation was examined The tissue and stage specificity of ACVRI expression in chimeric embryos generated by injection of Acvr1 null was examined in developing mouse embryos. Previous embryonic stem (ES) cells to wild-type blastocysts. The reports showed that Acvr1 (Alk2 or ActR-I) is only results show that ACVRI plays an important role in expressed in extraembryonic regions including visceral establishing L–R asymmetry in the node and is required endoderm in E6.5 embryos (Gu et al., 1999). ACVRI for expression of lefty1 in the midline to maintain L–R appears in a restricted part of the notochord at E8.0 asymmetry. (Yoshikawa et al., 2000). It was previously reported and S. Kishigami et al. / Developmental Biology 276 (2004) 185–193 187 was confirmed here that ACVRI is expressed in the node low (b30%)-, medium (30–80%)-, or high (N80%)-contri- and prospective notochord of E7.75 embryos in addition to bution chimeras. broad expression in the extraembryonic layer (Figs. 1A–E). In mouse embryos, the earliest morphological markers of However, Fig. 1 shows that ACVRI is highly expressed in L–R asymmetry are embryonic turning and rightward the entire node region (Fig. 1E), and in E8.5 embryos, looping of the linear heart tube. These morphologic features ACVRI is weakly expressed in myocardial cells, pharyngeal were examined in wild-type and Acvr1 mutant chimeras endoderm, and LPM (Figs. 1F–G). The expression of with low, medium, or high mutant contribution (Table 1). ACVRI was symmetric in all tissues and stages examined. Normal embryos settle with the tail pointing to the right These results are consistent with the possibility that BMP after completing embryonic turning at E9.5 (Fig. 2A). signaling through ACVRI plays a role in establishing L–R Although some Acvr1 chimeras turned normally (Fig. 2B), asymmetry in the node and in tissues derived from the node. other Acvr1 chimeras underwent reverse turning or failed to turn (Figs. 2C, D; Gu et al., 1999; Mishina et al., 1999). In Acvr1 chimeras show defects in lateral pattern formation summary, 31% (5 of 16) of high-contribution chimeras failed to turn, and 25% (4 of 16) turned in a reverse Previous studies show that Acvr1 null mutants do not orientation (Table 1); medium- and low-contribution Acvr1 complete gastrulation and die by E7.5 (Gu et al., 1999; chimeras turned normally or underwent reverse turning but Mishina et al., 1999). The gastrulation defect is rescued in did not completely fail to turn (Table 1). All chimeric chimeric embryos by the presence of the wild-type extra- embryos with b30% mutant ES cell contribution turned embryonic tissues. However, chimeric embryos with a high normally (0% chimeras: n = 13; b30% chimeras: n = 8). proportion of Acvr1 null ES cells survive to E10.5 with In wild-type embryos, the rostral part of the heart loops developmental defects including abnormal head structure towards the right and the caudal part twists to the left and shortening of the posterior (Gu et al., 1999; Mishina et beginning at around E8.5 and ending by E9.5. This process al., 1999). Chimeric Acvr1 embryos were used in the failed to occur in 12 of 19 high-contribution chimeras (Figs. following studies to analyze the role of ACVRI in L–R 2E, F). Three of 22 high-contribution chimeras showed pattern formation during mouse embryogenesis. incomplete fusion of the two cardiac primordia as reported Acvr1 chimeras were formed from wild-type blastocysts in Smad5 mutants (data not shown). This defect of fusion and Acvr1 homozygous mutant ES cells carrying a lacZ was not observed in medium- or low-contribution Acvr1 transgene from Rosa26 mice (Mishina et al., 1999). The chimeras (data not shown). Two of nine medium-contribu- lacZ transgene is used as a visual method (X-gal or Salmon- tion chimeras had a leftward looping heart (Fig. 2H), gal staining) to quantify and track the developmental fate of suggesting a link between abnormal heart looping and Acvr1 mutant ES cells in the chimeric embryo. The defective L–R patterning. Heart looping was normal in low- proportion of staining tissue was evaluated in each chimeric contribution and control chimeras (high-contribution control embryo, and the chimeras were classified as wild-type (0%), chimeras: n = 14) (Table 1). These results show that
Fig. 1. Detection of ACVRI protein during mouse development. Whole-mount immunohistochemistry was performed using mAb 2A7 to stain ACVRI protein at E7.75–8.0 (A, B) and E8.5 (F, G). Strong staining was observed in extraembryonic regions and in the midline region as observed in frontal (A, F) and bottom views of embryos (B, G). Transverse sections revealed limited expression of ACVR1 in the node and prospective notochord at E7.75 (C–E), and broader expression in the pharyngeal endoderm, heart, and lateral plate mesoderm and the notochord at E8.5 (H and I). Sectioned images are shown with anterior– posterior axis running from bottom to top. The line diagram indicates the level and angle of section shown in panel E. No signal was found in negative control (normal IgG, data not shown). Abbreviations: A, anterior; P, posterior; al, allantois; nd, node; nt, notochord; ht, heart; pe, pharyngeal endoderm; lpm, LPM. Scale bar = 100 Am. 188 S. Kishigami et al. / Developmental Biology 276 (2004) 185–193
Table 1 Embryo morphology in low, medium- and high-contribution Acvr1 chimeras Contribution Embryonic turning (E9.5) Total Normal (%) Opposite (%) No turning (%) N80% 7 (44) 4 (25) 5 (31) 16 30–80% 6 (67) 3 (33) 0 (0) 9 b30% 8 (100) 0 (0) 0 (0) 8 0% 13 (100) 0 (0) 0 (0) 13
Contribution Heart looping (E9.5) Total Rightward (%) Leftward (%) No looping (%) N80% 7 (37) 0 (0) 12 (63) 19a 30–80% 6 (66) 2 (22) 1 (11) 9 b30% 10 (100) 0 (0) 0 (0) 10 0% 23 (100) 0 (0) 0 (0) 23 a Embryos with incomplete fusion of the two cardiac primordia were not included. abnormalities of L–R patterning occur more frequently and in the left LPM; however, aberrant ectopic expression of with increasing severity in chimeric embryos with a greater these markers was observed in the right LPM (Figs. 3C, F, proportion of Acvr1 null cells. I). The frequency of ectopic expression of Pitx2 and nodal decreased as h-galactosidase activity decreased and as the Acvr1 chimeras express left-side markers bilaterally in LPM proportion of mutant ES cells contributing to the chimera decreased (Table 2). L–R asymmetry was examined at the molecular level in Expression of lefty2 was also examined in Acvr1 the Acvr1 chimeras by measuring expression of left-side chimeras. lefty2 is a TGF-h family member that is expressed marker genes Pitx2 and nodal in the LPM. These genes are moderately in the left LPM and weakly in the PFP (Meno et required for L–R patterning in the mouse (Kitamura et al., al., 1998). In high-contribution Acvr1 chimeras (n = 4), 1999; Lin et al., 1999; Lu et al., 1999; Lowe et al., 2001). aberrant expression of lefty2 was observed, with bilateral Pitx2 and nodal expression was examined by in situ expression in the LPM and no expression in the PFP (Fig. hybridization, and h-galactosidase activity was measured 3K). In general, these results indicate that loss of Acvr1 to assess the fraction of cells that are deficient in Acvr1 disrupts the normal asymmetric patterns of gene expression (S.K., in preparation). In most of the high-contribution associated with L–R patterning during embryogenesis. The Acvr1 chimeras, Pitx2 and nodal were expressed normally extent of disruption of normal L–R patterning is propor-
Fig. 2. Abnormal embryonic turning and heart looping in Acvr1 chimeras. Chimeric embryos were dissected at E9.5 (A–F) and E8.5 (G, H). Embryos were stained with X-gal (blue) (A–F) or Salmon-gal (magenta) (G, H). Staining pattern correlates to the proportion of ES-derived cells contributing to the chimera. (A–D) The position of tail tips (arrowheads) after turning indicates the direction of turning. High-contribution chimeras of Acvr1 heterozygous ES cells (control chimeras) display normal axial turning (A), but high-contribution chimeras of Acvr1 null ES cells (called Acvr1 chimeras) display randomization of axial turning (B, C) or failure to turn (D). (E, F) The heart tube (side view) of a control chimera and an Acvr1 chimera. The Acvr1 chimera does not initiate looping. (G, H) Randomized looping of the heart in medium-contribution Acvr1 chimeras. S. Kishigami et al. / Developmental Biology 276 (2004) 185–193 189
Fig. 3. Bilateral expression of left-side marker genes in LPM. Embryos were stained for h-galactosidase with Salmon-gal (magenta) and for left marker gene expression by whole-mount in situ hybridization (dark purple). Expression of Pitx2 (A–F), nodal (G–I, L), or lefty2 (J, K) was measured at E8.2 (A–C, G–L) or E8.5–9.0 (D–F) in Acvr1 chimeric embryos. (A, D, G, J) Gene expression in the left LPM in no-contribution chimeras (closed arrowheads). (B, E, H) Gene expression in medium-contribution chimeras which tend to lack right-sided gene expression (closed arrowheads). (C, F, I, K, L) Gene expression in high- contribution chimeras tends to show bilateral gene expression in the LPM (closed arrowheads). lefty2 is expressed in the midline (open arrowheads) and in the left LPM in a no-contribution chimera (J), but lefty2 is not expressed in the midline of high-contribution Acvr1 chimera (open arrowhead, K). (L) Some high- contribution chimeras only express nodal bilaterally in the posterior half of the LPM (closed arrowhead). Expression in the node region is indicated by an open arrowhead. Abbreviations: A, anterior; P, posterior. tional to the number of Acvr1 null ES cells in the chimeric Shh and lefty1 in the Acvr1 chimeras. SHH is essential for L– embryos (Figs. 3B, E, H). Interestingly, some chimeras R patterning at the midline (Izraeli et al., 1999; Meyers and (three of eight) only expressed nodal bilaterally in the Martin, 1999; Tsukui et al., 1999), and SHH- and IHH- posterior half of the LPM (Fig. 3L). These results are dependent signaling is required for expression of nodal in the consistent with observations of aberrant gene expression node and lefty1 in the PFP (Zhang et al., 2001). It has been associated with defective L–R patterning in Smad5-deficient proposed that expression of lefty1 is required to form a mouse embryos (Chang et al., 2000). bmidline barrierQ (Meno et al., 1998). In high-contribution Acvr1 chimeras (n = 7) from E8.0 to E8.5, expression of Shh AVCRI is essential for midline function was normal in the node and midline (Fig. 4D and data not shown). However, expression of lefty1 (Fig. 4C) and lefty2 In wild-type embryos, ACVRI is highly expressed in the (Fig. 3K) was completely lost in the midline of high- node and the midline (Fig. 1). It is possible that deficiency of contribution embryos (n = 5). In medium-contribution ACVRI in the node and the prospective notochord (midline) chimeras, lefty1 was expressed weakly (two of four) or not could cause L–R patterning defects such as abnormal at all (two of four) (Fig. 4B). These data indicate that ACVRI expression of left-side markers, as described above in Acvr1 signaling in the midline is essential for induction of lefty1 and chimeras. This idea was tested by examining expression of lefty2 in the PFP and to form the bmidline barrier.Q 190 S. Kishigami et al. / Developmental Biology 276 (2004) 185–193
Table 2 Expression of left marker genes in Acvr1 mutant chimeric embryos Contribution Left (%) Right (%) Bilateral (%) Absent (%) Total Expression of Pitx2 (E8.5–E9.0) N80% 2 (29) 0 (0) 5 (71) 0 (0) 7 30–80% 8 (61) 1 (7) 4 (31) 0 (0) 13 b30% 9 (100) 0 (0) 0 (0) 0 (0) 9 0% 21 (100) 0 (0) 0 (0) 0 (0) 21
Expression of Nodal in the LPM (three- to four-somite stage) N80% 0 (0) 0 (0) 8 (100)a 0 (0) 8 30–80% 3 (38) 1 (13) 4 (50) 0 (0) 8 b30% 6 (100) 0 (0) 0 (0) 0 (0) 6 0% 23 (100) 0 (0) 0 (0) 0 (0) 23
Expression of Nodal in perinode (three- to four-somite stage) Contribution Left N Right (%) Left = Right (%) Left b Right (%) Total N80% 1 (9) 9 (82) 1 (9) 11 30–80% 4 (100)b 0 (0) 0 (0) 4 b30% 13 (93) 1 (7) 0 (0) 14 0% 16 (94) 1 (6) 0 (0) 17 a In three embryos, right-sided expression was restricted to the caudal half of the LPM. b One embryo showed bilateral expression of nodal in the LPM.
In lefty1 mutant embryos, unlike in Acvr1 chimeras, genesis. Earlier studies indicated that ACVR1 negatively nodal is not expressed bilaterally in the LPM at the two- to regulates expression of left-side determinants in Xenopus three-somite stage (Meno et al., 1998), and at later stages, embryos, suggesting that ACVRI function is evolutionarily bilateral expression of nodal is limited to the anterior half of conserved from Xenopus to mouse. the embryo (Meno et al., 1998). Thus, lefty1 mutants are The downstream events of the ACVR1 signaling path- phenotypically distinct from Acvr1 chimeras, suggesting way and their role in L–R pattern formation have not yet that loss of lefty1 expression in the midline is not sufficient been described in detail. However, because Smad5 mutant to account for the disruption of L–R patterning in Acvr1 embryos are phenotypically similar to Acvr1 chimeric chimeras (Figs. 3I, L). nodal expression and ACVR1 embryos, it is possible that Smad5 acts downstream of function were also examined in the node of Acvr1 chimeras. ACVR1 during L–R pattern formation in vivo. It has also Asymmetric expression of nodal in the perinode region is been reported that Smad5 can transduce BMP6 and BMP7 temporally limited to the two- to three-somite stage during signals through ACVRI in vitro (Ebisawa et al., 1999; embryogenesis in wild-type embryos (Fig. 4E, Collignon et Macias-Silva et al., 1998). Bmp7 is only the known BMP al., 1996; Lowe et al., 1996). In high-contribution Acvr1 member that is expressed in the node (Solloway and mutant chimeras (9 of 11), nodal was expressed nearly Robertson, 1999), but Bmp7 null embryos are proficient symmetrically at the two- to three-somite stage (Fig. 4G, in L–R pattern formation (Dudley et al., 1995; Luo et al., Table 2). In some chimeras, nodal was expressed at a higher 1995). Thus, ligands of ACVRI that are functionally level in the right side than in the left side of the perinode, important during L–R patterning remain to be identified, which was not observed in wild-type or low-contribution but it is likely that mouse BMPs play a role in this process. chimeras (Figs. 4E–G). These results were confirmed in Recent studies of tetraploid chimeric embryos containing embryo sections (Figs. 4I–L). In addition, in high-contribu- Bmp4 null ES cells revealed that BMP4 positively regulates tion Acvr1 chimera (n = 3) that only expresses nodal left-side patterning (Fujiwara et al., 2002). BMP4 null bilaterally in the posterior half of the LPM, nodal chimeric embryos are morphologically normal in the node expression was incompletely down-regulated in the anterior region, but expression of nodal is patchy in the perinode, part of the peripheral node (Fig. 4H, arrowhead). These data and expression of nodal and lefty2 is suppressed in the left indicate that ACVR1-dependent signaling is required for LPM (Fujiwara et al., 2002). Thus, the phenotype of Bmp4 asymmetric expression of nodal in the node. null tetraploid chimeric embryos is the opposite of the phenotype of Acvr1 null chimeras, with respect to patterns of gene expressions along the L–R axis. Since Bmp4 is not Discussion expressed at the node region or notochord, BMP4 is not likely to play a role in establishing asymmetric expression This study demonstrates that ACVRI signaling is of nodal at the node. Bmp4 is expressed in the extraem- essential for normal L–R patterning during mouse embryo- bryonic region in pre-/early streak stage embryos and in the S. Kishigami et al. / Developmental Biology 276 (2004) 185–193 191
Fig. 4. Function of ACVRI in the node and the midline. (A–C) lefty1 expression in no-, medium-, and high-contribution Acvr1 chimeras in the midline (open arrowhead). (D) Shh expression at E8.0 in high-contribution Acvr1 chimera in the midline (open arrowhead) and the node (closed arrowhead). (E–H) nodal expression at the three- to four-somite stage in the node (closed arrowhead) of no-contribution, medium-contribution, and two types of high-contribution Acvr1 chimeras; complete bilateral expression in the LPM and bilateral expression in the posterior half of the LPM (open arrowhead and closed arrow, respectively). (I–L) Transverse sections of the node region of no-, medium-, and high-contribution Acvr1 chimeras. K and L are from the same high-contribution chimera with 14-Am distance. The insets in B and D show high magnification of midline area. Insets in E to H show the node in posterior view. Abbreviations: A, anterior; P, posterior. Bar = 50 Am. most posterior mesoderm of the primitive streak. It is likely L–R patterning in the mouse embryo (Fig. 5). However, it is that expression of Bmp4 in the primitive streak influences formally possible that ACVRI signaling is also important expression of nodal at the perinode, but this interaction is for maintaining L–R asymmetry in the LPM in the mouse not likely to involve ACVR1 signaling. embryo. Additional studies, such as tissue-specific disrup- Other evidence suggests that multiple BMPs are required tion of Acvr1, are needed to define temporal and spatial to establish and maintain L–R asymmetry during mouse variations in ACVRI signaling during mouse development embryogenesis. Thus, different BMPs may play different and L–R patterning. roles in different tissues and at different developmental Defects in lateral pattern formation may be secondary to stages, and these ligands may stimulate downstream events defects in anterior–posterior polarity (i.e., malformation of through different receptors. For example, studies in chick midline structure; Dufort et al., 1998). However, the midline embryos suggest that BMP2, BMP4, and BMP7 can forms normally in Acvr1 chimeras as indicated by normal stimulate expression of nodal in the LPM (Piedra and expression of Shh (Fig. 4), and a normal notochord forms in Ros, 2002; Schlange et al., 2002). In addition, mouse tetraploid Acvr1 null chimeric embryos (Mishina et al., embryos cultured with Noggin do not express nodal in the 1999). Thus, it is likely that the primary defect in Acvr1 null LPM (Fujiwara et al., 2002). These findings, opposite to the chimeric embryos is in L–R patterning and not in anterior– ones in Acvr1 chimeras, suggest that BMPs also may act as posterior patterning. a positive facilitator of the left-sided molecular cascade in This study demonstrates that ACVRI signaling is the LPM signaling through other type I receptors. Although required to induce expression of lefty1 and form the ACVRI is weakly expressed in the LPM (Fig. 1), the results bmidline barrier.Q Recent studies of mouse embryos with presented here suggest that ACVRI signaling plays primary an LPM-specific deletion of Foxh1 defined a positive roles in the node and the midline but not in the LPM during regulatory loop involving spatially separate expression of 192 S. Kishigami et al. / Developmental Biology 276 (2004) 185–193
show that ACVRI is required for asymmetric expression of nodal in the node. Although expression of nodal in the node is essential for L–R patterning (Brennan et al., 2002), there is no evidence that asymmetric expression of nodal is required for L–R patterning. Nevertheless, our finding that the loss of ACVRI signaling results in aberrant asymmetric expression of nodal in the perinode suggests that ACVRI regulates nodal expression in the node and that this is important for L–R patterning. BMP antagonists including noggin, chordin, and Bmp7 are expressed in the node (Solloway and Robertson, 1999). Interestingly, noggin and chordin double mutant embryos show a randomization of heart looping (Bachiller et al., 2000). Since these molecules are expressed in the node, their role in ACVRI signaling should be studied further. In addition, HH signaling acts as a left determinant in the node, although it is expressed symmetrically (Zhang et al., 2001). Fig. 5. Model for function of ACVRI L–R patterning in the mouse embryo. The interaction between BMPs and SHH in Hensen node ACVRI signaling in the node modifies nodal expression induced by HH. plays a key role in L–R patterning in the chick (Monsoro- This results in asymmetric expression of nodal. ACVRI signaling acts as upstream of Smad5; Smad5 is required to induce expression of lefty1 in the Burq and Le Douarin, 2001). It is tempting to speculate that left side of the midline (blue region). Shh is normally expressed in the BMP signaling through ACVRI in the node functions midline of Acvr1 chimeras (Fig. 4D), suggesting that ACVRI signaling acts antagonistically to HH signaling and negatively regulates in parallel with Shh signaling in the midline. BMPs can induce nodal nodal expression during L–R patterning (Fig. 5). expression at the LPM in mouse embryos, but this process may involve multiple BMP receptor(s) (blue arrow).
Acknowledgments left-side markers. In particular, Nodal protein is expressed in the node and migrates to the LPM where it induces We thank Dr. Michael Kuehn for helpful comments on additional expression of Nodal in the left LPM. The Nodal the manuscript and advice for in situ hybridization as well as protein expressed in the LPM migrates to the PFP, where it nodal probe; Drs. Christopher Wright and Manas Ray for induces expression of lefty1 (Yamamoto et al., 2003). helpful comments on the manuscript; Drs. Brigid Hogan and However, in Acvr1 chimeras, nodal is expressed in the Takeshi Fujiwara for helpful discussion; Drs. Allan Bradley LPM, which indicates that ACVRI signaling is involved in and Robert Crombie for Acvr1 mutant mice; Drs. Hiroshi expression of lefty1 at the midline in parallel to nodal. Since Hamada and Chikara Meno for lefty and Pitx2 probes; and ACVRI was detected in notochord, but not in the PFP by the Dr. Andrew McMahon for Shh probe. We also thank Greg time of E8.5 (Fig. 1), it is reasonable to speculate that an Scott and Toni Ward for assistance in the mouse activity and ACVRI-dependent signal from notochord may be essential Kuniko Kishigami for encouragement. S. K. was a recipient for expression of lefty1 (and lefty2, see below) in the PFP of a fellowship from the Japan Society for the Promotion of (Fig. 5). Furthermore, the serial sections of Shh- and nodal- Science. stained chimeras revealed that Shh was expressed in notochord, but not yet in the PFP in Acvr1 chimeras, and is as comparable to the wild-type embryos at E8.0, without References any overt morphological abnormalities in the notochord and the PFP (Fig. 4 and data not shown). Thus, midline Bachiller, D., Klingensmith, J., Kemp, C., Belo, J.A., Anderson, R.M., May, S.R., McMahon, J.A., McMahon, A.P., Harland, R.M., Rossant, structures including the notochord and the PFP normally J., De Robertis, E.M., 2000. The organizer factors Chordin and Noggin developed in the Acvr1 chimeras, suggesting that the lack of are required for mouse forebrain development. Nature 403, 658–661. lefty1 expression in the PFP is not caused by abnormal Brennan, J., Lu, C.C., Norris, D.P., Rodriguez, T.A., Beddington, R.S., structure of the PFP, but rather lacking the induction signals Robertson, E.J., 2001. Nodal signaling in the epiblast patterns the early from notochord. Interestingly, expression of lefty2 is up- mouse embryo. Nature 411, 965–969. Brennan, J., Norris, D.P., Robertson, E.J., 2002. Nodal activity in the node regulated in lefty1 null embryos (Meno et al., 1998), but governs left–right asymmetry. Genes Dev. 16, 2339–2344. lefty2 expression is suppressed at the midline of the high- Capdevila, J., Vogan, K.J., Tabin, C.J., Izpisua Belmonte, J.C., 2000. contribution Acvr1 chimeras. The fact that smad5 mutant Mechanisms of left–right determination in vertebrates. Cell 101, 9–21. embryos also suppress expression of lefty1 and lefty2 in the Chang, H., Zwijsen, A., Vogel, H., Huylebroeck, D., Matzuk, M.M., 2000. midline (Chang et al., 2000) suggests that ACVRI signaling Smad5 is essential for left–right asymmetry in mice. Dev. Biol. 219, 71–78. may regulate their expression through smad5 (Fig. 5). Collignon, J., Varlet, I., Robertson, E.J., 1996. Relationship between The function of ACVRI signaling in the node is not yet asymmetric nodal expression and the direction of embryonic turning. clear, and this question remains a subject of debate. Here, we Nature 381, 155–158. S. Kishigami et al. / Developmental Biology 276 (2004) 185–193 193
Dudley, A.T., Lyons, K.M., Robertson, E.J., 1995. A requirement for bone Meyers, E.N., Martin, G.R., 1999. Differences in left–right axis morphogenetic protein-7 during development of the mammalian kidney pathways in mouse and chick, functions of FGF8 and SHH. Science and eye. Genes Dev. 9, 2795–2807. 285, 403–406. Dufort, D., Schwartz, Lois, Harpal, K., Rossant, J., 1998. The transcription Mishina, Y., Crombie, R., Bradley, A., Behringer, R.R., 1999. Multiple factor HNF3b is required in visceral endoderm for normal primitive roles for activin-like kinase-2 signaling during mouse embryogenesis. streak morphogenesis. Development 125, 3015–3025. Dev. Biol. 213, 314–326. Ebisawa, T., Tada, K., Kitajima, I., Tojo, K., Sampath, T.K., Kawabata, M., Monsoro-Burq, A., Le Douarin, N.M., 2001. BMP4 plays a key role in left– Miyazono, K., Imamura, T., 1999. Characterization of bone morphoge- right patterning in chick embryos by maintaining Sonic hedgehog netic protein-6 signaling pathways in osteoblast differentiation. J. Cell asymmetry. Mol. Cell 7, 789–799. Sci. 112, 3519–3527. Nomura, M., Li, E., 1998. Smad2 role in mesoderm formation, left–right Fujiwara, T., Dehart, D.B., Sulik, K.K., Hogan, B.L.M., 2002. Distinct patterning and craniofacial development. Nature 393, 786–790. requirements for extra-embryonic and embryonic bone morphogenetic Piedra, M.E., Ros, M.A., 2002. BMP signaling positively regulates Nodal protein 4 in the formation of the node and primitive streak and expression during left right specification in the chick embryo. coordination of left–right asymmetry in the mouse. Development 129, Development 129, 3431–3440. 4685–4696. Ramsdell, A.F., Yost, H.J., 1999. Cardiac looping and the vertebrate left– Gu, Z., Reynolds, E.M., Song, J., Lei, H., Feijen, A., Yu, L., He, W., right axis: antagonism of left-sided Vg1 activity by a right-sided ALK2- MacLaughlin, D.T., van den Eijnden-van Raaij, J., Donahoe, P.K., Li, dependent BMP pathway. Development 126, 5195–5205. E., 1999. The type I serine/threonine kinase receptor ActRIA (ALK2) is Rodriguez-Esteban, C., Capdevila, J., Economides, A.N., Pascual, J., Ortiz, required for gastrulation of the mouse embryo. Development 126, A., Izpisua-Belmonte, J.C., 1999. The novel Cer-like protein Caronte 2551–2561. mediates the establishment of embryonic left–right asymmetry. Nature Hamada, H., Meno, C., Watanabe, D., Saijoh, Y., 2002. Establishment of 401, 243–251. vertebrate left–right asymmetry. Nat. Rev., Genet. 3, 103–113. Sakuma, R., Ohnishi, Y., Meno, C., Fujii, H., Juan, H., Takeuchi, J., Ogura, Izraeli, S., Lowe, L.A., Bertness, V.L., Kuehn, M.R., 1999. The SIL gene is T., Li, E., Miyazono, K., Hamada, H., 2002. Inhibition of Nodal required for mouse embryonic axial development and left–right signalling by Lefty mediated through interaction with common specification. Nature 399, 691–694. receptors and efficient diffusion. Genes Cells 7, 401–412. Kitamura, K., Miura, H., Miyagawa-Tomita, S., Yanazawa, M., Katoh-Fukui, Schlange, T., Arnold, H.H., Brand, T., 2002. BMP2 is a positive regulator Y., Suzuki, R., Ohuchi, H., Suehiro, A., Motegi, Y., Nakahara, Y., Kondo, of Nodal signaling during left–right axis formation in the chicken S., Yokoyama, M., 1999. Mouse Pitx2 deficiency leads to anomalies of embryo. Development 129, 3421–3429. the ventral body wall, heart, extra- and periocular mesoderm and right Solloway, M.J., Robertson, E.J., 1999. Early embryonic lethality in pulmonary isomerism. Development 126, 5749–5758. Bmp5;Bmp7 double mutant mice suggests functional redundancy Lin, C.R., Kioussi, C., O’Connell, S., Briata, P., Szeto, D., Liu, F., Izpisua- within the 60A subgroup. Development 126, 1753–1768. Belmonte, J.C., Rosenfeld, M.G., 1999. Pitx2 regulates lung asymme- Tsukui, T., Capdevila, J., Tamura, K., Ruiz-Lozano, P., Rodriguez-Esteban, try, cardiac positioning and pituitary and tooth morphogenesis. Nature C., Yonei-Tamura, S., Magallon, J., Chandraratna, R.A., Chien, K., 401, 279–282. Blumberg, B., Evans, R.M., Belmonte, J.C., 1999. Multiple left–right Lowe, L.A., Supp, D.M., Sampath, K., Yokoyama, T., Wright, C.V., Potter, asymmetry defects in Shh(À/À) mutant mice unveil a convergence of S.S., Overbeek, P., Kuehn, M.R., 1996. Conserved left–right asymmetry the shh and retinoic acid pathways in the control of Lefty-1. Proc. Natl. of nodal expression and alterations in murine situs inversus. Nature 381, Acad. Sci. U. S. A. 96, 11376–11381. 158–161. Wright, C.V., 2001. Mechanisms of left–right asymmetry: what’s right and Lowe, L.A., Yamada, S., Kuehn, M.R., 2001. Genetic dissection of what’s left? Dev. Cell 1, 179–186. nodal function in patterning the mouse embryo. Development 128, Yamamoto, M., Mine, N., Mochida, M., Sakai, Y., Saijoh, Y., Meno, C., 1831–1843. Hamada, H., 2003. Nodal signaling induces the midline barrier by Lu, M.F., Pressman, C., Dyer, R., Johnson, R.L., Martin, J.F., 1999. activating Nodal expression in the lateral plate. Development 130, Function of Rieger syndrome gene in left–right asymmetry and 1795–1804. craniofacial development. Nature 401, 276–278. Yokouchi, Y., Vogan, K.J., Pearse II, R.V., Tabin, C.J., 1999. Antagonistic Luo, G., Hofmann, C., Bronckers, A.L., Sohocki, M., Bradley, A., Karsenty, signaling by Caronte, a novel Cerberus-related gene, establishes left– G., 1995. BMP-7 is an inducer of nephrogenesis, and is also required for right asymmetric gene expression. Cell 98, 573–583. eye development and skeletal patterning. Genes Dev. 9, 2808–2820. Yoshikawa, S., Aota, S., Shirayoshi, Y., Okazaki, K., 2000. The ActR-I Macias-Silva, M., Hoodless, P.A., Tang, S.J., Buchwald, M., Wrana, J.L., activin receptor protein is expressed in notochord, lens placode and 1998. Specific activation of smad1 signaling pathways by the BMP7 pituitary primordium cells in the mouse embryo. Mech. Dev. 91, type I receptor, ALK2. J. Biol. Chem. 273, 25628–25636. 439–444. McMahon, J.A., Takada, S., Zimmerman, L.B., Fan, C.M., Harland, R.M., Yoshioka, H., Meno, C., Koshiba, K., Sugihara, M., Itoh, H., Ishimaru, Y., McMahon, A.P., 1998. Noggin-mediated antagonism of BMP signaling Inoue, T., Ohuchi, H., Semina, E.V., Murray, J.C., Hamada, H., Noji, S., is required for growth and patterning of the neural tube and somite. 1998. Pitx2, a bicoid-type homeobox gene, is involved in a lefty- Genes Dev. 12, 1438–1452. signaling pathway in determination of left–right asymmetry. Cell 94, Meno, C., Shimono, A., Saijoh, Y., Yashiro, K., Mochida, K., Ohishi, 299–305. S., Noji, S., Kondoh, H., Hamada, H., 1998. lefty-1 is required for Zhang, X.M., Ramalho-Santos, M., McMahon, A.P., 2001. Smoothened left–right determination as a regulator of lefty-2 and nodal. Cell 94, mutants reveal redundant roles for Shh and Ihh signaling including 287–297. regulation of L/R symmetry by the mouse node. Cell 106, 781–792.