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Twisted gastrulation mutation suppresses skeletal defect phenotypes in Crossveinless 2 mutant mice
MECHANISMS OF DEVELOPMENT
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Twisted gastrulation mutation suppresses skeletal defect phenotypes in Crossveinless 2 mutant mice Makoto Ikeyaa,*, Tetsuya Nosakab, Kumi Fukushimaa, Masako Kawadaa, Yasuhide Furutac, Toshio Kitamurad, Yoshiki Sasaia a
Organogenesis and Neurogenesis Group, RIKEN, Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo, Kobe 650-0047, Japan b Department of Microbiology, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan c Department of Biochemistry and Molecular Biology, M.D. Anderson Cancer Center, University of Texas, Houston, TX 77030, USA d Division of Cellular Therapy, The Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan
A R T I C L E I N F O
A B S T R A C T
Article history:
Bone morphogenetic protein (BMP) signaling controls various aspects of organogenesis,
Received 11 January 2008
including skeletal development. We previously demonstrated that the pro-BMP function
Received in revised form
of Crossveinless 2 (Cv2) is required for axial and non-axial skeletal development in mice.
25 June 2008
Here, we showed that skeletal defects in the Cv2-null mutant were reversed by the addi-
Accepted 26 June 2008
tional deletion of Twisted gastrulation (Tsg). Whereas the Cv2/ mutant lacks a substantial
Available online 3 July 2008
portion of the lumbar vertebral arches, Cv2/;Tsg/ mice have almost normal arches. Suppression of Cv2/ phenotypes is also seen in the non-axial skeleton, including the ribs,
Keywords:
humerus, skull, and laryngeal and tracheal cartilages. In contrast, the Tsg/ phenotype in
Mouse
the head is not significantly affected by the Cv2 mutation. These findings demonstrate that
Crossveinless 2 (Bmper)
Tsg mutation is epistatic to Cv2 mutation in the major skeletal phenotypes, suggesting that
Twisted gastrulation
the pro-BMP activity of Cv2 is, at least in part, dependent on Tsg. We also present genetic
BMP7
evidence for the context-dependent functional relationship between Tsg and Cv2 during
pro-BMP
mouse development.
Suppression
Ó 2008 Elsevier Ireland Ltd. All rights reserved.
Skeleton Genetic interaction
1.
Introduction
BMP signaling plays important regulatory roles in a variety of aspects of vertebrate and invertebrate development, including skeletal development (reviewed in Wan and Cao, 2005). In addition, BMP signals regulate cell-type specification, maturation, cell growth, apoptosis, and dorsal–ventral axis determination (De Robertis and Sasai, 1996; Hogan, 1996; Massague and Chen, 2000; Hammerschmidt and Mullins, 2002; De Robertis and Kuroda, 2004; Yamamoto and Oelgeschla¨ger, 2004; De Robertis, 2006). A number of factors
that negatively regulate BMP signals in the extracellular space have been identified (anti-BMP factors). A typical example is a class of secreted antagonist proteins, including Noggin, Chordin, Follistatin, Cerberus, and Gremlin, that bind to, and inactivate, BMP proteins (Smith and Harland, 1992; Lamb et al., 1993; Sasai et al., 1994; Sasai et al., 1995; Hemmati-Brivanlou et al., 1994; Glinka et al., 1997; Hsu et al., 1998). In contrast to these anti-BMP factors, several extracellular BMP-interacting proteins are reported to modulate BMP signaling in a positive manner. For instance, we previously reported in a study of knockout mice for Crossveinless 2 (Cv2),
* Corresponding author. Tel.:+81 78 306 1845; fax +81 78 306 1854. E-mail address: [email protected] (M. Ikeya). 0925-4773/$ - see front matter Ó 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mod.2008.06.011
MECHANISMS OF DEVELOPMENT
that Cv2 functions as an enhancer molecule of BMP signaling (pro-BMP factor) in mouse skeletal development (Ikeya et al., 2006). Cv2 is a BMP-binding protein that was first identified in a fly mutant study as being required for the formation of crossveins in the fly wing (Garcia-Bellido and de Celis, 1992). Genetic studies in flies have shown that the formation of these veins requires high BMP-signaling activity (involving Dpp and Gbb) and that Cv2 is essential for enhancing the local BMP signal near the receiving cells (Conley et al., 2000; O’Connor et al., 2006; Serpe et al., 2008). In mice, the Cv2 mutant phenotype in the skeleton is enhanced in the BMP4+/ background, showing that Cv2 and BMP4 work together in the same direction for skeletal development (Ikeya et al., 2006). This idea is consistent with non-mammalian and in vitro studies that suggested pro-BMP activity of Cv2 (Conley et al., 2000; Coles et al., 2004; Kamimura et al., 2004; Ralston and Blair, 2005; Rentzsch et al., 2006; Moser et al., 2007; Serpe et al., 2008). A similar pro-BMP role has been reported for another Cv2-class molecule, kielin/chordin-like protein (KCP) in kidney repair (Lin et al., 2005). Interestingly, these factors (Cv2 and KCP) contain multiple cystein-rich repeats homologous to those in Chordin. Since a characteristic feature of BMP signals is their function as a morphogen (generating an activity gradient and evoking multiple-threshold responses) (Gurdon and
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Bourillot, 2001), fine spatial control of BMP signals by these pro- and anti-BMP factors is important for tissue formation to occur in the right place when the tissue is at the right size. In addition, these pro- and anti-BMP factors themselves are known to regulate one another in an intricate manner. The activity of the anti-BMP factor Chordin is itself regulated by multiple interacting factors, such as Twisted gastrulation (Tsg) (Harland, 2001), Xolloid-related protease (Ge and Greenspan, 2006) and Sizzled (Kimelman and Szeto, 2006). These interactions may be regulated in a contextdependent fashion, and their quantitative control remains elusive at the molecular level. For example, whether Tsg acts as a pro- or anti-BMP factor has been addressed through multiple approaches but has not yet been totally clarified (see Section 2.5). Similarly, it is still unclear whether Chordin in mice has any pro-BMP role, which has been suggested for Sog (fly Chordin homolog) and Chordino in Drosophila and zebrafish, in addition to its anti-BMP role (Zusman et al., 1988; Ashe and Levine, 1999; Decotto and Ferguson, 2001; Hammerschmidt and Mullins, 2002; Rentzsch et al., 2006). As for Cv2, a previous study in zebrafish and in vitro studies suggested that Cv2 could also function as an anti-BMP factor, at least under certain artificial conditions (Moser et al., 2003; Binnerts et al., 2004; Rentzsch et al., 2006; Serpe et al., 2008).
Fig. 1 – Genetic crossing of Cv2 and Tsg mutants. (A) Analysis of genotypes by allele-specific genomic PCR. WT, wild type allele; MT, mutant allele. (B) Gross skeletal phenotypes of the compound mutants at E18.5. Skeletal preparations were stained with Alcian Blue and Alizarin Red. va, vertebral arch.
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Therefore, the extracellular regulation of BMP signaling involves multiple factors that interact positively and negatively in an intricate fashion; the careful investigation of such interactions by loss-of-function approaches is essential for understanding the actual in vivo functions. In an effort to elucidate the dynamic regulatory network of BMP signaling during mammalian organogenesis, we performed a gene interaction analysis of Cv2 and Tsg mutations in this study. Since our previous study clearly demonstrated the pro-BMP role of Cv2 in skeletal development (positive genetic interaction between Cv2 and BMP4 (Ikeya et al., 2006); a similar interaction between Cv2 and BMP7 is also shown later in Sections 2.1 and 2.4), we focused here on skeletal phenotypes. We demonstrate here that the Tsg mutation has a strong suppressive effect on the bone and cartilage defects caused by a Cv2-null mutation in axial and non-axial structures. In the majority of skeletal phenotypes, the epistatic relationship of Tsg mutation and Cv2 mutation is unidirectional. Interestingly, our detailed analysis of minor skeletal phenotypes suggested that the genetic interaction between these two mutations could be different in some other contexts. We discuss the possible roles and modes of action of these BMP modulators in skeletal development.
2.
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Results and discussion
2.1. Axial skeletal phenotypes of the Cv2-null mutant are suppressed by the Tsg mutation To examine the possible genetic interaction between Cv2 and Tsg, we crossed these mutant mice (Fig. 1A). As previously reported, Tsg-null mice were born alive and grew, and both male and female mutants were fertile (Nosaka et al., 2003; Petryk et al., 2004; Zakin and De Robertis, 2004). They showed moderate phenotypes in the skeleton at embryonic day 18.5 (E18.5), such as a short tail and partial loss of the cervical vertebral arches (Figs. 1B, 2B and 2F). Cv2+/;Tsg/ mice showed phenotypes indistinguishable from those of Tsg null mutants (data not shown), and the expression of Cv2 in the Tsg/ mutant was not substantially affected at E14.5 (Supplementary Fig. 1A and B). As described previously, Cv2/ mice died at, or shortly after, birth (Ikeya et al., 2006). They showed multiple skeletal defects that were much more severe than those found in Tsg/ mice (Figs. 1B and 2D, H, L, P, T, X); in the axial skeleton, unlike in Tsg/ mice, the dorsal parts of the arches of the entire vertebral column were missing (similar vertebral arch defects are observed in the conditional
Fig. 2 – Axial skeletal phenotypes of compound Cv2 and Tsg mutants at E18.5. Vertebral defects in Cv2/ mice were reversed in Tsg/;Cv2/ compound mutant mice. (A–D) Atlas; (E–H) third cervical vertebra; (I–L) first thoracic vertebra; (M–P) fourth lumbar vertebra; (Q–T) dorsal views and (U–X) lateral views of the anterior lumbar region. Note that the reversions were observed not only in the vertebral arches (black arrows in A–T and black arrowheads in U–X), but also in the lateral processes (white arrowheads in H) and in the proximal part of the first ribs (white arrows in L). C, cervical vertebrae; Th, thoracic vertebrae; L, lumbar vertebrae; arrowhead in U–X, ossifying part of the vertebral arch.
MECHANISMS OF DEVELOPMENT
Bmpr1a-null mutant (Bmpr1aCKO) in the Bmpr1b+/ background (Yoon et al., 2006)). In addition, the lateral processes of the vertebral body were missing from the mid-cervical to the anterior thoracic region (Fig. 2H and L) and the lateral ossifying portions of the vertebral arches were reduced in size (Fig. 2H, L, and X). The expression of Tsg in the Cv2-null mutant was not substantially changed at E12.5 and E14.5 (Supplementary Fig. 1C–E), and Cv2/;Tsg+/ mice had the same phenotypes as those of Cv2 null mutants (data not shown). Thus the loss of one Cv2 or Tsg allele did not mutually enhance the null mutant phenotypes of shown by the other, and the null mutation of either gene did not affect the other’s expression. Six Tsg/;Cv2/ neonates at postnatal day 0 (P0) were recovered by crossing of Tsg/;Cv2+/ and Tsg+/–;Cv2+/ mice (from total 80 neonates, which is slightly below the expected Mendelian ratio 12.5%). Among the six Tsg/;Cv2/ neonates, one double homozygote mutant escaped the respiratory defects constantly observed in Cv2/ mutants (n > 40) and survived for at least 12 hours after birth (died by P1). At E18.5, the same crossings resulted in Tsg/;Cv2/ embryos at close to the expected Mendelian ratio (n = 14, total n = 126; 11.1% versus the expected 12.5%) and the Cv2/; Tsg/ embryos exhibited axial skeletal phenotypes that resembled those of the Tsg/ mutant and were distinct from those of the Cv2/ mutant (Figs. 1B and 2). In the C1 (atlas bone) and C3 vertebrae, although the Cv2/, Tsg/, and Cv2/;Tsg/ mutants all exhibited loss of the dorsal vertebral arches, the Cv2/;Tsg/ phenotypes were similar to those of the Tsg/ mutant and less severe than those of the Cv2/ mutant (Fig. 2A–H). The Th1 vertebra in the Cv2/; Tsg/ mutant was also much less defective than in the Cv2/ mutant (Fig. 2K and L), but its phenotype did not show complete reversion to the Tsg/ phenotype, which showed a small gap in the dorsal-most part of the arch (Fig. 2J). In the lumbar region, as previously reported, the Tsg/ mutant had no obvious defects in the vertebral arches (Fig. 2N, R, and V). In the Cv2/ mutant, as described above, the lumbar vertebrae lacked dorsal arches (Fig. 2P and T) and had smaller ossifying portions in the lateral arches (Fig. 2X). In contrast, the Cv2/;Tsg/ mutant had no substantial defects in the lumbar arches (Fig. 2O, S, and W). These findings demonstrate that the Tsg mutation suppresses the major defective phenotypes of the Cv2/ mutant in the axial skeleton. This reversal of the Cv2/ phenotypes by the Tsg mutation contrasted with the previously reported enhancement of the vertebral defects of the Cv2/ mutant by the deletion of one allele of the BMP4 gene (Ikeya et al., 2006; also shown in Fig. 3D, lanes 4–6). A similar enhancement of the Cv2/ phenotype in the lumbar vertebral arches and the lateral processes of the vertebral bodies was also observed in the BMP7+/ background (Fig. 3A–C; also shown in Fig. 3D, lanes 1–3), indicating that Cv2 cooperates positively with both BMP4 and BMP7 in axial skeletal development. This is consistent with the results of previous in vitro studies showing that vertebrate Cv2 protein binds to BMP2, 4, 6, and 7 with high affinity (Moser et al., 2003; Coles et al., 2004; Rentzsch et al., 2006). The phenotypic suppression by the Tsg mutation suggests that Tsg functions against the pro-BMP role of Cv2 in
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Fig. 3 – Axial skeletal phenotypes in compound Cv2 and BMP7mutants at E18.5. (A–C) First lumbar vertebrae. Axial skeletal defects in the lumbar vertebral region of the Cv2/ mutant were enhanced in the BMP7+/;Cv2/ mutant. L1, first lumbar vertebrae; arrow, agenesis of the vertebral arch; arrowhead, missing of the lateral process of the vertebral body. (D) Percentages of compound BMP7; Cv2 and BMP4; Cv2 embryos possessing defective vertebral arches. Black column, normal; gray column, dorsal portion of vertebral arch has been lost; white column, both dorsal portion of vertebral arch and lateral process of vertebral body have been lost.
the context of vertebral arch development (the pro- and anti-BMP roles of Tsg in other contexts are discussed further in Sections 2.3 and 2.4).
2.2. Non-axial skeletal phenotypes of the Cv2 null mutant are also suppressed by the Tsg mutation We next examined whether the Tsg mutation would interact with Cv2 mutant phenotypes in non-axial skeletal development. As described previously (Ikeya et al., 2006), defects in the bone and cartilage of the Cv2 mutant were also seen in various non-axial regions, including the ribs (absence of the 13th rib; compare Fig. 4A and D), scapula (small or with a hole in the middle; arrowhead in Fig. 4H), humerus (absence of deltoid tuberosity; arrow in Fig. 4H), skull (loss of the retrotympanic process of the squamosal bone and a wider unossified area of the metopic suture; Fig. 4L, and Table 1), and laryngeal and tracheal cartilages (deformity of the small hyoid, thyroid, and cricoid cartilages, and absence of tracheal cartilages; Fig. 4P and T). These bones and cartilages were not affected in the Tsg/ mutant (Fig. 4B, F, J, N, and R), except for the metopic suture, which was affected in approximately half of the Tsg/ mutants (Table 1). As in the case of the vertebral arches, the nonaxial skeletal defects of the Cv2 mutant were suppressed by the additional Tsg mutation (Fig. 4C, G, K, O, and S and Table 1).
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Fig. 4 – Non-axial bone and cartilage phenotypes in compound Cv2 and Tsg mutants at E18.5. (A–D) Lateral views of the rib cage. (E–H) Forelimb. Arrow, absence of deltoid tuberosity; arrowhead, whole in scapula. (I–L) Side views of the skull. st, squamosal temporalis; rp, retrotympanic process of the squamosal bone. (M–P) Lateral and (Q–T) frontal views of the laryngeal and bronchial cartilages. Hy, hyoid cartilage; th, thyroid cartilage; cr, cricoid cartilage; tr, tracheal cartilage.
These findings indicate that the Tsg mutation is also epistatic to the non-axial skeletal phenotypes of the Cv2 mutation. The frequencies of occurrence of the axial and nonaxial defects in each genetic background are shown in Table 1.
2.3. The skull phenotype of the Tsg mutant is not substantially affected by the Cv2 mutation As reported previously, although many Tsg/ mutants were born alive and grew to adulthood, a minor population of Tsg/ neonates exhibited substantial defects in the head structure, including the upper jaw (Fig. 5B), lower jaw (Fig. 5C) and skull bones (Fig. 5D). Interestingly, head defects similar to those of the Tsg mutants have been reported in Chordin/;Noggin+/– mutants (regarded as deficient in BMP antagonism) (Stottmann et al., 2001; Anderson et al., 2002), suggesting the possibility that Tsg and the BMP antagonists (especially, Chordin) work in the same direction in this developmental context. These defects were not present in the Cv2null mutant (Fig. 5E, lane 2).
We next examined whether the Cv2 mutation would in turn affected these Tsg mutant phenotypes. The defects in the Tsg/ head were not significantly altered in either the Cv2+/– or the Cv2/ background (Fig. 5E, lanes 3–5; P > 0.05, Kruskal–Wallis test), showing that Cv2 mutation neither suppressed nor enhanced the Tsg mutant phenotypes, whereas the Cv2 mutant phenotypes were suppressed by the Tsg mutation (Figs. 2 and 4). In this regard, the epistatic relationship between the Tsg mutation and the Cv2 mutation appears to be unidirectional. However, the situation with the Tsg mutant phenotypes in the head may be more complicated with respect to the mode of BMP modulation. It was previously reported that head defects in the Tsg mutant are enhanced in the BMP4+/ background (Zakin and De Robertis, 2004). This genetic interaction indicates that Tsg and BMP4 function cooperatively; Tsg is a pro-BMP factor in this context. This relationship is apparently distinct from that of Tsg and Cv2 in the axial skeletal phenotypes, in which Cv2 clearly has a pro-BMP role and Tsg counteracts it (anti-BMP). Taken together, these findings suggest
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Table 1 – Penetrance of the skeletal abnormalities in Cv2;Tsg mutant mice Tsg+/;Cv2+ (n = 51)
Tsg/ (n = 26)
Tsg/;Cv2/ (n = 20)
Cv2/ (n = 25)
Normal Lost (cervical) Lost (entire body axis) Normal Malformed Normal Ribs are not attached Normal Malformed Normal Lost Normal Less ossified Normal
100 (51) 0 (0) 0 (0) 100 (51) 0 (0) 100 (51) 0 (0) 100 (51) 0 (0) 100 (51) 0 (0) 100 (51) 0 (0) 100 (51)
0 (0) 100 (26) 0 (0) 100 (26) 0 (0) 100 (26) 0 (0) 100 (26) 0 (0) 100 (26) 0 (0) 45 (9) 55 (11) 100 (20)
0 (0) 85.0 (17) 15.0 (3) 100 (20) 0 (0) 100 (20) 0 (0) 100 (20) 0 (0) 95.0 (19) 5.0 (1) 44 (7) 56 (9) 100 (16)
0 (0) 0 (0) 100 (25) 0 (0) 100 (25) 0 (0) 100 (25) 56.0 (14) 44.0 (11) 0 (0) 100 (25) 0 (0) 100 (24) 0 (0)
Lost Normal Malformed Normal Lost
0 (0) 100 (51) 0 (0) 100 (51) 0 (0)
0 (0) 100 (26) 0 (0) 100 (26) 0 (0)
0 (0) 100 (20) 0 (0) 100 (20) 0 (0)
100 (24) 0 (0) 100 (25) 0 (0) 100 (25)
Phenotypes Neural arch
Rib cage Rib (T13) Scapula Deltoid tuberosity Metopic suture(*) Retrotympanic process of the squamosal bone(*) Laryngeal cartilage Bronchial cartilage
* Because of severe craniofacial defects, we omit six, one and four pups of Tsg/, Cv2/, and Tsg/;Cv2/, respectively, from the number.
Fig. 5 – Cv2 mutation does not affect head phenotypes in Tsg/ mutants. (A–D) Side views of skull. (A) Normal; (B) reduced jaw phenotype; (C) agnathic phenotype; and (D) truncation phenotype. (E) Percentages of embryos with each skull phenotype.
that the in vivo role of Tsg depends on the developmental context. One possibility is that the Tsg mutation counteracts the Cv2 mutation only in the context where Tsg has an anti-BMP role under certain situations (e.g., in the presence or absence of co-factors).
2.4. Phenotypic enhancement of the Cv2 mutation by Tsg in the ossification of the thoracic vertebral bodies Since our findings suggested that the genetic relationship between the Cv2 and Tsg mutations depends on the developmental context for the tissue, we revisited the axial skeletal phenotypes to see whether enhancement, rather than suppression, of the Cv2 phenotype by the Tsg mutation was detectable in some circumstances. Ossification of the cervical vertebral bodies is reported to be substantially delayed in the Tsg/ mutant (Petryk et al., 2004), and we also observed re-
duced ossification of the cervical vertebral bodies of the Tsg/ mutant (E18.5; Fig. 6B, F, and I, lane 3). A similar phenotype was seen in the Cv2 mutant (Fig. 6D, H, and I, lane 2). The Cv2 mutation also caused a moderate reduction in the size of the cervical and thoracic vertebral bodies (Fig. 6D and H), but only a marginal size reduction was seen in the Tsg mutant (Fig. 6B and F). Interestingly, Cv2/;Tsg/ mice exhibited a very severe reduction in the ossification of the cervical and thoracic vertebral bodies (Fig. 6C, G, and I, lane 5), indicating that, in this context, the Cv2 and Tsg mutations synergistically enhanced their defective phenotypes. This enhancement of reduced ossification in the vertebral bodies over that in Cv2/ embryos was also seen in Cv2/; BMP7+/ (Fig. 6J, K, L and M, lanes 1–3) and Cv2/;BMP4+/ embryos (Fig. 6M, lanes 4–6; Ikeya et al., 2006), suggesting that Cv2 plays a pro-BMP role in promoting the ossification of the vertebral bodies as well as of other parts of the developing
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skeleton. Therefore, we can infer that, in this particular context, Cv2 and Tsg are both required to enhance local BMP activity, although it remains elusive whether this pro-BMP role of Tsg is direct or mediated by some complicated regula-
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tory network. In addition, since BMP signaling is required during bone development in multiple steps that lead to ossification (reviewed in Canalis et al., 2003; Deng et al., 2008), future investigations are required to clarify whether
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Cv2 and Tsg are required at the same step or sequentially during the process.
2.5. Context-dependent genetic interactions between Cv2 and Tsg in BMP signaling control Our results demonstrated that the major skeletal defects in Cv2 mutants are suppressed by the Tsg mutation, but head defects in the Tsg mutant are not generally affected by the Cv2 mutation. Interestingly, the enhancement of the Cv2 phenotypes by the Tsg mutation is seen in a certain aspect of skeletal development, i.e., the ossification of the vertebral body. The mechanism by which mammalian Cv2 enhances BMP signals in vivo remains to be clarified. A recent study suggested that the Cv2-related protein KCP enhances binding of the BMP protein to its receptor by forming a tertiary complex (Lin et al., 2005) and the same mechanism is advocated for Drosophila crossvein formation (Serpe et al., 2008). However, other reports using Biacore and crystal structure analyses do not support this mechanism (Zhang et al., 2007, 2008). Thus, more extensive analysis may be necessary in the future. In the developing skeletal tissues of the mouse embryo, Cv2 is produced by the tissues that are impaired in the null mutant, including the vertebral arches and bodies, ribs, skull, and laryngeal and tracheal cartilages (Ikeya et al., 2006). This suggests that the pro-BMP factor Cv2 has tissue-autonomous (i.e. short-range) effects. Tsg expression has a relatively wide distribution in the early mouse embryo, including in the skeletal tissues (Nosaka et al., 2003; Zakin and De Robertis, 2004). Tsg, first isolated in Drosophila, is known as a binding co-factor of Chordin/Sog. In Xenopus, the Chordin–Tsg complex binds BMP4 more efficiently than does Chordin alone (Oelgeschla¨ger et al., 2000). This enhanced segregation of BMP4 is thought to play an anti-BMP role (Larrain et al., 2001). Interestingly, a fly study suggested that when Dpp is bound to the Sog–Tsg complex, it is more efficiently transported and released by the Sog/Chordin-degradating enzyme Tolloid than when Dpp is bound to Sog alone (Shimmi et al., 2005; Wang and Ferguson, 2005). This mechanism is suggested to contribute to the pro-BMP role of Sog and Tsg in the formation of the amnioserosa (the dorsal-most extraembryonic epithelial tissue) (Ashe, 2005). In the mouse, our understanding of the regulation of tissue development during mouse organogenesis by the binary (antiand pro-BMP) functions of Chordin and Tsg is only fragmentary. A previous study (Bachiller et al., 2003) showed that the vertebral defect in the null Chordin mutant, at least in some aspects, is similar to the defects found in Cv2/ and BMP7/ mu-
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tants (e.g., in the skull, laryngeal cartilages, and cervical vertebral arches) (Jena et al., 1997; Ikeya et al., 2006), raising the possibility that Chordin has a pro-BMP role. However, the exact nature of the mode of action of Chordin must await detailed gene interaction analyses in future. Previous mouse studies demonstrated that Tsg has a positive genetic interaction with BMP4 and BMP7, suggesting possible pro-BMP roles for this Chordin-binding co-factor. As discussed in Section 2.3, the head defect in the Tsg mutant is enhanced by a one-allele deletion of BMP4 (Zakin and De Robertis, 2004), although the appearance of this enhancement is dependent on the genetic background (Petryk et al., 2004). Conversely, the phenotype of the BMP7 null mutant is enhanced in the Tsg+/ background (e.g. sirenomelia, microphthalmia, hindlimb polydactyly, and sternebral defects) (Zakin et al., 2005). In skeletal development, a Tsg deficiency prevents chondrocytes from transitioning from their resting state to their proliferating state, and therefore, Tsg is thought to act preferentially in the same direction as BMP during the cartilage formation (Nosaka et al., 2003). Defective cartilage development is also observed in the dorsal part of the vertebral arches from the mid-cervical to the thoracic regions in the Tsg-null embryo (Zakin and De Robertis, 2004) and the Cv2-null embryo (Ikeya et al., 2006), suggesting a common role for Cv2 and Tsg in this developmental context. However, in the Cv2/ background, the presence of Tsg (Cv2/ versus Cv2/;Tsg/) facilitates the loss of the dorsal and lateral portions of the lumber vertebral arches (compare Fig. 2P, T, and X with Fig. 2O, S, and W). Since the development of this region is positively regulated by BMP4 and BMP7 in the Cv2/ background (Fig. 3), it is likely that, in these posterior dorsal regions, Tsg functions as an anti-BMP factor that counteracts the pro-BMP factor Cv2. Interestingly, the phenotype of the dorsal most part of the arch in Cv2/ was not completely reversed by crossing with Tsg/ mice at the thoracic region (Fig. 2J, K, and L), in contrast to the complete reversal found at the lumber region (Fig. 2R, S, and T). These observations raise the intriguing possibility that, although the major functions of Cv2 during skeletal development are Tsg-dependent, the relationship between Tsg, Cv2, and BMPs depends on the regional context and differs along the rostro-caudal axis even within the axial skeleton; the Cv2 function is mostly dependent on Tsg in the formation of dorsal part of the lumber vertebra (Fig. 2Q, 2R, 2S, and 2T), whereas Cv2 also has Tsg-independent roles in the formation of dorso-lateral part of the Th1 (Fig. 2J and Fig. 2K). Two interesting questions to be addressed in future studies are: (1) how much of the function of Tsg and Cv2 depends on Chordin in mammals? and (2) what factors change
b Fig. 6 – Enhancement of the reduced ossification phenotype of the Cv2 null mutant by Tsg or BMP7 mutation. (A–D) Ventral views of cervical and thoracic regions of the Cv2; Tsg mutants. C2, axis; Th1, first thoracic vertebra. (E–H) High-magnification view of vertebral bodies in the first to fourth thoracic vertebral region. (I) Percentages of embryos possessing reduced ossification centers in the cervical and thoracic vertebral body at E18.5. Black column, ossification centers formed in the cervical vertebral bodies; gray column, ossification centers not formed in the cervical vertebral bodies but formed in the thoracic vertebral bodies; white column, ossification centers not formed both in the cervical and thoracic vertebral bodies. (J–L) Ventral views of the lumbar region in Cv2; BMP7 mutants. Arrow, ossification of vertebral bodies. (M) Percentages of the compound BMP7; Cv2 and BMP4; Cv2 embryos possessing reduced ossification centers in the lumbar vertebral bodies at E18.5. Black column, ossification centers formed normally; gray column, smaller ossification centers; white column, ossification centers not formed.
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3.
Experimental procedures
Inomata for invaluable comments and discussion. MI is thankful to Ayumi Ikeya for constant encouragement and support during this study. This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Kobe Cluster Project and the Leading Project (Y.S.) and by Special Postdoctral Researchers Program from RIKEN (M.I.).
3.1.
Mutant mice and crossings
Appendix A. Supplementary data
the regional response to Tsg, Cv2, and BMPs in the head, cervical, and caudal axial domains? The intricate extracellular regulatory network is an attractive topic for research on dynamic multiple-factor interactions in the complex mammalian organogenetic program.
Mice carrying mutations in Cv2, Tsg, and BMP7 were described previously (Luo et al., 1995; Nosaka et al., 2003; Ikeya et al., 2006). Here, we used the nLacZ-knock-in line of the Cv2 mutant. Compound heterozygous mutants were initially generated by crossing the Cv2 heterozygotes with Tsg or BMP7 heterozygotes. No obvious defects were observed in these compound heterozygote mice, and we used them for further crossing. To increase the yield of double (Tsg/;Cv2/) mutants, we crossed Tsg/;Cv2+/ mice, which are viable and fertile, to compound heterozygotes. Animals were housed in environmentally controlled rooms in accordance with the institutional (RIKEN) guidelines for animal experiments.
3.2.
Genotyping
To detect the nLacZ-knock-in allele of Cv2, genomic PCR was performed as described previously (Ikeya et al., 2006). To detect the Tsg mutant allele, primers were designed for the sequences upstream and downstream of the start codon of Tsg (Tsg-5 0 and Tsg-3 0 , respectively), and for the sequence of the neomycin-resistant gene (Tsg-neo). The lengths of the amplified cDNA fragments were 521 bp for the wild type and 367 bp for the mutant. Their sequences were as follows: Tsg5 0 , 5 0 -CCCTGCCTCCTGAGTACCGGAATTT-3 0 ; Tsg-3 0 , 5 0 -TTACA GCTCTGGGACACGGGGAGAC-3 0 ; and Tsg-neo, 5 0 -TGCGTGC AATCCATCTTGTTCAATG-3 0 . To detect the Bmp7 mutant allele, primers were designed against the sequences of intron 5 and exon 6 of Bmp7 and against the sequence of the PGK promoter. A 250-bp fragment in the wild type and a 300-bp fragment in the BMP7 mutant were amplified. Their sequences were as follows: Bmp7 Intron 5F, 5 0 -TAGCCTGA GCCTATGTCCACTG-3 0 ; Bmp7 Exon 6R, 5 0 -ACCCACCAGT GTCTGGACGATG-3 0 ; and PGK R2, 5 5 0 -CCACCAAAGAACGGA GCCGGTTG-3 0 .
3.3.
Skeletal specimen preparation
Skeletons were prepared as described previously (Ikeya et al., 2006). Briefly, E18.5 mice were eviscerated, skinned, fixed in 80% alcohol, and stained with Alcian Blue and Alizarin Red. Bright field pictures were taken under a dissecting microscope (Olympus SZX12) with a digital camera system Axio Cam (Carl Zeiss).
Acknowledgments We are grateful to the staff of the Laboratory of Animal Resources and Genetic Engineering at Center for Developmental Biology for their help with mouse husbandry, and to Dr. H.
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.mod.2008.06.011.
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available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/modo
Twisted gastrulation mutation suppresses skeletal defect phenotypes in Crossveinless 2 mutant mice Makoto Ikeyaa,*, Tetsuya Nosakab, Kumi Fukushimaa, Masako Kawadaa, Yasuhide Furutac, Toshio Kitamurad, Yoshiki Sasaia a
Organogenesis and Neurogenesis Group, RIKEN, Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo, Kobe 650-0047, Japan b Department of Microbiology, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan c Department of Biochemistry and Molecular Biology, M.D. Anderson Cancer Center, University of Texas, Houston, TX 77030, USA d Division of Cellular Therapy, The Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan
A R T I C L E I N F O
A B S T R A C T
Article history:
Bone morphogenetic protein (BMP) signaling controls various aspects of organogenesis,
Received 11 January 2008
including skeletal development. We previously demonstrated that the pro-BMP function
Received in revised form
of Crossveinless 2 (Cv2) is required for axial and non-axial skeletal development in mice.
25 June 2008
Here, we showed that skeletal defects in the Cv2-null mutant were reversed by the addi-
Accepted 26 June 2008
tional deletion of Twisted gastrulation (Tsg). Whereas the Cv2/ mutant lacks a substantial
Available online 3 July 2008
portion of the lumbar vertebral arches, Cv2/;Tsg/ mice have almost normal arches. Suppression of Cv2/ phenotypes is also seen in the non-axial skeleton, including the ribs,
Keywords:
humerus, skull, and laryngeal and tracheal cartilages. In contrast, the Tsg/ phenotype in
Mouse
the head is not significantly affected by the Cv2 mutation. These findings demonstrate that
Crossveinless 2 (Bmper)
Tsg mutation is epistatic to Cv2 mutation in the major skeletal phenotypes, suggesting that
Twisted gastrulation
the pro-BMP activity of Cv2 is, at least in part, dependent on Tsg. We also present genetic
BMP7
evidence for the context-dependent functional relationship between Tsg and Cv2 during
pro-BMP
mouse development.
Suppression
Ó 2008 Elsevier Ireland Ltd. All rights reserved.
Skeleton Genetic interaction
1.
Introduction
BMP signaling plays important regulatory roles in a variety of aspects of vertebrate and invertebrate development, including skeletal development (reviewed in Wan and Cao, 2005). In addition, BMP signals regulate cell-type specification, maturation, cell growth, apoptosis, and dorsal–ventral axis determination (De Robertis and Sasai, 1996; Hogan, 1996; Massague and Chen, 2000; Hammerschmidt and Mullins, 2002; De Robertis and Kuroda, 2004; Yamamoto and Oelgeschla¨ger, 2004; De Robertis, 2006). A number of factors
that negatively regulate BMP signals in the extracellular space have been identified (anti-BMP factors). A typical example is a class of secreted antagonist proteins, including Noggin, Chordin, Follistatin, Cerberus, and Gremlin, that bind to, and inactivate, BMP proteins (Smith and Harland, 1992; Lamb et al., 1993; Sasai et al., 1994; Sasai et al., 1995; Hemmati-Brivanlou et al., 1994; Glinka et al., 1997; Hsu et al., 1998). In contrast to these anti-BMP factors, several extracellular BMP-interacting proteins are reported to modulate BMP signaling in a positive manner. For instance, we previously reported in a study of knockout mice for Crossveinless 2 (Cv2),
* Corresponding author. Tel.:+81 78 306 1845; fax +81 78 306 1854. E-mail address: [email protected] (M. Ikeya). 0925-4773/$ - see front matter Ó 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mod.2008.06.011
MECHANISMS OF DEVELOPMENT
that Cv2 functions as an enhancer molecule of BMP signaling (pro-BMP factor) in mouse skeletal development (Ikeya et al., 2006). Cv2 is a BMP-binding protein that was first identified in a fly mutant study as being required for the formation of crossveins in the fly wing (Garcia-Bellido and de Celis, 1992). Genetic studies in flies have shown that the formation of these veins requires high BMP-signaling activity (involving Dpp and Gbb) and that Cv2 is essential for enhancing the local BMP signal near the receiving cells (Conley et al., 2000; O’Connor et al., 2006; Serpe et al., 2008). In mice, the Cv2 mutant phenotype in the skeleton is enhanced in the BMP4+/ background, showing that Cv2 and BMP4 work together in the same direction for skeletal development (Ikeya et al., 2006). This idea is consistent with non-mammalian and in vitro studies that suggested pro-BMP activity of Cv2 (Conley et al., 2000; Coles et al., 2004; Kamimura et al., 2004; Ralston and Blair, 2005; Rentzsch et al., 2006; Moser et al., 2007; Serpe et al., 2008). A similar pro-BMP role has been reported for another Cv2-class molecule, kielin/chordin-like protein (KCP) in kidney repair (Lin et al., 2005). Interestingly, these factors (Cv2 and KCP) contain multiple cystein-rich repeats homologous to those in Chordin. Since a characteristic feature of BMP signals is their function as a morphogen (generating an activity gradient and evoking multiple-threshold responses) (Gurdon and
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Bourillot, 2001), fine spatial control of BMP signals by these pro- and anti-BMP factors is important for tissue formation to occur in the right place when the tissue is at the right size. In addition, these pro- and anti-BMP factors themselves are known to regulate one another in an intricate manner. The activity of the anti-BMP factor Chordin is itself regulated by multiple interacting factors, such as Twisted gastrulation (Tsg) (Harland, 2001), Xolloid-related protease (Ge and Greenspan, 2006) and Sizzled (Kimelman and Szeto, 2006). These interactions may be regulated in a contextdependent fashion, and their quantitative control remains elusive at the molecular level. For example, whether Tsg acts as a pro- or anti-BMP factor has been addressed through multiple approaches but has not yet been totally clarified (see Section 2.5). Similarly, it is still unclear whether Chordin in mice has any pro-BMP role, which has been suggested for Sog (fly Chordin homolog) and Chordino in Drosophila and zebrafish, in addition to its anti-BMP role (Zusman et al., 1988; Ashe and Levine, 1999; Decotto and Ferguson, 2001; Hammerschmidt and Mullins, 2002; Rentzsch et al., 2006). As for Cv2, a previous study in zebrafish and in vitro studies suggested that Cv2 could also function as an anti-BMP factor, at least under certain artificial conditions (Moser et al., 2003; Binnerts et al., 2004; Rentzsch et al., 2006; Serpe et al., 2008).
Fig. 1 – Genetic crossing of Cv2 and Tsg mutants. (A) Analysis of genotypes by allele-specific genomic PCR. WT, wild type allele; MT, mutant allele. (B) Gross skeletal phenotypes of the compound mutants at E18.5. Skeletal preparations were stained with Alcian Blue and Alizarin Red. va, vertebral arch.
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MECHANISMS OF DEVELOPMENT
Therefore, the extracellular regulation of BMP signaling involves multiple factors that interact positively and negatively in an intricate fashion; the careful investigation of such interactions by loss-of-function approaches is essential for understanding the actual in vivo functions. In an effort to elucidate the dynamic regulatory network of BMP signaling during mammalian organogenesis, we performed a gene interaction analysis of Cv2 and Tsg mutations in this study. Since our previous study clearly demonstrated the pro-BMP role of Cv2 in skeletal development (positive genetic interaction between Cv2 and BMP4 (Ikeya et al., 2006); a similar interaction between Cv2 and BMP7 is also shown later in Sections 2.1 and 2.4), we focused here on skeletal phenotypes. We demonstrate here that the Tsg mutation has a strong suppressive effect on the bone and cartilage defects caused by a Cv2-null mutation in axial and non-axial structures. In the majority of skeletal phenotypes, the epistatic relationship of Tsg mutation and Cv2 mutation is unidirectional. Interestingly, our detailed analysis of minor skeletal phenotypes suggested that the genetic interaction between these two mutations could be different in some other contexts. We discuss the possible roles and modes of action of these BMP modulators in skeletal development.
2.
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Results and discussion
2.1. Axial skeletal phenotypes of the Cv2-null mutant are suppressed by the Tsg mutation To examine the possible genetic interaction between Cv2 and Tsg, we crossed these mutant mice (Fig. 1A). As previously reported, Tsg-null mice were born alive and grew, and both male and female mutants were fertile (Nosaka et al., 2003; Petryk et al., 2004; Zakin and De Robertis, 2004). They showed moderate phenotypes in the skeleton at embryonic day 18.5 (E18.5), such as a short tail and partial loss of the cervical vertebral arches (Figs. 1B, 2B and 2F). Cv2+/;Tsg/ mice showed phenotypes indistinguishable from those of Tsg null mutants (data not shown), and the expression of Cv2 in the Tsg/ mutant was not substantially affected at E14.5 (Supplementary Fig. 1A and B). As described previously, Cv2/ mice died at, or shortly after, birth (Ikeya et al., 2006). They showed multiple skeletal defects that were much more severe than those found in Tsg/ mice (Figs. 1B and 2D, H, L, P, T, X); in the axial skeleton, unlike in Tsg/ mice, the dorsal parts of the arches of the entire vertebral column were missing (similar vertebral arch defects are observed in the conditional
Fig. 2 – Axial skeletal phenotypes of compound Cv2 and Tsg mutants at E18.5. Vertebral defects in Cv2/ mice were reversed in Tsg/;Cv2/ compound mutant mice. (A–D) Atlas; (E–H) third cervical vertebra; (I–L) first thoracic vertebra; (M–P) fourth lumbar vertebra; (Q–T) dorsal views and (U–X) lateral views of the anterior lumbar region. Note that the reversions were observed not only in the vertebral arches (black arrows in A–T and black arrowheads in U–X), but also in the lateral processes (white arrowheads in H) and in the proximal part of the first ribs (white arrows in L). C, cervical vertebrae; Th, thoracic vertebrae; L, lumbar vertebrae; arrowhead in U–X, ossifying part of the vertebral arch.
MECHANISMS OF DEVELOPMENT
Bmpr1a-null mutant (Bmpr1aCKO) in the Bmpr1b+/ background (Yoon et al., 2006)). In addition, the lateral processes of the vertebral body were missing from the mid-cervical to the anterior thoracic region (Fig. 2H and L) and the lateral ossifying portions of the vertebral arches were reduced in size (Fig. 2H, L, and X). The expression of Tsg in the Cv2-null mutant was not substantially changed at E12.5 and E14.5 (Supplementary Fig. 1C–E), and Cv2/;Tsg+/ mice had the same phenotypes as those of Cv2 null mutants (data not shown). Thus the loss of one Cv2 or Tsg allele did not mutually enhance the null mutant phenotypes of shown by the other, and the null mutation of either gene did not affect the other’s expression. Six Tsg/;Cv2/ neonates at postnatal day 0 (P0) were recovered by crossing of Tsg/;Cv2+/ and Tsg+/–;Cv2+/ mice (from total 80 neonates, which is slightly below the expected Mendelian ratio 12.5%). Among the six Tsg/;Cv2/ neonates, one double homozygote mutant escaped the respiratory defects constantly observed in Cv2/ mutants (n > 40) and survived for at least 12 hours after birth (died by P1). At E18.5, the same crossings resulted in Tsg/;Cv2/ embryos at close to the expected Mendelian ratio (n = 14, total n = 126; 11.1% versus the expected 12.5%) and the Cv2/; Tsg/ embryos exhibited axial skeletal phenotypes that resembled those of the Tsg/ mutant and were distinct from those of the Cv2/ mutant (Figs. 1B and 2). In the C1 (atlas bone) and C3 vertebrae, although the Cv2/, Tsg/, and Cv2/;Tsg/ mutants all exhibited loss of the dorsal vertebral arches, the Cv2/;Tsg/ phenotypes were similar to those of the Tsg/ mutant and less severe than those of the Cv2/ mutant (Fig. 2A–H). The Th1 vertebra in the Cv2/; Tsg/ mutant was also much less defective than in the Cv2/ mutant (Fig. 2K and L), but its phenotype did not show complete reversion to the Tsg/ phenotype, which showed a small gap in the dorsal-most part of the arch (Fig. 2J). In the lumbar region, as previously reported, the Tsg/ mutant had no obvious defects in the vertebral arches (Fig. 2N, R, and V). In the Cv2/ mutant, as described above, the lumbar vertebrae lacked dorsal arches (Fig. 2P and T) and had smaller ossifying portions in the lateral arches (Fig. 2X). In contrast, the Cv2/;Tsg/ mutant had no substantial defects in the lumbar arches (Fig. 2O, S, and W). These findings demonstrate that the Tsg mutation suppresses the major defective phenotypes of the Cv2/ mutant in the axial skeleton. This reversal of the Cv2/ phenotypes by the Tsg mutation contrasted with the previously reported enhancement of the vertebral defects of the Cv2/ mutant by the deletion of one allele of the BMP4 gene (Ikeya et al., 2006; also shown in Fig. 3D, lanes 4–6). A similar enhancement of the Cv2/ phenotype in the lumbar vertebral arches and the lateral processes of the vertebral bodies was also observed in the BMP7+/ background (Fig. 3A–C; also shown in Fig. 3D, lanes 1–3), indicating that Cv2 cooperates positively with both BMP4 and BMP7 in axial skeletal development. This is consistent with the results of previous in vitro studies showing that vertebrate Cv2 protein binds to BMP2, 4, 6, and 7 with high affinity (Moser et al., 2003; Coles et al., 2004; Rentzsch et al., 2006). The phenotypic suppression by the Tsg mutation suggests that Tsg functions against the pro-BMP role of Cv2 in
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Fig. 3 – Axial skeletal phenotypes in compound Cv2 and BMP7mutants at E18.5. (A–C) First lumbar vertebrae. Axial skeletal defects in the lumbar vertebral region of the Cv2/ mutant were enhanced in the BMP7+/;Cv2/ mutant. L1, first lumbar vertebrae; arrow, agenesis of the vertebral arch; arrowhead, missing of the lateral process of the vertebral body. (D) Percentages of compound BMP7; Cv2 and BMP4; Cv2 embryos possessing defective vertebral arches. Black column, normal; gray column, dorsal portion of vertebral arch has been lost; white column, both dorsal portion of vertebral arch and lateral process of vertebral body have been lost.
the context of vertebral arch development (the pro- and anti-BMP roles of Tsg in other contexts are discussed further in Sections 2.3 and 2.4).
2.2. Non-axial skeletal phenotypes of the Cv2 null mutant are also suppressed by the Tsg mutation We next examined whether the Tsg mutation would interact with Cv2 mutant phenotypes in non-axial skeletal development. As described previously (Ikeya et al., 2006), defects in the bone and cartilage of the Cv2 mutant were also seen in various non-axial regions, including the ribs (absence of the 13th rib; compare Fig. 4A and D), scapula (small or with a hole in the middle; arrowhead in Fig. 4H), humerus (absence of deltoid tuberosity; arrow in Fig. 4H), skull (loss of the retrotympanic process of the squamosal bone and a wider unossified area of the metopic suture; Fig. 4L, and Table 1), and laryngeal and tracheal cartilages (deformity of the small hyoid, thyroid, and cricoid cartilages, and absence of tracheal cartilages; Fig. 4P and T). These bones and cartilages were not affected in the Tsg/ mutant (Fig. 4B, F, J, N, and R), except for the metopic suture, which was affected in approximately half of the Tsg/ mutants (Table 1). As in the case of the vertebral arches, the nonaxial skeletal defects of the Cv2 mutant were suppressed by the additional Tsg mutation (Fig. 4C, G, K, O, and S and Table 1).
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Fig. 4 – Non-axial bone and cartilage phenotypes in compound Cv2 and Tsg mutants at E18.5. (A–D) Lateral views of the rib cage. (E–H) Forelimb. Arrow, absence of deltoid tuberosity; arrowhead, whole in scapula. (I–L) Side views of the skull. st, squamosal temporalis; rp, retrotympanic process of the squamosal bone. (M–P) Lateral and (Q–T) frontal views of the laryngeal and bronchial cartilages. Hy, hyoid cartilage; th, thyroid cartilage; cr, cricoid cartilage; tr, tracheal cartilage.
These findings indicate that the Tsg mutation is also epistatic to the non-axial skeletal phenotypes of the Cv2 mutation. The frequencies of occurrence of the axial and nonaxial defects in each genetic background are shown in Table 1.
2.3. The skull phenotype of the Tsg mutant is not substantially affected by the Cv2 mutation As reported previously, although many Tsg/ mutants were born alive and grew to adulthood, a minor population of Tsg/ neonates exhibited substantial defects in the head structure, including the upper jaw (Fig. 5B), lower jaw (Fig. 5C) and skull bones (Fig. 5D). Interestingly, head defects similar to those of the Tsg mutants have been reported in Chordin/;Noggin+/– mutants (regarded as deficient in BMP antagonism) (Stottmann et al., 2001; Anderson et al., 2002), suggesting the possibility that Tsg and the BMP antagonists (especially, Chordin) work in the same direction in this developmental context. These defects were not present in the Cv2null mutant (Fig. 5E, lane 2).
We next examined whether the Cv2 mutation would in turn affected these Tsg mutant phenotypes. The defects in the Tsg/ head were not significantly altered in either the Cv2+/– or the Cv2/ background (Fig. 5E, lanes 3–5; P > 0.05, Kruskal–Wallis test), showing that Cv2 mutation neither suppressed nor enhanced the Tsg mutant phenotypes, whereas the Cv2 mutant phenotypes were suppressed by the Tsg mutation (Figs. 2 and 4). In this regard, the epistatic relationship between the Tsg mutation and the Cv2 mutation appears to be unidirectional. However, the situation with the Tsg mutant phenotypes in the head may be more complicated with respect to the mode of BMP modulation. It was previously reported that head defects in the Tsg mutant are enhanced in the BMP4+/ background (Zakin and De Robertis, 2004). This genetic interaction indicates that Tsg and BMP4 function cooperatively; Tsg is a pro-BMP factor in this context. This relationship is apparently distinct from that of Tsg and Cv2 in the axial skeletal phenotypes, in which Cv2 clearly has a pro-BMP role and Tsg counteracts it (anti-BMP). Taken together, these findings suggest
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Table 1 – Penetrance of the skeletal abnormalities in Cv2;Tsg mutant mice Tsg+/;Cv2+ (n = 51)
Tsg/ (n = 26)
Tsg/;Cv2/ (n = 20)
Cv2/ (n = 25)
Normal Lost (cervical) Lost (entire body axis) Normal Malformed Normal Ribs are not attached Normal Malformed Normal Lost Normal Less ossified Normal
100 (51) 0 (0) 0 (0) 100 (51) 0 (0) 100 (51) 0 (0) 100 (51) 0 (0) 100 (51) 0 (0) 100 (51) 0 (0) 100 (51)
0 (0) 100 (26) 0 (0) 100 (26) 0 (0) 100 (26) 0 (0) 100 (26) 0 (0) 100 (26) 0 (0) 45 (9) 55 (11) 100 (20)
0 (0) 85.0 (17) 15.0 (3) 100 (20) 0 (0) 100 (20) 0 (0) 100 (20) 0 (0) 95.0 (19) 5.0 (1) 44 (7) 56 (9) 100 (16)
0 (0) 0 (0) 100 (25) 0 (0) 100 (25) 0 (0) 100 (25) 56.0 (14) 44.0 (11) 0 (0) 100 (25) 0 (0) 100 (24) 0 (0)
Lost Normal Malformed Normal Lost
0 (0) 100 (51) 0 (0) 100 (51) 0 (0)
0 (0) 100 (26) 0 (0) 100 (26) 0 (0)
0 (0) 100 (20) 0 (0) 100 (20) 0 (0)
100 (24) 0 (0) 100 (25) 0 (0) 100 (25)
Phenotypes Neural arch
Rib cage Rib (T13) Scapula Deltoid tuberosity Metopic suture(*) Retrotympanic process of the squamosal bone(*) Laryngeal cartilage Bronchial cartilage
* Because of severe craniofacial defects, we omit six, one and four pups of Tsg/, Cv2/, and Tsg/;Cv2/, respectively, from the number.
Fig. 5 – Cv2 mutation does not affect head phenotypes in Tsg/ mutants. (A–D) Side views of skull. (A) Normal; (B) reduced jaw phenotype; (C) agnathic phenotype; and (D) truncation phenotype. (E) Percentages of embryos with each skull phenotype.
that the in vivo role of Tsg depends on the developmental context. One possibility is that the Tsg mutation counteracts the Cv2 mutation only in the context where Tsg has an anti-BMP role under certain situations (e.g., in the presence or absence of co-factors).
2.4. Phenotypic enhancement of the Cv2 mutation by Tsg in the ossification of the thoracic vertebral bodies Since our findings suggested that the genetic relationship between the Cv2 and Tsg mutations depends on the developmental context for the tissue, we revisited the axial skeletal phenotypes to see whether enhancement, rather than suppression, of the Cv2 phenotype by the Tsg mutation was detectable in some circumstances. Ossification of the cervical vertebral bodies is reported to be substantially delayed in the Tsg/ mutant (Petryk et al., 2004), and we also observed re-
duced ossification of the cervical vertebral bodies of the Tsg/ mutant (E18.5; Fig. 6B, F, and I, lane 3). A similar phenotype was seen in the Cv2 mutant (Fig. 6D, H, and I, lane 2). The Cv2 mutation also caused a moderate reduction in the size of the cervical and thoracic vertebral bodies (Fig. 6D and H), but only a marginal size reduction was seen in the Tsg mutant (Fig. 6B and F). Interestingly, Cv2/;Tsg/ mice exhibited a very severe reduction in the ossification of the cervical and thoracic vertebral bodies (Fig. 6C, G, and I, lane 5), indicating that, in this context, the Cv2 and Tsg mutations synergistically enhanced their defective phenotypes. This enhancement of reduced ossification in the vertebral bodies over that in Cv2/ embryos was also seen in Cv2/; BMP7+/ (Fig. 6J, K, L and M, lanes 1–3) and Cv2/;BMP4+/ embryos (Fig. 6M, lanes 4–6; Ikeya et al., 2006), suggesting that Cv2 plays a pro-BMP role in promoting the ossification of the vertebral bodies as well as of other parts of the developing
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skeleton. Therefore, we can infer that, in this particular context, Cv2 and Tsg are both required to enhance local BMP activity, although it remains elusive whether this pro-BMP role of Tsg is direct or mediated by some complicated regula-
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tory network. In addition, since BMP signaling is required during bone development in multiple steps that lead to ossification (reviewed in Canalis et al., 2003; Deng et al., 2008), future investigations are required to clarify whether
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Cv2 and Tsg are required at the same step or sequentially during the process.
2.5. Context-dependent genetic interactions between Cv2 and Tsg in BMP signaling control Our results demonstrated that the major skeletal defects in Cv2 mutants are suppressed by the Tsg mutation, but head defects in the Tsg mutant are not generally affected by the Cv2 mutation. Interestingly, the enhancement of the Cv2 phenotypes by the Tsg mutation is seen in a certain aspect of skeletal development, i.e., the ossification of the vertebral body. The mechanism by which mammalian Cv2 enhances BMP signals in vivo remains to be clarified. A recent study suggested that the Cv2-related protein KCP enhances binding of the BMP protein to its receptor by forming a tertiary complex (Lin et al., 2005) and the same mechanism is advocated for Drosophila crossvein formation (Serpe et al., 2008). However, other reports using Biacore and crystal structure analyses do not support this mechanism (Zhang et al., 2007, 2008). Thus, more extensive analysis may be necessary in the future. In the developing skeletal tissues of the mouse embryo, Cv2 is produced by the tissues that are impaired in the null mutant, including the vertebral arches and bodies, ribs, skull, and laryngeal and tracheal cartilages (Ikeya et al., 2006). This suggests that the pro-BMP factor Cv2 has tissue-autonomous (i.e. short-range) effects. Tsg expression has a relatively wide distribution in the early mouse embryo, including in the skeletal tissues (Nosaka et al., 2003; Zakin and De Robertis, 2004). Tsg, first isolated in Drosophila, is known as a binding co-factor of Chordin/Sog. In Xenopus, the Chordin–Tsg complex binds BMP4 more efficiently than does Chordin alone (Oelgeschla¨ger et al., 2000). This enhanced segregation of BMP4 is thought to play an anti-BMP role (Larrain et al., 2001). Interestingly, a fly study suggested that when Dpp is bound to the Sog–Tsg complex, it is more efficiently transported and released by the Sog/Chordin-degradating enzyme Tolloid than when Dpp is bound to Sog alone (Shimmi et al., 2005; Wang and Ferguson, 2005). This mechanism is suggested to contribute to the pro-BMP role of Sog and Tsg in the formation of the amnioserosa (the dorsal-most extraembryonic epithelial tissue) (Ashe, 2005). In the mouse, our understanding of the regulation of tissue development during mouse organogenesis by the binary (antiand pro-BMP) functions of Chordin and Tsg is only fragmentary. A previous study (Bachiller et al., 2003) showed that the vertebral defect in the null Chordin mutant, at least in some aspects, is similar to the defects found in Cv2/ and BMP7/ mu-
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839
tants (e.g., in the skull, laryngeal cartilages, and cervical vertebral arches) (Jena et al., 1997; Ikeya et al., 2006), raising the possibility that Chordin has a pro-BMP role. However, the exact nature of the mode of action of Chordin must await detailed gene interaction analyses in future. Previous mouse studies demonstrated that Tsg has a positive genetic interaction with BMP4 and BMP7, suggesting possible pro-BMP roles for this Chordin-binding co-factor. As discussed in Section 2.3, the head defect in the Tsg mutant is enhanced by a one-allele deletion of BMP4 (Zakin and De Robertis, 2004), although the appearance of this enhancement is dependent on the genetic background (Petryk et al., 2004). Conversely, the phenotype of the BMP7 null mutant is enhanced in the Tsg+/ background (e.g. sirenomelia, microphthalmia, hindlimb polydactyly, and sternebral defects) (Zakin et al., 2005). In skeletal development, a Tsg deficiency prevents chondrocytes from transitioning from their resting state to their proliferating state, and therefore, Tsg is thought to act preferentially in the same direction as BMP during the cartilage formation (Nosaka et al., 2003). Defective cartilage development is also observed in the dorsal part of the vertebral arches from the mid-cervical to the thoracic regions in the Tsg-null embryo (Zakin and De Robertis, 2004) and the Cv2-null embryo (Ikeya et al., 2006), suggesting a common role for Cv2 and Tsg in this developmental context. However, in the Cv2/ background, the presence of Tsg (Cv2/ versus Cv2/;Tsg/) facilitates the loss of the dorsal and lateral portions of the lumber vertebral arches (compare Fig. 2P, T, and X with Fig. 2O, S, and W). Since the development of this region is positively regulated by BMP4 and BMP7 in the Cv2/ background (Fig. 3), it is likely that, in these posterior dorsal regions, Tsg functions as an anti-BMP factor that counteracts the pro-BMP factor Cv2. Interestingly, the phenotype of the dorsal most part of the arch in Cv2/ was not completely reversed by crossing with Tsg/ mice at the thoracic region (Fig. 2J, K, and L), in contrast to the complete reversal found at the lumber region (Fig. 2R, S, and T). These observations raise the intriguing possibility that, although the major functions of Cv2 during skeletal development are Tsg-dependent, the relationship between Tsg, Cv2, and BMPs depends on the regional context and differs along the rostro-caudal axis even within the axial skeleton; the Cv2 function is mostly dependent on Tsg in the formation of dorsal part of the lumber vertebra (Fig. 2Q, 2R, 2S, and 2T), whereas Cv2 also has Tsg-independent roles in the formation of dorso-lateral part of the Th1 (Fig. 2J and Fig. 2K). Two interesting questions to be addressed in future studies are: (1) how much of the function of Tsg and Cv2 depends on Chordin in mammals? and (2) what factors change
b Fig. 6 – Enhancement of the reduced ossification phenotype of the Cv2 null mutant by Tsg or BMP7 mutation. (A–D) Ventral views of cervical and thoracic regions of the Cv2; Tsg mutants. C2, axis; Th1, first thoracic vertebra. (E–H) High-magnification view of vertebral bodies in the first to fourth thoracic vertebral region. (I) Percentages of embryos possessing reduced ossification centers in the cervical and thoracic vertebral body at E18.5. Black column, ossification centers formed in the cervical vertebral bodies; gray column, ossification centers not formed in the cervical vertebral bodies but formed in the thoracic vertebral bodies; white column, ossification centers not formed both in the cervical and thoracic vertebral bodies. (J–L) Ventral views of the lumbar region in Cv2; BMP7 mutants. Arrow, ossification of vertebral bodies. (M) Percentages of the compound BMP7; Cv2 and BMP4; Cv2 embryos possessing reduced ossification centers in the lumbar vertebral bodies at E18.5. Black column, ossification centers formed normally; gray column, smaller ossification centers; white column, ossification centers not formed.
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1 2 5 ( 2 0 0 8 ) 8 3 2 –8 4 2
3.
Experimental procedures
Inomata for invaluable comments and discussion. MI is thankful to Ayumi Ikeya for constant encouragement and support during this study. This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Kobe Cluster Project and the Leading Project (Y.S.) and by Special Postdoctral Researchers Program from RIKEN (M.I.).
3.1.
Mutant mice and crossings
Appendix A. Supplementary data
the regional response to Tsg, Cv2, and BMPs in the head, cervical, and caudal axial domains? The intricate extracellular regulatory network is an attractive topic for research on dynamic multiple-factor interactions in the complex mammalian organogenetic program.
Mice carrying mutations in Cv2, Tsg, and BMP7 were described previously (Luo et al., 1995; Nosaka et al., 2003; Ikeya et al., 2006). Here, we used the nLacZ-knock-in line of the Cv2 mutant. Compound heterozygous mutants were initially generated by crossing the Cv2 heterozygotes with Tsg or BMP7 heterozygotes. No obvious defects were observed in these compound heterozygote mice, and we used them for further crossing. To increase the yield of double (Tsg/;Cv2/) mutants, we crossed Tsg/;Cv2+/ mice, which are viable and fertile, to compound heterozygotes. Animals were housed in environmentally controlled rooms in accordance with the institutional (RIKEN) guidelines for animal experiments.
3.2.
Genotyping
To detect the nLacZ-knock-in allele of Cv2, genomic PCR was performed as described previously (Ikeya et al., 2006). To detect the Tsg mutant allele, primers were designed for the sequences upstream and downstream of the start codon of Tsg (Tsg-5 0 and Tsg-3 0 , respectively), and for the sequence of the neomycin-resistant gene (Tsg-neo). The lengths of the amplified cDNA fragments were 521 bp for the wild type and 367 bp for the mutant. Their sequences were as follows: Tsg5 0 , 5 0 -CCCTGCCTCCTGAGTACCGGAATTT-3 0 ; Tsg-3 0 , 5 0 -TTACA GCTCTGGGACACGGGGAGAC-3 0 ; and Tsg-neo, 5 0 -TGCGTGC AATCCATCTTGTTCAATG-3 0 . To detect the Bmp7 mutant allele, primers were designed against the sequences of intron 5 and exon 6 of Bmp7 and against the sequence of the PGK promoter. A 250-bp fragment in the wild type and a 300-bp fragment in the BMP7 mutant were amplified. Their sequences were as follows: Bmp7 Intron 5F, 5 0 -TAGCCTGA GCCTATGTCCACTG-3 0 ; Bmp7 Exon 6R, 5 0 -ACCCACCAGT GTCTGGACGATG-3 0 ; and PGK R2, 5 5 0 -CCACCAAAGAACGGA GCCGGTTG-3 0 .
3.3.
Skeletal specimen preparation
Skeletons were prepared as described previously (Ikeya et al., 2006). Briefly, E18.5 mice were eviscerated, skinned, fixed in 80% alcohol, and stained with Alcian Blue and Alizarin Red. Bright field pictures were taken under a dissecting microscope (Olympus SZX12) with a digital camera system Axio Cam (Carl Zeiss).
Acknowledgments We are grateful to the staff of the Laboratory of Animal Resources and Genetic Engineering at Center for Developmental Biology for their help with mouse husbandry, and to Dr. H.
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.mod.2008.06.011.
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