Tài liệu Báo cáo khoa học: Bone morphogenetic proteins in the early development of zebrafish pptx

MINIREVIEW Bone morphogenetic proteins in the early development of zebrafish Mariko Kondo* Department of Biological Sciences, The University of Tokyo, Japan Introduction Bone morphogenetic proteins (BMPs), now widely known for their involvement in many biological pro- cesses, were first described for their bone morpho- genetic activity, and thus were given their names. Four proteins were initially identified, and one of them, BMP1 is a metalloproteinase. The other three (BMP2, 3 and 4) are members of the transforming growth fac- tor b (TGF-b) superfamily of secreted signaling mole- cules. Subsequently, molecular cloning studies have identified more than 20 members of the BMP sub- group in the TGF-b family, from various species. Examples of these members are decapentaplegic (Dpp) and 60A from Drosophila, Xenopus Vg1, and BMP5-7. Although it is not known whether all of the members of this subgroup are involved in bone differentiation, they control a wide range of biological processes in various cell types, such as differentiation, cell prolifer- ation, migration, and apoptosis. The signaling cascade of BMPs has been inten- sively studied, and the players have been identified to a great extent (Fig. 1). Signals from BMPs are mediated by BMP receptors, which also comprise a gene family, the TGF-b receptor family. Functionally active BMPs form dimers, which are secreted and bind to the type I and type II receptors on the cell surface. These receptors are serine ⁄ threonine kinase receptors with a single transmembrane domain. Bind- ing of the ligand to the receptor complex induces the type II receptor to phosphorylate the type I receptor, which then leads to activation of the type I receptor. The signal is passed on to the substrates of the type I receptor kinase, receptor-activated Smad proteins (R-Smads, Smad1, -2, -3, -5 and -8), which, upon phosphorylation, are activated and bind to a common mediator Smad (Co-Smad, Smad4). The complexes move into the nucleus and act as regulators of transcription. The activity of BMP is regulated by the binding of extracellular inhibitors. The roles and functions of BMPs in embryogenesis, from insects to mammals, mostly during the early sta- ges, have attracted the interest of many scientists. In this review, I mainly focus on the recent findings using Keywords bone morphogenetic protein (BMP); dorsoventral patterning; embryogenesis; zebrafish Correspondence M. Kondo, Graduate School of Frontier Sciences, The University of Tokyo, Chiba, Japan E-mail: konmari@biol.k.u-tokyo.ac.jp (Received 30 November 2006, accepted 27 February 2007) doi:10.1111/j.1742-4658.2007.05838.x Bone morphogenetic proteins (BMPs) are known to be widely involved in various biological processes. Many of the members of the BMP family, as well as related factors, receptors and molecules in the BMP signaling path- way, have been isolated, but their precise functions are still unclear. In addition to the ‘classical’ model organism Xenopus, zebrafish, Danio rerio, is now considered to be a suitable model organism to study the roles of the BMP signaling pathway during embryogenesis. Mutagenesis screens have identified a number of mutants in the pathway. Although they do not cover the entire members of the BMP signaling cascade that are currently known, they serve as a powerful tool to broaden our understanding of BMP func- tions, in combination with other experimental techniques. Abbreviations ADMP, anti-dorsalizing morphogenetic protein; BMP, bone morphogenetic protein; TGF-b, transforming growth factor b. *Correction added after online publication 22 May 2007: An author name has been removed at the request of the individual. 2960 FEBS Journal 274 (2007) 2960–2967 ª 2007 The Author Journal compilation ª 2007 FEBS zebrafish, Danio rerio . Zebrafish has established the position as a major model species for early develop- mental studies during the past decade. With zebrafish, large-scale mutagenesis screens have been performed, strains carrying mutation for genes involved in early development have been identified, and much know- ledge, mostly about the function of genes and the rela- tionships among them, is being accumulated. Members of the BMP family in zebrafish The roles of BMP proteins in mesoderm induction and dorsoventral patterning have been studied in detail using Xenopus [1]. Xenopus bmp2, bmp4 and bmp7 tran- scripts are maternally expressed. bmp4 is expressed in the ventral marginal zone during gastrulation but is excluded from the organizer region, and is considered to play the major role in specifying dorsal–ventral struc- tures. Techniques such as animal cap assays and mRNA injection, and, recently, morpholino knock- downs, have been and are the major tools for identifica- tion and functional assays of BMPs. Knockdowns of bmp2, bmp4 and bmp7 by morpholino injections into embryos result in mild dorsalizations, and of these three BMP proteins, BMP4 seems to be the most effective [2]. Members of the zebrafish BMP family involved in early embryogenesis have been likewise identified. In zebrafish, two bmp2 genes, bmp2a and bmp2b, bmp4 and bmp7 are expressed as early as 4 h after fertiliza- tion, in the sphere stage [3–5]. Expression patterns of bmp2 and bmp4 in general show high conservation to those homologs of mouse and Xenopus [6,7]. Zebrafish bmp2b and bmp4 are expressed in the ventral regions in gastrula embryos, indicating the involvement in regulation of dorsoventral patterning, whereas bmp2a cannot be detected by in situ hybridization. To be more precise, bmp2b is expressed in the presumptive mesoderm region exclusive of the embryonic shield but, in contrast, bmp4 is expressed at a low level in the embryonic shield (Fig. 2). Therefore it is likely that zebrafish bmp2b rather than bmp4 may be functionally homologous to Xenopus bmp4. Indeed, judging from the expression pattern in the shield, which corresponds to the Xenopus organizer, and from further studies using zebrafish mutant strains, it has been proven that bmp2b possesses ventralizing activity [8], like bmp4 of mouse and Xenopus. Zebrafish bmp7 was identified through the analysis of the mutant snailhouse (snh) (discussed below). bmp7 is expressed also like bmp2 and bmp4, from the sphere stage on [5,9]. At the sphere stage, bmp7 is expressed Fig. 1. Schematic drawing of the BMP signal transduction pathway. Extracellular BMP dimers bind to the type I and type II BMP receptor complexes at the cell membrane. The BMP type II receptor phos- phorylates the type I receptor, which transduces the signal by phos- phorylating an R-Smad protein. The phosphorylated R-Smad binds with a Co-Smad, and the complex is translocated into the nucleus, where it activates transcription of target genes. P, phosphorylations. Fig. 2. Expression domains of bmp genes and genes involved in BMP signaling. Expression of mRNA at shield stage is denoted by the shaded area. The drawings are based on data from whole mount in situ hybridizations [9,13–15,19]. All embryos are shown from the lateral view, with dorsal to the right. M. Kondo BMPs in the early development of zebrafish FEBS Journal 274 (2007) 2960–2967 ª 2007 The Author Journal compilation ª 2007 FEBS 2961 in the entire blastoderm except for the dorsalmost regions where the organizer will form. At shield stage, a graded expression is observed, with a high expression in the ventral half of the embryo (Fig. 2). Anti-dorsalizing morphogenetic protein (ADMP) [10] is another member of the BMP family. The admp transcripts are, unlike other bmps, expressed dorsally in the zebrafish organizer region. Contradictory to its dorsal expression, overexpression induces loss of dorsal fates, and likely acts as an antagonist of organizer function and inhibits head formation and promotes trunk formation [10]. Although admp may cooper- ate with bmp2b or bmp7 in establishing dorsoventral regionalization, ADMP appears to act through a different signaling pathway because, although the phenotypes from overexpression of these genes are similar at early stages, they are completely different at later stages [11]. BMP antagonists Dorsal development in Xenopus embryos is carried out by secreted peptides synthesized in the Spemann organizer, namely Follistatin, Chordin and Noggin [12]. These proteins are antagonists of BMP and other members of the TGF-b family, binding to and inhibit- ing these signaling molecules from binding to their receptors in the extracellular space, thus inhibiting ven- tralizing activities. Of these proteins, Chordin has a long-range effect. In Xenopus, expression of the chordin gene is localized to the dorsal marginal zone, but the protein is considered to diffuse laterally and ventrally, in a graded manner, hence producing a gradient in BMP activity in the marginal zone. Similarly, zebrafish chordin [13], follistatin [14] and noggin [15] genes have been isolated. These genes, when experimentally overexpressed in the embryo, cause dorsalization of the embryo. chordin,asinXen- opus embryos, is expressed in the dorsal embryonic shield [13] (Fig. 2), which corresponds to the organizer region in frog. In addition, the transcripts are detected in additional regions, such as paraxial mesoderm and ectoderm, which is different from what is observed in Xenopus. Three noggin homologs were identified [15], named noggin1, 2 and 3, and show different expression patterns, but have dorsalizing activities. noggin1 is the earliest of these noggin genes to be expressed in the embryo, and its transcripts are found in the organizer of gastrula embryos, later in the prechordal plate and axial mesoderm (Fig. 2). By contrast, noggin2 tran- scripts are detected at the end of gastrulation in the axial mesoderm, and noggin3 transcripts are limited to chondrogenic regions, and are not expressed in the organizer. Nevertheless, these three gene products have similar biological activities, being able to antagonize BMPs. The expressed regions combined together, cor- respond to that of single-copy noggin homologs in other vertebrates, and could be explained as reflecting the additional genome duplication and subfunctionali- zation of the genes in teleosts. follistatin displays an expression pattern clearly different from Xenopus:itis not expressed in the organizer [14]. It is not detected at early gastrula stages but is detected first at 60% epiboly, in dispersed presumptive mesodermal cells located in the hypoblast (Fig. 2), and then the paraxial expression domain expands, although excluded from anterior axial regions. This indicates that follistatin is not involved in organizer activity, different from the Xenopus homolog, but resembles the mouse homolog, which is also not present in the organizer. BMP inhib- iting activities in other domains or during later stages of dorsoventral patterning is more likely to be the bio- logical function of zebrafish Follistatin. BMP mutations affecting dorsoventral patterning Large-scale mutagenesis screens in zebrafish has been utilized for identification of genes essential for early embryogenesis [16], and a number of mutants with defects in dorsoventral patterning have been identified. Figure 3 shows examples of mutants with either dor- salized (C1–C5) or ventralized (V1–V4) phenotypes [8]. Of those mutant strains isolated from mutant screens, swirl (swr) and snailhouse (snh) [17] were identified as being mutants for bmp2b [8] and bmp7 [5,9], respect- ively. The swirl (bmp2b) mutant is severely dorsalized (C5 phenotype; Fig. 3), and lacks ventral structures, but the paraxial mesoderm and the neuroectoderm are expanded. This demonstrates that bmp2b is required for early dorsoventral patterning. BMP2 functions to maintain its expression in ventral regions during gastr- ulation, and also maintains bmp4 expression, thereby Fig. 3. Morphologies of normal (N), dorsalized (C1–C5) and ventral- ized (V1–V4) embryos, adapted from Kishimoto et al. [8]. V1 and C1 embryos are weakly ventralized or dorsalized, respectively, and the strongest phenotypes are V4 and C5. BMPs in the early development of zebrafish M. Kondo 2962 FEBS Journal 274 (2007) 2960–2967 ª 2007 The Author Journal compilation ª 2007 FEBS the expression is autoregulatory. snailhouse (bmp7) mutants show slightly weaker dorsalization (C4 pheno- type) compared to swirl, although the snh ty68 mutant is temperature sensitive and displays a stronger pheno- type at 33 °C than at lower temperatures, and the aub mutant is a stronger allele of snh and shows the C5 phenotype. Interestingly, bmp2b and bmp7 are only required during embryogenesis, as transient mutant embryos rescued during embryogenesis develop into adulthood and bred [5,18]. swr, snh double mutants are not stronger in phenotype compared to single mutants, indicating that bmp2b and bmp7 function in the same pathway, possibly as heterodimers [9], because the coinjection of bmp2b and bmp7 into the same blastomere, opposed to separate injections in adjacent cells, results in stronger ventralization of the embryo. BMP pathway analyses using zebrafish mutants for bmp genes The somitabun (sbn) mutant, which is strongly dorsal- ized, is caused by the mutation in smad5, a signaling molecule in the BMP pathway [19]. The sbn tc24 muta- tion is both dominant maternal (causing strong C4 dorsalization) and dominant zygotic (weak C1 dorsal- ization). The phenotype is rescued by smad5, bmp2b and bmp4. The early expression of bmp2b in sbn is not affected, but that in early gastrula is severely reduced. This expression also is rescued by smad5, showing that the autoregulatory maintenance of bmp2b expression is mediated by smad5. The sbn phenotype is also rescued by human smad4 RNA injection. Thus, the interaction with Smad4 could account for the sbn mutant phenotype. In addition, the loss of smad5 activity might lead to the inhibition of other Smads, such as Smad1, involved in the Smad signaling pathway, and also lead to the pheno- type. Overexpression of smad1 and smad5 by mRNA injection into wild-type embryos resulted in ventrali- zation (causing up to V4 phenotypes), with smad1 being more effective than smad5 [20]. smad5 mRNA injection rescues sbn and snh (bmp7), but not swr (bmp2b). By contrast, smad1 rescues swr. smad1 is expressed ventrally in gastrula as is bmp2b, and the expression is expanded dorsally and at a higher level by bmp2b overexpression, but is lost in swr embryos, and strongly reduced in sbn embryos. There is a dif- ference between smad1 and smad5 expression, which may cause the difference in function. Smad5 acts very early in development, and its mRNA is supplied maternally and expressed throughout the embryo at a basal level (Fig. 2), whereas smad1 transcripts are only detected from 80% epiboly. As sbn and swr [17], and sbn and snh [5], interact genetically, the bmp2b and bmp7 signals should converge at the level of Smad5 [5]. Based upon these observations, a model for dorsoventral patterning in three phases is suggested [5,19,20]. In the first phase, likely by mater- nal components, the organizer is induced and the ini- tial dorsoventral pattern is set up. bmp2b expression is initiated. The putative dorsoventral BMP gradient is set up in the second phase, where bmp2b expres- sion is maintained through a positive feedback by BMP2B itself and Smad5, but BMP is inhibited by Chordin from the organizer. During this phase, smad1 starts to be expressed. smad1 is also positively autoregulated. bmp7 is also required in this phase to establish the BMP gradient. In the third phase, dorsoventral patterning is carried out by BMP signa- ling, which is mediated by Smad1. By contrast to the second phase, dorsoventral patterning is independent of Smad5 and BMP7. The BMP morphogen gradient established along the ventrolateral axis is explained to induce different cell types [18]. The morphogen concentration is high in the ventral and lower in the dorsal domain. The steepness of the gradient is determined by how much BMP there is available. The different concentration induces differential gene expression according to the threshold needed for induction and leads to cell dif- ferentiation. Depending on the threshold, the dorso- ventral position and quantity of a certain cell type is determined. In swr embryos, no gradient develops due to the lack of bmp2b (morphogen). sbn embryos have a low, and snh embryos have a moderate, morphogen concentration. Therefore, in these mutants, the morphogen gradient is less steep than wild-type embryos, and not sufficient to induce genes at the normal position and quantity in the embryo or can- not induce these genes at all, thus resulting in mutant phenotypes. There is only one report of a mutant strain of the BMP receptor. The dorsalized mutant lost-a-fin (laf) (C2 phenotype) is a mutant of the alk8, the gene cod- ing a member of the ALK1 subgroup of BMP type I receptors [21,22]. This mutant could not be rescued by overexpression of bmp2b or bmp7, but could be rescued by smad5. Moreover, alk8 mRNA injected wild-type embryos developed normally, whereas the constitutive active version of the receptor induced strong ventralization (V4 phenotype) [22], resembling the bmp2, bmp4 or bmp7 overexpressed embryos [4,5,9]. These show that ALK8 acts as a BMP2B ⁄ 7 receptor, and that Smad5 is positioned downstream of this pathway of dorsoventral patterning. M. Kondo BMPs in the early development of zebrafish FEBS Journal 274 (2007) 2960–2967 ª 2007 The Author Journal compilation ª 2007 FEBS 2963 Mutations in BMP regulators Mutation in the BMP binding protein Chordin causes the dino mutant [23,24]. dino mutants show a ventral- ized phenotype (V2), with a small head and a large tail. Other ventral and posterior structures are also enlarged and gene expression is altered: for example, gata2, which is normally expressed in the ventral half of the animal region, is expressed much broader and stronger. The generation of dino-swirl mutant [25] showed that swirl (bmp2b) is epistatic to dino (chordin). This is in agreement with the function of Chordin as a suppressor of BMP. Tolloid is a metalloprotease that acts to cleave Chordin, and to inhibit its activity. In Xenopus,itis considered to counteract the negative regulation of BMP by Chordin. The tolloid gene product of zebra- fish was also shown to possess Chordin cleavage activity [26]. The transcripts are detected throughout the early gastrula stage embryo but toward the end of gastrulation, the accumulation of transcripts around the blastopore and expression in the ectoderm flanking the anterior neural plate is detected. mini fin (mfn), the mutant for tolloid-like 1 (tll1, renamed from tolloid, tld ), show a very weak dorsalized phe- notype (C1) [17], although the allele strengths differed [27]. Analyses using swr or din mutants revealed that tld (tll1) gene expression is positively regulated by bmp2b and negatively by chordin. In some mfn embryos, chordin expression is expanded posteriorly and laterally, but not always in all mutant embryos. The wide range of mfn phenotype may be caused by the degree of chordin expression. Nevertheless, Tld (Tll1) positively regulates bmp4 and tld (tll1) (itself), and negatively regulates chordin. Taken together with the autoregulatory expression of bmp2b and bmp4, these factors form a transcriptional feedback regula- tory loop. Although Tld (Tll1) is involved in regulating BMP expression and BMP activity, mini fin mutants show a relatively weak phenotype, and no effects on dorsoven- tral patterning could be observed. This makes it poss- ible to think that Chordin cleavage may not be so important, but there is evidence for the redundancy in cleavage enzymes, and Chordin is cleaved in mfn [28]. Moreover, subdorsalizing knockdown of Tsg (twisted gastrulation, tsg) by a low dose of tsg-MO induces a stronger phenotype of mfn (C3), indicating that Tld (Tll1) also functions to pattern dorsoanterior struc- tures [29]. Twisted gastrulation (Tsg), a BMP-binding protein, initially found in Drosophila, is involved in dorsal– ventral patterning, The role of twisted gastrulation is not clearly understood because there are reports on its functions in opposite directions in the BMP pathway. The function of Tsg as a BMP antagonist is repor- ted to be conserved in Xenopus and zebrafish [30–32]. Injection of antisense morpholino against tsg1 blocking the Tsg function weakly ventralizes zebrafish embryos, whereas overexpression of tsg1 mRNA dorsalizes embryos [31]. Tsg inhibits BMP activity synergistically with Chordin, as shown by the enhancement of ventral features caused by coinjection of subinhibitory doses of morpholinos against these two factors [31]. Accord- ing to Ross et al. [31], there are three molecular func- tions of Tsg: (a) synergistic inhibition of BMP by forming a tripartite complex between Tsg, Chordin and BMP; (b) enhancement of the Tld ⁄ BMP-1 medi- ated cleavage rate of Chordin, which may change the preference of site utilization; and (c) promotion of the dissociation of Chordin cysteine-rich (CR)-containing fragments from the ligand. They suggest that the first of these three is the primary function in zebrafish. The function of Tsg as a BMP agonist has also been reported [29,33]. Tsg is considered to ventralize Xeno- pus embryos by blocking the CR1 domain of the Chor- din protein that blocks BMP. Tsg competes with CR-1, but not full-length Chordin, to bind BMP, and releases BMP, forming a complex between BMP and Tsg [33]. In zebrafish, knockdown experiments with tsg-MO resulted in moderately to moderately strong dorsalization (C3–C4) [28,29] by contrast to the previ- ous study [31] that reports ventralization. Quite sur- prisingly, with higher doses of Tsg, again, embryos are dorsalized (C1–C3) [28]. In the low-dosage injected embryos, chordin expression is expanded laterally [29]. The dual activities of tsg was also shown from injec- tion experiments into ventralized dino or sizzled mutants, where partial rescue of the phenotype was observed upon either tsg-MO or tsg mRNA [29]. In another study, chordin mRNA injection with tsg- MO injection resulted in an increase of rescued dino embryos compared to without tsg-MO [28]. Thus, Tsg decreases the effectiveness of Chordin as a BMP inhib- itor. swr heterozygous embryos, which normally are phenotypically wild-type, are dorsalized (C1–C4) with a subdorsalizing dose of tsg-MO (less that that required for dorsalizing wild-type embryo), suggesting the interaction of Tsg with BMP2B for dorsalization [29]. The ventralizing activity of Tsg is not completely Chordin dependent because, as mentioned above, the ventralized phenotype of din mutants could be partially rescued by tsg-MO injection [29]. According to the model proposed by Larrain et al. [34], based mainly on studies from Xenopus, Tsg acts BMPs in the early development of zebrafish M. Kondo 2964 FEBS Journal 274 (2007) 2960–2967 ª 2007 The Author Journal compilation ª 2007 FEBS in two steps, first by binding to BMP and forming a ternary complex with full-length Chordin, thus inhibit- ing BMP to bind to its receptor. When this complex is cleaved by Tolloid (in the case of Xenopus, Xolloid), Tsg is able to free BMP from the Chordin complex and to destabilize the Chordin proteolytic products that still possess anti-BMP activity, thus functioning to enhance BMP signaling [34]. The involvement of other unidentified factors in this process is also suggested [28,29,35]. The debate regarding the function of tsg in the BMP pathway probably will remain unresolved for some time. Other factors affecting the BMP pathway Factors inhibiting BMP2b, other than direct binding proteins such as Noggin and Chordin, also affect dorsoventral patterning. One example is found from the mutant bozozok (boz) with a mutation in the homeodomain protein coding dharma ⁄ nieuwkoid gene [36]. In wild-type embryos, bmp2b is first expressed ubiquitously in the blastoderms but just before gastru- lation, the transcripts disappear from the dorsal side. However, in boz mutants, this clearing of bmp2b tran- scripts does not happen, and the expression is detected in the dorsal margin and dorsal yolk syncytial layer [37]. Misexpression of dharma mRNA reduced the level of bmp2b expression, thereby indicating that dharma is responsible for the bmp2b down-regulation in pregastrula embryos. The level in which dharma regulates bmp2b is not yet clear. sizzled is the gene responsible for the ogon (allelic to mercedes and short tail) mutant, a ventralized mutant (V1) [38,39]. swr, snh and lost-a-fin are epistatic to ogon [40,41]. sizzled is expressed in late blastula to 24 hours after fertilization embryos, located in the ventral side of the wild-type embryo, but the expres- sion is expanded dorsally in ogon and din, and is down-regulated in swr embryos [38,39]. This suggests that the expression of sizzled is regulated itself in a BMP2B-dependent manner. Sizzled is a homolog of Xenopus Secreted Frizzled, which is a putative Xwnt8 antagonist, and is a secreted protein with similarity with the Frizzled receptor, but lacks the transmem- brane domain. However, zebrafish Sizzled does not inhibit the function of Wnt8 [39], but rather acts as a BMP inhibitor. Although overexpressed sizzled dorsal- izes wild-type embryos, the din mutant is not rescued. Expression of chordin is induced by Sizzled in embryos with Chordin, but not in the absence of Chordin. These results indicate that Chordin is required for dor- salization through Sizzled. Recently, the mechanism by which Sizzled, with BMP2b, BMP7, Chordin, Tll1, and BMP1a (a new member of the Tolloid-family metalloproteinase), con- trols dorsoventral polarity, was proposed in zebrafish [42] and similarly in Xenopus [43]. The dorsalizing activity of Sizzled is Chordin dependent and, on the other hand, sizzled suppresses the ventralization by tll1 and bmp1a. Sizzled stabilizes Chordin protein, whereas the Chordin-cleavage activity of BMP1a and Tll1 are suppressed. Thus Sizzled, positively regulated by BMP2b and BMP7, negatively regulates BMP1a and Tll1, which in turn, inhibit Chordin, which neg- atively regulates BMP activity. If Sizzled diffuses from the ventral side where the mRNA is expressed to the dorsal side, this mechanism could create the gradient of BMP and Chordin along the dorsoventral axis. Other aspects of BMP signaling, besides dorsoventral patterning, are reported. BMP signaling is involved in endodermal patterning, and this is also regulated by the same mechanism as the dorsoventral patterning, the morphogen activity gradient, only that dorsal corres- ponds to anterior, and ventral to posterior endoderm [44]. The gradient of BMP2b and Chordin define the regional expression of her5 , which is an endodermal marker controlling endodermal cell patterning. her5 is negatively regulated by BMP2b, which in turn, is ant- agonized by Chordin. Thus similar feedback loops, perhaps by other unidentified proteins might be respon- sible for the regulation. Conclusions The study of BMPs during embryogenesis has been extensively carried out using Xenopus but, during recent years, zebrafish has also contributed much to gaining new knowledge through the use of genetics. However, in spite of extensive and thorough screening that have been done in the zebrafish system, currently, there are no reported bmp4 mutant or mutants of other many factors involved in the BMP pathway. This may be explained by assuming that there is a func- tional overlap or redundancy among the different BMP-signaling genes, and that the function could be compensated by other factors or by other members of BMPs; thus, no apparent mutation could be detected in the screens. It may also be explained by the addi- tional whole genome duplication event that occurred in teleosts. There is still much to be elucidated in the BMP pathway during embryogenesis, Many important ques- tions are unanswered, such as the how the earliest dorsoventral pattern is formed or how specific the M. Kondo BMPs in the early development of zebrafish FEBS Journal 274 (2007) 2960–2967 ª 2007 The Author Journal compilation ª 2007 FEBS 2965 threshold of BMP as a morphogen is to induce cell fate speciation. In addition, the biological functions of some (or many) of the factors involved in modifying BMP signaling are still undetermined or may be vari- able depending on the situation of the cell or the tim- ing during embryogenesis. Nevertheless, the availability of mutant strains, gen- etic crosses, knockout and overexpression systems make zebrafish a very powerful tool in studying early development and, taken together with the findings from Xenopus, would give us a clearer view of the BMP signaling pathway. 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MINIREVIEW Bone morphogenetic proteins in the early development of zebrafish Mariko Kondo* Department of Biological Sciences, The University of Tokyo,