Mesoderm induction: from caps to chips

Mesoderm induction: from caps to chips David Kimelman Abstract Vertebrate mesoderm induction is one of the classical problems in developmental biology. Various developmental biology approaches, particularly in Xenopus and zebrafish, have identified many of the key factors that are involved in this process and have provided major insights into how these factors interact as part of a signalling and transcription-factor network. These data are beginning to be refined by high-throughput approaches such as microarray assays. Future challenges include understanding how the prospective mesodermal cells integrate the various signals they receive and how they resolve this information to regulate their morphogenetic behaviours and cell-fate decisions. Blastula A stage during which the embryo undergoes cleavage to become multicellular. The late blastula stage precedes gastrulation. Gastrula A stage during which the embryo undergoes major morphogenetic changes, which positions the endoderm on the inside, the mesoderm in the middle and the ectoderm on the outside. Organizer A signalling centre in a vertebrate embryo comprising a group of cells that secrete signalling factors or inhibitors of signalling factors, which changes the fate of the surrounding cells. Department of Biochemistry, Box 357350, University of Washington, Seattle, Washington 98195-7350, USA e-mail: kimelman@ u.washington.edu doi:10.1038/nrg1837 The vertebrate mesoderm produces a wide range of tissues including the muscles, heart, vasculature, blood, kidney, gonads, dermis and cartilage, and it also has a major role in the morphogenetic movements of gastrulation. The study of mesoderm formation originated with ieter Nieuwkoop s classical experiments in the amphibian embryo, nearly 40 years ago. Nieuwkoop showed that explanted tissue from the bottom of the blastula-stage embryo (the vegetal cap) could convert prospective ectodermal cells taken from the top of the embryo (the animal cap) into mesodermal tissues that normally reside at the equator, demonstrating that the mesoderm is formed by a mechanism called induction 1. Induction refers to a process in which extracellular signals bring about a change from one cell fate to another in a particular group of cells. In the classical view, induction occurs when one group of cells signal to a different set of cells that respond by changing fate. In practice, it can be more complex than this. In zebrafish, for example, the prospective mesodermal cells are involved in sending and receiving the inducing signals. Before Nieuwkoop s seminal experiments, the first inductive event in the amphibian embryo was thought to involve gastrula-stage signals that originate in the organizer and pattern the mesoderm and ectoderm (reviewed in REF. 2). Although subsequent experiments have led to the subdivision of mesoderm induction into initiation, maintenance and patterning events, here I use mesoderm induction in a broad sense to encompass all these steps because all three take place concurrently in the late blastula/early gastrula embryo. Nieuwkoop s original assay provided a valuable tool for studying embryonic signalling that is still in constant use today. Because animal caps can be easily isolated and grown in a simple buffer solution, and because conversion of animal cap explants to mesoderm can be readily monitored at the macroscopic level, this assay provided a valuable means for identifying the first mesoderm-inducing factors (reviewed in REF. 3). With the development of more sophisticated methods for measuring gene expression and manipulating the amount of endogenous signalling in Xenopus, the utility of the animal cap-explant assay for understanding the basic biology of inductive processes has continued to expand. Although the field of mesoderm induction began with studies in amphibians, today it is impossible to discuss this topic without including the genetic studies in zebrafish that have contributed important new discoveries to this area. Comparison of the two species, which is also the primary focus of this review, provides interesting insights into conserved aspects of mesoderm induction, as well as changes in the induction mechanisms to accommodate different embryological features. Studies in Xenopus and zebrafish also provide a valuable foundation for understanding mesoderm induction in other species, such as birds and mammals. We can now begin to develop a clear understanding of the molecular interactions that underlie this essential process in embryonic development. Here I first introduce the principal signalling factors that are involved in mesoderm induction, with emphasis on members of the family, which function to initiate the formation of the mesoderm. I discuss how these factors function at different times and in various combinations to regulate different regions within the embryo. I conclude by examining some new approaches that are 360 MAY 2006 VOLUME 7 www.nature.com/reviews/genetics

Tail somites Xenopus Trunk somites Ventral Notochord ronephros Blood Blood Head Notochord A rod-shaped structure that runs along the dorsal axis of the embryo, separating the muscle blocks. It is one of the defining features of the phylum Chordata, to which vertebrates belong. Fate map A map that shows which tissues are likely to develop from different regions of the embryo. Animal Vegetal Dorsal Zebrafish Atrium Ectoderm Mesoderm Endoderm YSL Tail somites Trunk somites Notochord ronephros Head Erythroid Ventricle Myeloid Figure 1 Fate maps of Xenopus and zebrafish embryos at the late blastula/early gastrula stage. rospective mesodermal territories are shown in red. Note that in zebrafish the bottom layer of the mesoderm sits on top of the extra-embryonic yolk syncytial layer (YSL), and that mesodermal cells are intermixed with endodermal cells (green). The fate maps are based on REFS 5,125,126. being used to gain further insights into the mechanism of mesoderm formation and regulation. Forming the mesoderm In both zebrafish and Xenopus embryos, mesoderm induction creates a zone of mesodermal cells at the equator of the embryo (often called the marginal zone). Whereas the mesoderm and endoderm are regionally distinct in Xenopus, in zebrafish the endoderm and mesoderm precursors are mixed (FIG. 1). In addition, whereas all the cells in the early Xenopus gastrula contribute to the final embryo, in zebrafish the embryo sits on top of a yolk cell that does not undergo cleavage. This difference in architecture between the two species has major consequences for the mechanism of mesoderm induction, as discussed below. In addition to establishing the mesodermal zone, inductive processes pattern the embryo along what is commonly called the dorsal ventral axis as well as the animal vegetal axis (FIG. 1). Because the dorsal side produces both anterior (head) and dorsal (notochord) fates, whereas the ventral side produces both posterior (tail) and ventral (for example, pronephros) fates, the equatorial axis in the pre-gastrula embryo might be better termed the dorsoanterior ventroposterior axis (for an excellent discussion of the complexities in labelling the pre-gastrula axis see REF. 4). Regardless of the nomenclature, complex morphogenetic movements during gastrulation bring mesodermal cells into their correct position within the post-gastrula embryo. Although fate maps are often drawn with the late blastula/early gastrula mesoderm demarcated into regions of defined fate, careful lineage labelling of zebrafish embryos at these stages has demonstrated that mesodermal derivatives outside the organizer region are highly intermixed 5,6. Similarly, a recent study in Xenopus has shown that more cells in the early gastrula embryo express a marker for a different domain than is predicted by the fate map 7. This result probably reflects the fact that embryonic cells are exposed to multiple signals (including signalling factors and their inhibitors) as they migrate in the embryo; therefore, their eventual fate might represent the sum of these external influences, rather than their position at a particular point in time. Signalling factors in mesoderm induction It is likely that all of the families of signalling factors that are important for mesoderm induction have been identified, although all of the crucial individual factors might not yet have been determined. A surprising feature is the complexity of signalling factors that are used by the embryo for inducing mesoderm (TABLE 1). From the perspective of the embryo, there might be evolutionary advantages to having multiple factors with overlapping functions. From the perspective of the experimentalist, however, this degree of redundancy can make loss-of-function studies much more challenging. As a first approximation one can say that: the family is involved in initiating mesoderm formation, FGFs (Fibroblast growth factors) and Wnts are involved in maintaining the mesodermal state, and BMs (Bone morphogenetic proteins) are involved in patterning the mesoderm. This, however, is an oversimplification of what these factors actually do. For example, in various experimental models, FGFs, BMs and Wnts have been shown to be sufficient for initiating mesoderm formation 8 13, and family members have been shown to be involved in patterning the mesoderm 14,15. These results indicate that extracellular signals do not necessarily have rigidly separated functions in mesoderm induction, and instead indicate that these signals might work in a partially overlapping way to form and pattern the mesoderm. Because the family functions as the main initiating stimulus for mesoderm induction 16, it is the main focus of the discussion below (for a more thorough discussion of the roles of Wnts, BMs and FGFs see REFS 17 19). A description of the intracellular signalling pathways that are used by each of these factors can be found in BOX 1. Models for mesoderm induction in fish and frogs Recent studies have revealed how the mesoderm-inducing signal described by Nieuwkoop 1 is localized to the vegetal hemisphere in Xenopus embryos. During oogenesis, transcripts that encode the T-box transcription factor VegT are localized to the vegetal pole through a complex process that involves specific RNA-binding factors and cytoskeletal elements (reviewed in REF. 20). At fertilization, VegT transcripts are released from the vegetal pole and slowly diffuse upwards. Because the third cleavage plane passes through the equator of the embryo before the transcripts leave the vegetal hemisphere, the VegT transcripts are trapped there, and therefore the subsequently translated VegT protein is restricted to the vegetal half. At the start of zygotic transcription at the 4,000-cell stage, which is called the mid-blastula transition, VegT activates the transcription of the Xenopus nodal related (Xnr) genes, which then initiate mesoderm formation 21 25 (FIG. 2). NATURE REVIEWS GENETICS VOLUME 7 MAY 2006 361

In zebrafish, the genes squint (sqt; also known as ndr1) and cyclops (cyc; also known as ndr2) (BOX 1) initiate mesoderm formation, but their transcription is activated by an unknown signal originating in the extraembryonic yolk syncytial layer (YSL; reviewed in REF. 26) (FIG. 2). The zebrafish VegT orthologue has been identified, and although it has an important role in forming the trunk musculature as discussed below, it is not maternal and it does not activate nodal gene expression 27. It is still unclear how a YSL-specific signal is actually produced, although it seems unlikely to use the same mechanism as is used by Xenopus VegT. The YSL does not even form as a distinct entity until approximately the 1,000-cell stage, and the YSL nuclei and cytoplasm have the same origin as the overlying embryonic cells (called the blastomeres) that form the mesendoderm. Therefore, if a transcript were restricted to the vegetal pole during oogenesis and released at fertilization, it would probably be inherited by both the YSL and the blastomeres. It is possible that there is a mechanism that anchors a transcript (or protein) to the yolk and keeps it inactive until the YSL is formed; at that point the factor could be released into the YSL and activated to initiate mesoderm formation. signalling: are gradients important? As described below, the embryo potentially has gradients of activity along the dorsal ventral and animal vegetal axes. The existence, significance and role of these gradients in mesoderm induction continue to be hot topics of debate. In Xenopus, several lines of evidence indicate that signalling is stronger on the dorsal than on the ventral side of the pre-gastrula embryo. In Xenopus embryos, a dorsalizing activity moves from the vegetal pole towards one side of the embryo soon after fertilization (reviewed in REF. 28), and evidence from experiments in which the early vegetal cytoplasm was removed from the embryo indicates that there is a similar mechanism in zebrafish 29,30. The net result of this movement is the stabilization of β-catenin, an intracellular Wnt pathway component (BOX 1), on the future dorsal side of the embryo (FIG. 3). This asymmetrical stabilization of β-catenin is essential for the formation of all dorsal and anterior structures (REFS 31,32; reviewed in REFS 28,33). Among the many targets of β-catenin are members of the gene family called the Xenopus nodal-related factors; for this reason, the Xnr genes are expressed earlier and/or at a higher level on the dorsal side of the embryo (FIG. 3). For example, Xnr5 and Xnr6 are transcribed on the dorsal side of the embryo in response to β-catenin as early as the 256-cell stage, well before most embryonic genes begin to be transcribed at the mid-blastula transition 34. These two Xnr genes, as well as Xnr1, Xnr2 and Xnr4, which are first transcribed at the mid-blastula transition, are expressed in a dorsal ventral gradient in the late blastula stages 35,36. Consequently, signalling is at least transiently enriched dorsally, as indicated by increased phosphorylation (and so activation) of the intracellular factor Smad2 (BOX 1) on the dorsal Table 1 Candidate zygotic signalling molecules in mesoderm induction* Signal family Xenopus Mouse Chick Zebrafish (mutant/morpholino phenotype) Xnr1 Sqt (cyclopia/dorsal mesoderm defects) Xnr2 Gdf3 Vg1 Cyc (cyclopia) Xnr4 Vg1 (N.D.) Xnr5 Xnr6 Activin Derriére Vg1 FGF Fgf3 Fgf3 Fgf8? Fgf3 (N.D.) Fgf4 Fgf9 Fgf4 Fgf8 (cerebellum and mild posterior defects) Fgf8 Fgf8 Fgf24 (pectoral fin defect ) BM Bmp2 Bmp7 Bmp2 Bmp2b (severe dorsalization) Bmp4 Bmp2 Bmp4 Bmp4 (N.D.) Bmp7 Bmp4 Bmp7 Bmp7 (severe dorsalization) Admp Admp Admp (dorsalized) Wnt Wnt8 Wnt8 Wnt8c Wnt8 (posterior mesoderm and neural defects) Wnt3 Wnt3a (no effect # ) Wnt3a *There is no horizontal relationship between the genes listed in this table. Zebrafish with mutations in these genes have been identified. sqt;cyc double mutants have no head or trunk mesoderm, and no endoderm. fgf24 MO in fgf8 mutants causes loss of the most posterior mesoderm. Members of the canonical (β-catenin stabilizing) family of Wnts are listed. # Loss of wnt3a enhances loss of wnt8. Admp, Anti-dorsalizing morphogenetic protein; BM, Bone morphogenetic protein; Cyc, Cyclops; FGF, Fibroblast growth factor; Gdf3, Growth/differentiation factor; N.D., not determined; Sqt, Squint; Xnr, Xenopus nodal related. 362 MAY 2006 VOLUME 7 www.nature.com/reviews/genetics

Box 1 Intercellular signalling pathways Many of the essential details of the intracellular pathways that are used by the four signalling factors discussed in this review have been identified and are shown here (see figure). Below I discuss key aspects of each of the main pathways; for more extensive discussions of these pathways see REFS 119,120,121. and BM Both the and BM (bone morphogenetic protein) pathways are activated when a ligand binds a specific heterotetramer that is composed of two type I and two type II receptors, as is the case with all members of the Transforming growth factor-β (TGFB) family. In zebrafish, mutants have been identified in two ligands: squint (sqt) and cyclops (cyc), and in two BM ligands: swirl (bmp2b) and snailhouse (bmp7). The type II receptor phosphorylates a cytoplasmic domain on the type I receptor, activating the type I receptor to phosphorylate a Smad factor. Whereas Smad2 and Smad3 are specific mediators of (including Activin and Vg1) pathways, Smad1, Smad5 (Somitabun in zebrafish) and Smad8 are mediators of the BM pathway. When these Smads are phosphorylated they bind Smad4 and translocate to the nucleus where they bind to specific DNA-binding factors. The Smad factors also have a weak DNAbinding ability, and it is the combination of the DNA-binding specificity of the molecular partner together with the DNA-binding specificity of the Smads that allows the transcriptional activation of specific targets. For example, in the zebrafish pathway, the binding of Smads to the trancription factor Bonnie (Bon) is an essential step in endoderm formation, whereas the binding of Smads to Schmalspur (Sur) is important for mesoderm formation. The pathway is unique in that it requires an extracellular membrane-bound EGF CFC cofactor called One-eyed pinhead (Oep) in zebrafish, Xcr1/Frl1, Xcr2 and Xcr3 in frogs, Cryptic in chicks and Cripto and Cryptic in mice 122,123. Although these factors are required for and Vg1 signalling 124, it remains unclear why and Vg1 specifically require these cofactors to signal whereas related ligands such as Activin do not. Canonical Wnt The Wnt pathway is divided into at least two main branches, including the canonical (β-catenin-dependent) pathway and the non-canonical pathway(s), which is independent of β-catenin. In the canonical Wnt pathway, the Wnt ligand binds to a Frizzled/LR heterodimer, which mediates the intracellular response. Exactly how these receptors activate the downstream pathway is still unclear, but it involves G-protein signalling, LR phosphorylation on its cytosolic C terminus, and the activity of Dishevelled (Dsh). The net result of these factors is the disruption of a large protein machine called the β-catenin destruction complex which phosphorylates β-catenin on its N terminus causing it to be ubiquitylated and then rapidly degraded by the proteasome. The destruction complex is composed of many proteins, including the central scaffolding protein Axin (Masterblind in zebrafish), the Adenomatous polyposis coli (AC) protein, and the kinase Glycogen synthase kinase 3 (Gsk3). Several studies have suggested that Wnt signalling disrupts the complex by causing one or more proteins to leave the complex, preventing the phosphorylation and ubiquitylation of β-catenin. When β-catenin is not degraded, it accumulates and translocates to the nucleus, where it binds members of the Tcf/LEF1 family of DNA binding factors (including Headless (Hdl) in zebrafish), and recruits transcriptional activators to the promoter. FGF Binding of the ligand to the Fibroblast growth factor (FGF) receptor (Fgfr) results in receptor dimerization and transphosphorylation of the receptor s cytosolic domain. The phosphorylated receptor recruits proteins that activate the G-protein Ras, which then activates the kinase Raf. Raf phosphorylates and activates Mek, which subsequently phosphorylates and activates MA kinase (Mapk). Mapk enters the nucleus where it phosphorylates and activates target transcription factors. BM Wnt FGF BM Wnt FGF Type I Type II EGF CFC Type I Type II Frizzled LR Dsh G Fgfr Fgfr Ras Raf Smad2,3 Smad4 Smad1,5,8 Smad4 AC Gsk3 Axin β-catenin Mapk Mek Smad2,3 Smad4 Smad1,5,8 Smad4 β-catenin Tcf/LEF Mapk NATURE REVIEWS GENETICS VOLUME 7 MAY 2006 363

Xenopus Xnrs VegT Morpholinos Antisense oligonucleotides that are stable and are commonly used in zebrafish and Xenopus to inhibit either the translation or splicing of mrnas. Spemann s organizer A signalling centre in amphibians that is created on the dorsal side of the late blastula embryo. The equivalent centre in fish is called the shield. YSL Zebrafish sqt and cyc Figure 2 Models for activation of signalling in Xenopus and zebrafish. In Xenopus, the vegetally localized transcription factor VegT (blue) activates the transcription of the Xenopus nodal-related genes (Xnrs) in the vegetal hemisphere, which then initiate mesoderm formation. In zebrafish, signals from the yolk syncytial layer (YSL; blue) activate the transcription of the genes squint (sqt) and cyclops (cyc), which then initiate mesoderm formation. side of the late blastula embryo 37,38. However, by the early gastrula stages, phosphorylated Smad2 levels are equal across the dorsal ventral axis 37,38. Therefore, there is a period of time during which signalling is asymmetrical across the dorsal ventral axis, and this could be important for establishing asymmetrical gene expression along this axis. In zebrafish, the gene sqt has been proposed to be directly activated by β-catenin, resulting in dorsal expression of sqt for a brief time before its expression throughout the mesendoderm 39. Whether or not this causes a transient dorsal ventral asymmetry in phosphorylated Smad2 levels has not yet been determined. Another potential mechanism for regulating asymmetrical zebrafish nodal expression has recently been reported. Maternal sqt transcripts seem to be transported to the dorsal side of the zebrafish embryo as early as the 4-cell stage, presumably allowing the Sqt protein to accumulate dorsally before the onset of zygotic transcription 40. Morpholino-mediated knockdown experiments indicate that a loss of maternal and zygotic Sqt causes a more severe phenotype than loss of the zygotic Sqt alone. However, embryos that genetically lack maternal and zygotic Sqt were reported to have no more severe defects than embryos that lack only zygotic Sqt 41, and embryos that lack only maternal Sqt show no early patterning defects (S. Dougan, personal communication). Therefore, the importance of localized maternal sqt might depend on the genetic background of the embryo and/or epigenetic effects. Several elegant experiments in Xenopus using Activin as a pathway stimulant have shown that variations in the level of activity can regulate dorsal ventral patterning, with low levels inducing ventral fates and high levels inducing dorsal fates 42 44. These results are consistent with the idea that activity is higher dorsally than ventrally, at least in the pre-gastrula embryo. Nevertheless, it has been difficult to ascertain whether the pathway actually regulates dorsal ventral patterning. Studies in frogs using different doses of a synthetic -specific inhibitor (the truncated Cerberus protein) seemed to indicate that ventral mesoderm was most readily eliminated by small reductions in signalling, whereas dorsal mesoderm was only eliminated with higher doses of antagonist 35. These are the only data so far that support the importance of a dorsal ventral gradient in Xenopus. Similarly, no evidence has yet been presented that a reduction of signalling on the dorsal side results in a respecification of dorsal to ventral fates. Surprisingly, reduction of signalling using various sqt and cyc mutants in zebrafish produced the opposite result; the dorsal region was the most sensitive to a loss of signalling 45. In this case, reduced signalling resulted in a reduction in dorsal mesoderm formation but not a respecification to ventral mesodermal fates. Therefore, there is no compelling evidence at this point that a temporal/spatial gradient of signalling regulates differences in dorsal ventral mesodermal fates in either fish or frogs. Studies in zebrafish have indicated that gradients are involved in patterning the mesoderm along the animal vegetal axis. Differences in levels are proposed to separate head mesoderm from the notochord within the organizer 14,45, and to separate ventricular and atrial myocardial precursors 46. This concept is generally consistent with Xenopus studies showing that partial or Activin inhibition causes head defects without affecting the notochord 47 50, and with the existence of a vegetal to animal gradient of phosphorylated Smad2, indicating that a gradient might also pattern the embryo along the animal vegetal axis in frogs 37,38. Further studies will need to determine to what extent differences in -signalling levels specify different mesodermal tissue fates in vivo. The enigmatic Vg1 The Xenopus Transforming growth factor-β (TGFB) family member Vg1 was originally identified as an excellent candidate to be the vegetally derived Nieuwkoop signal (REF. 51; reviewed in REF. 52). Vg1 transcripts are localized to the vegetal pole in a similar way to VegT. Moreover, the mature region of the Vg1 pro-protein attached to a different pro-domain can function as a potent mesoderminducing agent. However, the originally identified Vg1 pro-protein has no activity and endogenous mature Vg1 is not detectable in Xenopus embryos. Although there is evidence that Vg1 has an important role in chick mesoderm induction 53,54 and a Vg1-related protein Growth/differentiation factor 3 (Gdf3) is essential for mesoderm formation in the mouse 55, until recently Vg1 was shown to be crucial only for establishing left right asymmetry in Xenopus 56 58. However, Birsoy et al. showed that embryos with depleted Vg1 lack head and notochord structures, which is most likely due to a reduction in the expression of Spemann s organizer genes, particularly the BM and Wnt inhibitors 15. Moreover, the authors resolved the issue of the inactive Vg1 pro-protein by demonstrating the existence of a second Vg1 allele, which is active as an inducing agent 15. 364 MAY 2006 VOLUME 7 www.nature.com/reviews/genetics

ostfertilization Blastula re-gastrula Xenopus Zebrafish? Dorsalizing activity β-catenin activity Maternal squint Figure 3 Establishing the dorsal ventral axis. In Xenopus (left panels) a dorsalizing activity moves from the vegetal pole to one side of the embryo after fertilization, and a similar mechanism seems to occur in zebrafish (right panels). During the blastula stages, this dorsalizing activity stabilizes β-catenin on what will be the future dorsal side of the embryo. When zygotic transcription of the Xenopus genes begins, the β-catenin creates an asymmetry in expression, which results in elevated phosphorylated Smad2 levels on the dorsal side of the pre-gastrula embryo. In zebrafish, maternal squint transcripts are localized to the future dorsal side by the 4-cell stage, and zygotic squint is initially transcribed dorsally. It is not yet clear whether this creates a significant asymmetry in phosphorylated Smad2 levels. Although this work has re-established Vg1 as an important member of the family of mesoderm inducers, it raises the question as to why so many members of this family are used in Xenopus (TABLE 1). In zebrafish, the vg1 transcript is maternal but not localized 59, and so far there is no evidence that it is involved in either mesoderm induction or left right patterning. Therefore, if zebrafish Vg1 is essential for signalling as in frogs, chicks and mice, it either functions as a ubiquitous factor or the processing of the pro-protein to the mature ligand is spatially regulated. Beyond s: maintenance and patterning Evidence from both fish and frogs demonstrates that the head (anterior) is regulated differently from the trunk and tail (posterior). For example, inhibition of FGF signalling in both systems eliminates the trunk and tail without causing severe head defects, indicating that these two regions of the body use different regulatory circuits 60,61 (FIG. 4). Mesoderm induction in the head activates various transcription factors and secreted inhibitors of the BM, Wnt and pathways that are essential for patterning the embryo 33. Meanwhile, trunk mesoderm induction activates signalling factors of the FGF and Wnt families (BOX 1; TABLE 1), along with several transcription factors. Among the transcription factors are at least three members of the T-box family, Xbra (in frogs) or No tail (Ntl; in fish), VegT (in frogs) or Spadetail (Spt; in fish), and Tbx6 (in fish and frogs), which function combinatorially to regulate mesoderm formation 62. Whereas zebrafish that lack spt (also known as tbx16) or ntl function have major defects within the trunk or tail, respectively, fish that lack both T-box genes fail to form the trunk and tail mesoderm, demonstrating that these factors function early and redundantly in the initial stages of mesoderm induction 63. Among the targets of the T-box genes are FGFs, which function in an autoregulatory loop to maintain T-box gene expression 64 66 (FIG. 4). Zygotic Wnt signalling not only limits the size of the Spemann s organizer 67, it is also necessary for the maintained expression of the T-box genes 68 70 (FIG. 4). Why both FGFs and Wnts are needed to maintain the mesoderm is still not clear. In addition to s, Wnts and FGFs, several members of the BM family and their inhibitors are involved in mesoderm patterning (REFS 71,72; reviewed in REF. 73) (BOX 1; TABLE 1). Surprisingly, both the dorsal and ventral sides of the embryo simultaneously express BMs and BM inhibitors, indicating that the regulation of mesodermal patterning by BMs is complex. Zebrafish embryos that are mutant for bmp2b or frog embryos with depleted Bmp4 and Bmp7 fail to form tails 26,74 76, which is consistent with the proposal that BMs are essential regulators of tail development 77,78 (FIG. 4). Many lines of evidence indicate that the embryonic mesoderm of frogs and fish is subdivided into three principal domains: the head, trunk and tail. As discussed above, the trunk and tail require FGF signalling and functional T-box genes, whereas the head does not. The trunk and tail are also under separate regulatory control, although the regulation of tail formation seems to be different between fish and frogs. In Xenopus, the initiation of tail outgrowth involves the BM and Notch signalling pathways, coupled with distinct changes in gene expression as the tail forms 79 82. Although BM signalling is also required for tail formation in zebrafish 75,76,78, there is no evidence for Notch signalling in this process, nor are there any reported changes in gene expression when the tail begins to develop. However, zebrafish tail formation clearly involves a pathway that is distinct from head or trunk formation, because zebrafish embryos with deficient signalling form essentially normal tails, even though the head and trunk mesoderm are absent 83. Depletion of the dorsally expressed BM ligand Admp (Anti-dorsalizing morphogenetic protein) in Xenopus and zebrafish also results in embryos with defects in posterior development 84 86. Moreover, an important new analysis of Xenopus embryos that lack dorsal (Admp) and ventral (Bmp2, Bmp4 and Bmp7) BMs revealed that interactions between the BMs and NATURE REVIEWS GENETICS VOLUME 7 MAY 2006 365

Maternal β-catenin High Wnt Medium T-box genes FGF Low (frog) Low /YSL signal (fish) Wnt T-box genes Head Trunk Tail Figure 4 A model for patterning the embryonic body in Xenopus and zebrafish. The fish and frog body (shown here as a generic embryo) is divided into three principal domains: the head, trunk and tail, and some of the key signalling events are shown. In zebrafish, the yolk syncytial layer (YSL) signal seems to be redundant, with signalling inducing the formation of the tail. Note that the role of Wnt signalling within the trunk is uncertain. BM, Bone morphogenetic protein; FGF, Fibroblast growth factor. FGF BM their inhibitors on both the dorsal and ventral sides of the embryo are essential for establishing normal dorsal ventral patterning 86,87. It is still unclear how the posterior dorsal (tail mesoderm), posterior ventral (the posterior blood islands) and ventral (pronephros) fates (FIG. 1) are specified. Although BM signalling is necessary for the formation of the ventral mesodermal fates, there is no compelling evidence that a gradient of BM signalling distinguishes between the different tissues that form on the ventral side. This remains an important area of investigation. Temporal aspects of mesoderm induction Mesoderm induction occurs over several hours during which embryonic cells are exposed to shifting concentrations of inducing signals and their inhibitors. Even if a cell begins to head towards a particular fate decision, it might change fate if it receives new external influences. But once a cell becomes irreversibly committed to a specific fate it is said to be determined. An important area of research involves understanding when cells are able to respond to the different mesoderminducing signals and when they become committed to a particular fate. A detailed analysis of cell commitment in Xenopus showed that cells in the early gastrula embryo are labile, expressing genes that mark multiple germ layers and regions 88. By the end of gastrulation, however, cells seem to be more committed to a specific fate, as judged by gene expression. How and when cells become committed is still mostly unknown. One mechanism that limits the ability of -type signals to affect the mesoderm after the mid-gastrula stage in Xenopus involves phosphorylation of residues in the middle of the Smad2 protein, which keeps Smad2 out of the nucleus and therefore prevents it from activating gene expression 89. Many studies in Xenopus and zebrafish have examined how signalling pathways influence embryonic gene expression by using overexpression of specific signalling molecules, overexpression of dominant-negative receptors, morpholino oligonucleotides and zebrafish embryos that are mutant for one component of a particular signalling pathway. Although these results have been informative, they do not provide information about the important temporal requirement for the signalling pathway as all of these approaches alter the signalling pathway throughout the entire process of mesoderm induction, and therefore there is not much known in this area in general. Fortunately, new approaches are being developed that will allow the time requirement for different signalling factors to be addressed. Relatively specific drugs that inhibit the FGF (SU5402 (REF. 90)) and (SB505124 (REF. 91)) pathways are commercially available. For example, the FGF inhibitor has been used to examine a wide variety of processes, including the regulation of the T-box genes by FGF in the zebrafish mesoderm, Xenopus neural induction, Xenopus forebrain development, and zebrafish tooth development 64,92 94. As an alternative approach, transgenic zebrafish that express inhibitors of the canonical Wnt and BM pathways under heat-shock control have been developed 95,96. For example, this was used to show that the major role for BM signalling in patterning the mesoderm occurs during the late blastula/early gastrula period, but that BM signalling continues to operate during the mid late gastrula stages to specify ventral tail-fin formation 95. Similarly, studies with secreted natural inhibitors of FGF signalling in frogs indicated that the FGF-signalling pathway functions during the gastrula stages primarily to regulate mesodermal gene expression, whereas FGF signalling largely regulates morphogenesis after the gastrula stages 97. These types of temporal analysis provide a framework for examining changes in mesodermal gene expression in order to understand how and why the mesoderm changes its ability to respond to inducing signals. Combinatorial signalling Mesodermal cells respond to a cacophony of signals, but somehow they need to integrate this information to make a specific fate decision. How the different signalling pathways combine to regulate gene expression in the mesoderm is still poorly understood. Different signalling pathways might to some extent regulate each other s intracellular networks but, most likely, much of the interaction occurs through changes in gene expression. Although there is some evidence for genes that are directly regulated by multiple signalling pathways using promoter analysis (for example, Wnt and signalling regulating the Xenopus twin promoter 98 and BM and Wnt signalling regulating the zebrafish tbx6 promoter 11 ), for the most part it is not known how many mesodermal genes are regulated by just one pathway and how many are regulated by more than one pathway. romoter analysis is feasible in zebrafish and Xenopus using injected plasmid DNA, although it is best done using technology that allows transgenic Xenopus embryos to be produced in the F0 generation 99 ; nevertheless, these methods are still laborious. Moreover, with the exception of binding 366 MAY 2006 VOLUME 7 www.nature.com/reviews/genetics

a Vg1 and Wnt8c rimitive streak Vg1 and Wnt8c Cerberus Cerberus Hypoblast b 5.5 DC 6.5 DC Wnt3 AVE inhibitors Wnt inhibitors Wnt3 Brachyury roximal Anterior osterior AVE Distal Figure 5 Mesoderm induction in the chick and mouse. a In the chick embryo, Vg1 and Wnt8c cooperate to activate expression. activity is blocked initially by the secreted -binding protein Cerberus, which is expressed in the underlying hypoblast (green circles). The movement of the endoblast (white circles) displaces the Cerberus-expressing hypoblast, allowing to function, which causes mesoderm and endoderm cells to ingress through the primitive streak. Modified with permission from REF. 103 (2004) Company of Biologists Ltd. b In 5.5 day postcoitum (DC) mouse embryos, Wnt3 is initially expressed in the posterior extra-embryonic tissue (the visceral endoderm). The anterior visceral endoderm (AVE) initially forms in the distal region of the embryo. By 6.5 DC, the AVE, which secretes and Wnt inhibitors, has moved to the anterior side. Wnt3 is now expressed in the posterior epiblast, where the primitive streak forms, and in the posterior visceral endoderm. Wnt3 activates the expression of genes such as and Brachyury in the posterior epiblast. is also expressed throughout the epiblast at 5.5 DC (not shown). Modified with permission from REF. 105 (2005) Academic ress. rimitive streak The site of major morphogenetic movements during gastrulation in reptiles, birds and mammals. The mesoderm, as well as the endoderm, moves through this structure as it ingresses. sites for the Tcf factor (a transcriptional activator in the Wnt pathway; BOX 1), it is still not possible to easily recognize elements that respond to the FGF, or BM pathways because each of these pathways regulate several DNA-binding proteins and it is typically not clear which binding protein interacts with a specific promoter. For these reasons, few mesodermal promoters have been studied in any detail, leaving much of the molecular network of mesoderm induction incomplete. A recent paper has brought to light a new interesting example of combinatorial signalling in the mesoderm, which would not have been obviously revealed by promoter studies. As discussed above, the T-box transcription factor Xbra is a crucial regulator of posterior mesoderm formation. The specificity of Xbra-mediated regulation of gene expression is altered by its binding to Smad1, an intracellular mediator of the BM pathway 100 (BOX 1). Because BM signalling is high ventrally and low dorsally, targets of Xbramediated activation are likely to be different on the dorsal and ventral sides of the embryo. It will be interesting to see how general this type of mechanism is for regulating the activity of transcription factors in different regions of the embryo. Mesoderm induction in chicks and mice The basic process of mesoderm induction is generally conserved among all vertebrates, although the basic embryonic architecture is different between species (for a more extensive discussion comparing mesoderm induction in chick and mouse to fish and frogs see REF. 3). A direct comparison of chicks and mice to fish and frogs reveals interesting commonalities and differences. For example, in chicks, a combination of the TGFB factor Vg1 and canonical Wnt (Wnt8c) signalling has been proposed to initiate the formation of the initial axial structure, the primitive streak, albeit with the caveat that there are no loss-of-function data yet that test the exact role of Vg1 signalling in chicks 54 (FIG. 5a). Intriguingly, one of the earliest targets of Vg1 and Wnt8c is the chick gene 101, which parallels the observations in fish and frogs that expression is a key step in the NATURE REVIEWS GENETICS VOLUME 7 MAY 2006 367

Epiblast The portion of the mouse embryo that will become the definitive embryo (as opposed to extra-embryonic tissues). Amniotes Include reptiles, birds and mammals, which all have a protective membrane (the amnion) surrounding the embryo that prevents it from desiccating. mesoderm-induction process (see above). However, an important difference is that the chick extra-embryonic structure called the hypoblast, which initially lies under the future primitive streak, secretes the inhibitor Cerberus, which blocks function 102. Only when the hypoblast is physically displaced away from the posterior region of the embryo, where the primitive streak forms, is able to function in forming the primitive streak, together with Fgf8 and Chordin 103 (FIG. 5a). This mechanism of hypoblast displacement is proposed to be essential for ensuring that the primitive streak forms only in the posterior region of the embryo 102,103. It is also possible that the gradual movement of inhibitors away from the primitive streak establishes a gradient of activity within the streak itself 102. In mice, understanding mesoderm induction is complicated by the fact that the primitive streak forms on one side of the embryo (proximal to the site of implantation and in the region of the future posterior end of the embryo), whereas a separate crucial signalling centre, the anterior visceral endoderm (AVE), initially forms distal to the site of implantation and then moves to the opposite side from the primitive streak (the future anterior end of the embryo; FIG. 5b). A complex series of signalling interactions that involve, BM and Wnt signalling sets up these two centres (reviewed in REF. 104). Gdf3, a Vg1-like gene, is expressed in the very early mouse embryo, paralleling the very early expression of Vg1 in frogs, fish and chick. As with zebrafish vg1, but unlike Vg1 in frogs and chicks, Gdf3 expression is uniform within the cells that will give rise to the embryo 55. Surprisingly, Gdf3-null mutant mice are often viable, with approximately one-third showing a range of morphological defects, which can be explained by alterations in signalling 55. In general terms this result fits with loss-of-function experiments in frogs, indicating that Xenopus Vg1 works together with the factors 15, and it differs from studies in chicks, indicating that Vg1 is the key initiator of expression and primitive streak formation 53,54,103. These differences might reflect the ways in which the use of these separate TGFB factors has evolved to establish the mesoderm in embryos that have different topologies. A recent study challenges the view that the mouse primitive streak is initially established in a radial pattern and then refined to a localized site in the proximal posterior region of the epiblast, potentially through inhibitory activities of the AVE 105. Instead, the authors find that Wnt3, which is essential for the formation of the primitive streak and mesoderm and for expression 106, is initially expressed in tissues next to the proximal posterior region of the epiblast before the AVE has moved into the future anterior region, and not in a radial pattern throughout the proximal region as was previously thought. Wnt3 then regulates downstream genes such as and the T-box gene Brachyury (and potentially Wnt3 itself) in the adjacent proximal posterior epiblast (FIG. 5b). How Wnt3 expression is initially restricted to the proximal posterior region remains unanswered. In an interesting parallel to the function of the chick hypoblast, the AVE secretes inhibitors of signalling, Cerberus-like and Lefty1, that are essential for restricting the primitive streak to the posterior end of the embryo by limiting the region in which signalling can function 107,108 (FIG. 5b). Moreover, these inhibitors are essential for the normal patterning of the streak because mutants that lack both inhibitors have an expansion of the anterior mesoderm and a concomitant loss of posterior mesodermal tissues 108. The AVE also expresses inhibitors of Wnt signalling 109, which also limits the domain of Wnt3 signalling. Therefore, an essential role of the AVE is to secrete inhibitors that restrict the primitive streak to one side of the embryo. Because different experimental approaches are often used in different species, a direct comparison of mesoderm induction between vertebrates is complicated. Nonetheless, it is possible to make some basic generalizations. First, the same families of signalling factors are involved in all vertebrates, as are the T-box genes, although there are some significant differences in which molecular players are involved (TABLE 1). Second, localized maternal determinants have no role or a reduced role in axis specification and mesoderm induction in amniotes, and the polarity of the amniote embryo is not fixed until the beginning of gastrulation. Third, extra-embryonic structures have an important role in the early intercellular signalling processes in amniotes, whereas the fish extraembryonic structure, the YSL, initiates only the pathway, and frogs do not have extra-embryonic structures. Finally, the effect of inhibiting signalling pathways in the mouse embryo produces much more severe effects than in fish and frogs. For example, inhibition of BM signalling 110,111 or elimination of Wnt3 (REF. 106) in mouse embryos causes a failure of mesoderm formation, whereas inhibition of these pathways in fish and frogs results only in truncations of the body. Schier and Talbot have suggested that the differences in the roles of the signalling factors in these different species are due to a much greater role for cross-regulation between the signalling pathways in mouse embryos than in fish and frogs, so that if one pathway is inhibited, the others are affected as well 26. Future directions Many studies on mesoderm induction have involved identifying a new gene either through an unbiased screen or homology searches that are based on what is known from other species, and analysing its regulation by the signalling pathways that are discussed in this review. An alternative approach involves altering one of the signalling pathways by molecular or genetic methods and then analysing the expression and function of a handful of marker genes, typically by RT-CR. Although the zebrafish and Xenopus genomes are still not completely sequenced, it has recently become possible to use genomic methods, such as microarray screens and genome-based expression screens, to analyse mesoderm induction. For example, Xenopus embryos that lack the T-box transcription factor VegT were examined using microarrays, identifying 99 known and novel genes that are activated by VegT, of 368 MAY 2006 VOLUME 7 www.nature.com/reviews/genetics

+ 100 ng ml 1 Activin + 10 50 ng ml 1 Activin + 10 50 ng ml 1 Activin + RA + 5 10 ng ml 1 Activin 0.1 1 ng ml 1 Activin + Il3 0.1 1 ng ml 1 Activin + Scf 0.1 1 ng ml 1 Activin + Ang2 Heart and cartilage Notochord ronephros Muscle Lymphocyte Erythrocyte Vascular tissue Figure 6 roduction of different mesodermal cell types from animal cap explants. Animal caps are excised from Xenopus embryos at the mid-blastula stage and cultured in a saline solution. In the absence of inducers, the explants will form primitive epidermis. Addition of different factors will enhance the formation of specific mesodermal cell types. RA, Retinoic acid; Il3, Interleukin 3; Scf, Stem cell factor; Ang2, Angiopoietin 2. Modified with permission from REF. 114 (2003) Elsevier Science. which 13 were shown to be direct targets of VegT within the mesendoderm 112. Among these direct VegT targets, the transcription factors Snail, Hesr1 (hairy/enhancerof-split related) and Esr4 (enhancer-of-split related 4) were shown to be essential for proper embryonic morphology. A similar approach using the drug SU5402 to block FGF signalling in Xenopus identified 43 FGFregulated genes, of which 26 were novel 113. One of the 43 genes (Xmig6) was shown to be required for muscle differentiation, whereas a novel G-protein coupled receptor (encoded by Xgpcr4) seems to be involved in the morphogenesis of gastrulation. These approaches demonstrate the value of microarrays for finding novel genes that are regulated by the signalling and transcription factors that are involved in mesoderm induction, and for establishing regulatory relationships between known genes. Although initial experiments are likely to identify targets when a pathway is activated or blocked throughout the entire mesoderm-induction period (as with the elimination of VegT in the above example), the development of transgenic fish and frog embryos that contain inducible activators and inhibitors, as well as the use of specific pharmacological reagents, will facilitate the genome-wide examination of changes in gene expression when a signalling pathway is activated or blocked at a specific time in development. Because the functions of the different signalling factors are constantly changing over developmental time as discussed above, the use of microarray analysis combined with the ability to temporally regulate each of the signalling pathways will show how the transcriptional network changes to allow each of the signals to regulate different processes as embryogenesis proceeds. Microarray analysis is not without its drawbacks. One of the problems is that when whole embryos are analysed, many non-mesodermal cells contribute to the signal. For genes that are expressed broadly throughout the mesoderm this might not be a problem, but many mesodermal fates are represented by only a small subset of cells from the entire embryo. One probable solution is to return to Nieuwkoop s original assay. When animal caps are removed from embryos and treated with different factors such as Activin, which activates the signalling pathway, different cell fates are induced depending on the concentration of the factors used (reviewed in REF. 114) (FIG. 6). Although the cell populations in these explants typically are not made up of single cell types, they are enormously enriched in a particular tissue compared with the whole embryo. Moreover, because the explants can be maintained in culture for up to 2 months 115, not only can the early events in mesoderm induction be examined, but longer-term changes in gene expression can also be analysed. Although animal cap explants have occasionally been used in zebrafish, they are technically more challenging and it is not yet clear whether the same approach will be useful. In zebrafish the use of transgenic lines that express a fluorescent protein from a cell-type specific promoter combined with fluorescent cell sorting might be a more feasible alternative. Because mesoderm induction happens early in embryogenesis, it has been particularly easy to manipulate this process by injecting pools of synthetic RNAs into Xenopus embryos to identify genes that regulate mesoderm formation. revious attempts used pooled libraries of cdna that is attached to a promoter for the S6 RNA polymerase (reviewed in REF. 116). Although some interesting genes have been identified in this way, there were several problems with this approach. Even in the most highly normalized library many cdnas, especially abundant ones, were overrepresented. In addition, particularly for longer mrnas, many cdnas are not full length. Because of these problems, it was necessary to NATURE REVIEWS GENETICS VOLUME 7 MAY 2006 369