Nodal signaling and the evolution of deuterostome gastrulation

Authors

  • Helen K. Chea,

    1. Biology Department and Center for Developmental Biology, University of Washington, Seattle, Washington
    2. Friday Harbor Laboratories, Friday Harbor, Washington
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  • Christopher V. Wright,

    1. Department of Cell and Developmental Biology, Program in Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee
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  • Billie J. Swalla

    Corresponding author
    1. Biology Department and Center for Developmental Biology, University of Washington, Seattle, Washington
    2. Friday Harbor Laboratories, Friday Harbor, Washington
    3. Station Biologique, Roscoff, France
    • Biology Department, Box 351800, University of Washington, Seattle, WA 98195-1800
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Abstract

Chordates, including vertebrates, evolved within a group of animals called the deuterostomes. All holoblastic deuterostomes gastrulate at the vegetal pole and the blastopore becomes the anus, while a mouth is formed at the anterior or to the oral side. Nodal is a member of the TGF-β superfamily of signaling molecules that are important in signaling between cells during many embryonic processes in vertebrate embryos. Nodal has also been found in other invertebrate deuterostomes, such as ascidians and sea urchins, but, so far, is missing in protostomes. Nodal has been shown to be particularly important in determining left-right asymmetries in vertebrate embryos, but less information is available for its developmental role in the invertebrate deuterostomes. We review gastrulation in the deuterostomes, then examine nodal expression early during mesoderm formation and later during the establishment of asymmetries in both vertebrates and invertebrates. Nodal is expressed asymmetrically on the left side in chordates and on the presumptive oral side of the embryo in echinoid echinoderms. The expression of nodal is in different germ layers in embryos of different phyla. Expression is in the ectoderm in most of the invertebrate deuterostomes, and in the mesoderm in vertebrates. We summarize the work that has been published to date, especially nodal expression in the invertebrate deuterostomes, and suggest future experiments to better understand the evolution of nodal signaling and deuterostome gastrulation. Developmental Dynamics 234:269–278, 2005. © 2005 Wiley-Liss, Inc.

DEUTEROSTOME GASTRULATION

The evolution of gastrulation within the deuterostomes has allowed the formation of the various developmental and morphological differences seen in larval and adult deuterostomes today. The Deuterostomia group of animals contains two major clades (Fig. 1). One, a group called Ambulacraria, consists of the echinoderms and hemichordates (Zeng and Swalla,2005). These invertebrate deuterostomes gastrulate at the vegetal pole, and frequently form a feeding larva that captures small particles via a ciliary band (Strathmann,1987; Wray,1997). The other clade is made up of the chordates, which includes the Tunicata, Cephalochordata and Vertebrata (Fig. 1; Swalla,2004). When embryos of the deuterostomes are viewed at early gastrulation, it is clear that all of the holoblastic deuterostomes begin gastrulation at the vegetal pole (Fig. 1). In amphibians, the dorsal lip is offset from the vegetal pole somewhat by the very yolky cells in the vegetal hemisphere. Echinoderm and hemichordate embryos have much more space in the center of the gastrula, as they make a hollow blastula before gastrulating (Fig. 1). In contrast, chordate embryos are more compact, and have variable sizes of blastocoels (Fig. 1). In all deuterostomes, the blastopore becomes the anus (posterior) and a mouth is formed secondarily in another location, hence the name “deuterostome” or “second mouth.”

Figure 1.

Gastrulation in the deuterostomes. The animal pole (a) is marked by polar bodies; (v) is vegetal pole. (A) is future anterior; (P) is future posterior. In echinoderms and hemichordates, invagination of the endoderm begins at the vegetal pole and the early gastrula is radially symmetrical. The blastopore becomes the future anus of the larvae. In most echinoderms, the animal-vegetal axis becomes the larval A-P axis, except in echinoid and ophiuroid pluteus larvae. In chordates, invagination during gastrulation also begins at the vegetal pole. However, in the Chordata, the future anterior and posterior of the larva are determined by the point of sperm entry, which breaks the symmetry of the radial egg. The embryo posterior develops opposite the point of sperm entry.

Note that in chordates, the animal-vegetal (A-V) axis becomes the dorsal-ventral (D-V) axis of the embryo, and the anterior-posterior (A-P) axis forms at right angles to the D-V axis, which is determined by the organizer that forms opposite the site of sperm entry (Nishida,1994; Clavert,1962; Eyal-Giladi,1997). This is in contrast to hemichordates and echinoderms, where the A-V axis becomes the larval A-P axis and the D-V axis is less defined. In ophiuroid and echinoid echinoderms, a pluteus larva develops that has an oral-aboral axis, but the second axis is not exactly anterior-posterior and so is referred to as the A-V (Angerer et al.2001). However, in other echinoderm larvae, including basal crinoid larvae, it is clear that the egg A-V axis becomes the larval A-P axis (Nakano et al.2003). In hemichordates, the adult A-P axis is clearly identical to the larval A-P axis and the D-V axis is still being debated (Gerhart,2001; Henry et al.,2001). In summary, in echinoderms and hemichordates the egg A-V axis becomes the A-P axis of the larva, while in chordates that A-V axis becomes the D-V axis and the A-P axis forms at a right angle to the original axis. This divergence in adult axis orientation is due in part to differences in gastrulation, which sets up the rudimentary primary body axes of the embryo.

Although gastrulation is an amazingly complicated developmental process, involving wide-scale cell movements and signaling pathways, there are some common mechanisms that are beginning to emerge (Stern,2004). When one looks at cleavage patterns within the vertebrates, cleavage and gastrulation vary enormously, primarily due to the amount and distribution of yolk that is found in the egg (Fig. 2). Holoblastic cleavage, found in ascidians, lancelets, lamprey, and amphibians, is likely to be the ancestral cleavage pattern, but meroblastic cleavage has evolved independently in hagfish, teleost fishes, and the amniotes (Elinson, 1989) (Fig. 2). Mammals reverted back to holoblastic cleavage, probably due to the evolution of the placenta and internal fertilization (Fig. 2).

Figure 2.

Cleavage patterns within the Chordates. Ascidians, lancelets, and lamprey display holoblastic cleavage, suggesting that it is the ancestral cleavage pattern in chordates. Ascidians have bilateral cleavage, while lancelets display radial cleavage. Lamprey and amphibians have mesolecithal eggs, with larger, yolky cells confined to the vegetal half of the embryo. Present day teleost fish show meroblastic cleavage, which evolved independently in fish and amniotes. Mammals show a reversion to holoblastic cleavage, but exhibit rotational cleavage.

Gastrulation is precisely controlled by a complex array of transcription factors and signaling pathways. Nodal is a signaling molecule that is critical for mesoderm induction and the establishment of left-right asymmetry in many deuterostome phyla. In this review, we examine nodal expression in a few of the vertebrate model systems, then compare them to some invertebrate model systems, and see if there are any general conclusions that can be drawn from the available expression data.

NODAL IS PART OF THE TFG-β SUPERFAMILY OF SIGNALING MOLECULES

Many members of the transforming growth factor-β (TGF-β) superfamily of signaling molecules are involved in body plan determination during embryogenesis in vertebrates (Whitman,1998; Herpin et al.,2004). TGF-β growth factor ligands act through a signal transduction pathway and bind to specific cell surface receptors identified as type I and type II receptors (Massagúe,1998; Whitman,1998; Schier and Shen,2000; Mehra and Wrana,2002). TGF-β binds first with the type II activin receptor, a serine/threonine kinase, producing a subsequent phosphorylation interaction with the type I receptor (Massagúe,1998; Whitman,1998; Schier and Shen,2000; Mehra and Wrana,2002). It is interesting to note that the ligand is unable to bind directly with the type I receptor and that the phosphorylation process is essential for signal propagation (Wrana et al.,1994). In vertebrates, the type I receptor activates Smad2 or related Smad proteins, which act with transcription co-factors to effect transcription at the nuclear level, activating downstream genes (Massagúe,1998; Whitman,1998; Schier and Shen,2000; Wrana, and Pawson,1997). This signaling pathway, including the ligand, receptors, and Smad proteins, is highly conserved among all vertebrates studied (Whitman,2001) and is also highly conserved in invertebrates (Herpin et al.2004).

Nodal, a TGF-β ligand, associates with EGF-CFC cofactors to bind to its receptors (Cheng et al.,2004; Herpin et al.,2004). Nodal plays a regulatory role in cell interactions that are responsible for patterning the early embryo, including mesoderm induction and axis formation. Left-right axis determination in vertebrates is critical to the development and morphology of adult organs. Disruptions in proper left-right axis symmetry can cause severe deformities, such as improper cardiac looping and folding (Lohr et al.,1997). It has been confirmed through genetic and embryological studies that the developmental basis of left-right axis determination is conserved across many vertebrate species and, most recently, in invertebrate deuterostomes species as well (Yu et al.,2002; Morokuma et al.,2002; Duboc et al.,2004, and our own unpublished data). In addition, other asymmetrically expressed gene homologues involved in the nodal cascade, including Pitx2, a member of the Pitx homeobox gene family, have been isolated in a number of deuterostomes (Morokuma et al.,2002; Boorman and Shimeld,2002a; Yasui et al.,2000). The current data reviewed here suggests a conserved signaling pathway involved in mesoderm formation and left-right axis determination in all deuterostomes. However, changes in the timing and spatial expression of nodal are likely to have contributed to the evolution of a new signaling center during gastrulation, leading to the evolution of a shift in axis formation in chordates.

NODAL SIGNALING IN VERTEBRATES

Nodal was initially identified through a retrovirally induced insertional mutation in mouse embryos (Zhou et al.,1993). Embryos homozygous for the insertion at the nodal locus lack a primitive streak and fail to form most embryonic mesoderm, exhibiting developmental arrest at or prior to gastrulation (Zhou et al.,1993; Conlon et al.,1994). Single or multiple nodal-related genes have also been isolated in zebrafish, frogs, and chick, with similar nodal loss-of-function mutations and manipulations yielding results comparable to those observed in mouse embryos (Long et al.,2003). Additionally, molecular studies have revealed that the expression patterns of nodal homologues in these different animals share important conserved features. In all vertebrates studied, nodal is expressed in the organizer and in the left lateral plate mesoderm (LPM) during embryogenesis (Fig. 3). Such consistent data have led to the conclusion that the nodal signaling pathway plays a crucial and highly conserved role in the embryonic development of vertebrates, particularly in mesoderm induction and patterning, and axis determination (Whitman,2001).

Figure 3.

Comparison of vertebrate nodal and nodal-related gene expression to lancelets, ascidians, and sea urchins. Animals are shown at early gastrulation (top, A–K) and after completion of gastrulation (bottom, B–L). Areas of nodal expression are indicated by red shading. A: In mouse gastrulae, nodal is expressed bilaterally around the node. B: By early somite stages, nodal is expressed asymmetrically in the left lateral plate mesoderm and in the node where ingression is occurring. C: In early Xenopus gastrulae, nodal is expressed at the dorsal lip and later (D) in the left lateral plate mesoderm at late neurulation. E: Expression of cyc is localized to the hypoblast layer of the embryonic shield in early zebrafish embryos while sqt is expressed in the dorsal forerunner cells. F: During somitogenesis in zebrafish, cyc and spaw are both expressed in the left lateral plate mesoderm. G: In lancelets, nodal expression is observed in the hypoblast layer of the dorsal lip. H: At late neurulation, nodal is asymmetrically expressed on the left side of the paraxial mesoderm, gut endoderm, and presumptive oral ectoderm. I: In ascidians, nodal is expressed bilaterally within the presumptive endoderm, epidermis, and trunk lateral cells from the 32-cell stage to early gastrulation. J: By the initial tailbud stage, ascidian nodal is expressed in the left side of the epidermis. K: In sea urchins, nodal expression begins in the presumptive ectoderm at the 60-cell stage, but decreases once gastrulation begins. L:Nodal transcripts are localized in the presumptive oral ectoderm during prism and pluteus stages. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

A comparison of nodal expression and its putative functions in the development of various vertebrates is necessary to examine its role in the evolution of gastrulation. Nodal expression begins at different stages in different vertebrate embryos. During mouse development, nodal signaling becomes restricted to in the proximal epiblast prior to gastrulation and diminishes before neurulation is complete (Brennan et al.,2001). Signaling in the epiblast is critical in setting up the proximal-distal axis and in patterning the overlying primitive visceral endoderm (Varlet et al.,1997). Prior to overt streak formation, nodal expression becomes localized to the posterior region of the epiblast, marking the site of mesodermal gene expression and primitive streak formation (Thomas and Beddington,1996; Beddington and Robertson,1999; reviewed in Whitman,2001). As gastrulation proceeds, nodal is expressed around the node at the distal end of the embryo, which serves as the mouse organizer and gives rise to anterior mesendoderm via delamination (Fig. 3A; Collignon et al.,1996; Lowe et al.,1996). During somitogenesis, nodal expression becomes restricted to the left LPM, where it is believed to emit signals that coordinate heart and visceral organ positioning (Collignon et al.,1996; Lowe et al.,1996). Although mutant mouse embryos lacking nodal activity are still capable of producing posterior mesoderm, the failure of these cells to migrate properly suggests that nodal signaling is required for appropriate posterior mesoderm patterning during mouse development (Conlon et al.,1994). Indeed, recent data have shown that cell sorting within the epiblast is a Nodal-dependent process in which Nodal-deficient cells tend to localize to the anterior portion of the epiblast (Lu et al.,2004).

Nodal-related genes in the frog, Xenopus laevis, and in zebrafish are expressed in a comparable fashion to that of nodal in mouse embryos. Currently, six Xenopus nodal-related (Xnr) factors have been identified, five of which are involved in mesoderm induction and patterning during embryogenesis and are expressed in pre-gastrula mesendoderm (Jones et al.,1995; Lustig et al.,1996; Joseph and Melton,1997; Takahashi et al.,2000; Osada and Wright,1999). Xnr-1 and Xnr-2, the most well-studied of the Xenopus nodal-related genes, are first expressed within the vegetal hemisphere of late blastula and then in the marginal zone during gastrulation, with enrichment in the dorsal lip, or organizer (Fig. 3C; Jones et al.,1995). Xnr-1, but not Xnr-2, is detected in the left side of the LPM during late neurulae and tailbud stages (Lustig et al.,1996). Another Xenopus nodal-related factor, Xnr-3, has been observed to induce neural tissue in animal caps, but lacks mesoderm-inducing activity, although it appears to be necessary for cell migration and movement (Smith et al.,1995; Varlet,1997). In addition, Xnr-3 does not have the conserved cysteine residues characteristic of Nodal and Nodal-related proteins (Hansen et al.,1997).

Multiple nodal-related genes have also been identified in zebrafish: cyclops (cyc), squint (sqt), and southpaw (spaw) (Hatta et al.,1991; Feldman et al.,1998; Long et al.,2003). Sqt and cyc play distinct roles during gastrulation and mesendoderm induction, as mutants lack a medial floorplate and display defects in ventral brain development, structures that are usually specified by signals from mesoderm-derived notochord (Hatta et al,1991; Sampath et al.,1998; Feldman et al.,1998). Consistent with their roles, both sqt and cyc are expressed in the dorsal mesodermal progenitor cells of the margin at blastula stages (Feldman et al.,1998; Rebagliati et al.,1998; Sampath et al.,1998). Sqt is also expressed in the yolk syncytial layer during early gastrulation, a region implicated as a source of signals essential for organizer development and mesodermal induction (Erter et al.,1998). During gastrulation, cyc and sqt signaling divides the embryonic shield into two distinctive domains. Sqt is expressed in non-involuting dorsal forerunner cells, whereas cyc is expressed in the overlying dorsal hypoblast layer, which gives rise to the notochord and prechordal plate (Fig. 3E; Rebagliati et al.,1998). At the somite stages, cyc is transiently expressed in the left side of the lateral plate mesoderm and developing forebrain (Fig. 3F; Rebagliati et al.,1998). Spaw is also expressed in the left lateral plate mesoderm during somitogenesis and is currently the earliest asymmetrically expressed marker in zebrafish (Long et al.,2003). While cyc and sqt mutations have dire effects on mesoderm and endoderm formation, they have only a small effect on visceral left-right asymmetry. In contrast, a morpholino-based knock-down of spaw function causes a severe disruption in visceral left-right asymmetry, pointing to its role in axis patterning in the zebrafish embryo (Long et al.,2003). The separation of functions of nodal-related genes in zebrafish and Xenopus represents an evolutionary divergence after the duplication of a single ancestral nodal gene, as found in ascidians and lancelets (Dehal et al.,2002).

The dynamic expression of nodal throughout embryogenesis is a commonality in all vertebrate embryos studied, suggesting that multiple developmental processes are regulated by nodal signaling. The presence of nodal prior to the onset of gastrulation, as well as defects in mesoderm formation observed in nodal loss-of-function mutants, supports the idea that nodal plays a central role in the formation and maintenance of mesoderm in vertebrates. However, unlike mouse nodal, which is expressed throughout the epiblast prior to gastrulation, there is no such comparable expression reported in frogs or zebrafish. This is significant because it suggests a recent co-option of nodal in the formation of the anterior-posterior and proximal-distal axes through cell-cell signaling between the epiblast and extraembryonic ectoderm and visceral endoderm in mammalian embryos. Nodal participates in determining the anterior-posterior and dorsal-ventral body axes during gastrulation in other vertebrates as well, but does so by patterning the organizer, which sets up the anterior-posterior axis (Lu and Robertson,2004; Schier and Talbot,2001; De Robertis et al.,2000). The conserved expression of nodal and most of the nodal-related homologues in the left lateral plate mesoderm prior to organogenesis point to its role in establishing left-right asymmetry. This role is also supported by the fact that mutations that prevent the left-sided localization of nodal signals, such as iv and inv mice, result in embryos with randomized or reversed positioning of visceral organs (Okada et al.,1999; Supp et al.,1997). Thus, nodal signaling is critical in regulating mesoderm induction and axis determination in vertebrate embryos, processes that are essential during gastrulation.

NODAL SIGNALING IN INVERTEBRATE DEUTEROSTOMES

Nodal was first described in vertebrates, but recent evidence suggests that the nodal signaling cascade diverged before the deuterostome lineage because the entire signaling cascade has been found in all deuterostomes. Nodal homologues have now been characterized in cephalochordates, tunicates, and echinoderms (Fig. 1; Yu et al.,2002; Morokuma et al.,2002; Duboc et al.,2004). The downstream transcription factor, Pitx, has also been described in both cephalochordates and tunicates (Yasui et al.,2000; Boorman and Shimeld,2002a; Mororkuma et al.,2002; Christiaen et al.,2002; Tiozzo et al.,2005). The expression and likely functions of these nodal-related genes, and how they compare to vertebrate nodal homologues, is reviewed below. We then compare how axis formation during gastrulation may have evolved differently in the chordates than in the Ambulacraria.

Lancelets, or Branchiostoma, belong to the phylum Cephalochordata and are the closest living invertebrate relatives of the vertebrates (Fig. 1). Lancelet embryos display morphological asymmetry as early as the neurula stage, when the left and right anterior somite boundaries shift out of alignment by a half segment (Whittaker,1997). The most prominent display of left-right asymmetry occurs at the larval stage. During larval development, the mouth opens on the left side of the head and the gill slits form on the right (Whittaker,1997; Boorman and Shimeld,2002b). This radical and unique asymmetry lasts until metamorphosis, when the mouth repositions to the midline and new gill slits form, although other directional asymmetries exist in the adult (Whittaker,1997; Boorman and Shimeld,2002b). Recently, a lancelet nodal gene, AmphiNodal, was isolated from the species Branchiostoma floridae, and its zygotic expression has been described (Yu et al.,2002). Its expression pattern shares some similarities to the patterns observed in vertebrates, including expression in the organizer as well as a bias towards left-sided expression in the embryos. AmphiNodal expression is first detected in the invaginating hypoblast layer within the dorsal lip of the blastopore during early gastrulation (Fig. 3G; Yu et al.,2002). The dorsal lip is made up of presumptive notochord cells and has been proposed to be analogous to Spemann's organizer in vertebrates (Yu et al.,2002; Tung et al.,1962; Whittaker,1997). By late gastrulation, nodal expression is limited to the paraxial mesoderm and to overlying regions of the neural plate (Yu et al.,2002). During the late neurula stage, all neural and right-sided mesoderm expression disappears whereas expression on the left side remains and spreads ventrally to the left side of the gut endoderm (Fig. 3H; Yu et al.,2002). Expression is also localized to a region of ectoderm on the left side of the embryo where the larval mouth will eventually form (Yu et al.,2002). Thus, unlike vertebrate nodal, which is expressed primarily in the mesoderm, lancelet nodal is asymmetrically expressed in all three germ layers during development and left-sided expression occurs later in development.

A Pitx-related gene has been characterized in two lancelet species, Branchiostoma floridae (BfPitx) and Branchiostoma belcheri (BpPitx) (Yasui et al.,2000; Boorman and Shimeld,2002a). In vertebrates, Pitx2 is expressed asymmetrically in the left lateral plate mesoderm and is a downstream target of nodal signaling. Pitx is also expressed in the oral ectoderm and developing pituitary in vertebrates (Boorman and Shimeld,2002b). In lancelets, Pitx and nodal expression are closely correlated, with Pitx transcripts becoming asymmetrically localized in the anterior left endoderm, ectoderm, and mesoderm throughout neurulation (Yasui et al.,2000; Boorman and Shimeld,2002a). During the larval stages, Pitx is expressed in the pre-oral pit and in the ectoderm and endoderm surrounding the mouth, both asymmetric structures that open on the left side of the body (Yasui et al.,2000; Boorman and Shimeld,2002a). Ascidians, which are part of the subphylum Tunicata (Urochordata), are the smallest chordates (Zeng and Swalla,2005) (Fig. 1). Ascidians can be either solitary or colonial and most species develop as free-swimming larvae that metamorphose into sessile adults (Jeffery and Swalla,1997; Swalla,2004). Ascidian larvae show some signs of morphological asymmetry; for example, the two sensory pigment spots, the ocellus and the otolith, normally develop on the left side of the head and the adjoining brain vesicles tend to tilt to the right (Swalla,2004). As adults, the most distinguishable directional asymmetry is observed in the looping of the gut. To date, nodal has been reported in two solitary species, Halocynthia roretzi and Molgula oculata (Morokuma et al.,2002; Schumpert et al.,2000). The expression of nodal, beginning at the 32-cell stage, is bilaterally symmetric, with transcripts detected within the presumptive endodermal, epidermal, and trunk lateral cell lineages (Fig. 3I; Morokuma et al.,2002). This bilateral expression disappears shortly after the onset of gastrulation (Morokuma et al.,2002). At the initial tailbud stage, asymmetric nodal expression is detected on the left side of the body. Interestingly, this expression is restricted to epidermal cells only, as compared to the lateral plate mesoderm in vertebrates (Fig. 3J; Morokuma et al.,2002; Schumpert et al.,2000). Although nodal expression during later stages of ascidian development has yet to be examined, the expression of Pitx has been described up to blastogenesis, or bud formation, in the colonial species, Botryllus schlosseri (Tiozzo et al.,2005). In Halocynthia roretzi, Pitx transcripts are localized in the left epidermis at the same time as nodal and are also expressed in left-sided mesenchyme in Ciona intestinalis (Morokuma et al.,2002; Boorman and Shimeld,2002a). Furthermore, the over-expression of nodal in ascidian embryos causes a marked disruption of left-sided Pitx expression, resulting in right-sided or bilateral expression, indicating that nodal acts upstream of Pitx in ascidians as well as in vertebrates (Morokuma et al.,2002). During organogenesis in Botryllus schlosseri, Pitx signaling is observed in the neurohypophysal duct and in the pharyngeal epithelium on the future internal lips of the oral siphon (Tiozzo et al.,2005). In the metamorphosing larva, Pitx expression remains in the inner wall of the oral siphon (Tiozzo et al.,2005). During the adult stages, when buds form as thickenings in the peribranchial epithelium, Pitx is asymmetrically detected in two domains (Tiozzo et al.,2005). It is asymmetrically expressed in the left peri-branchial chamber, which is connected to the stomach and intestine, and also in the left side of the dorsal lamina (Tiozzo et al.,2005). Thus, as in lancelets, expression of pitx in ascidians occurs in all three germ layers during later development and marks the site of future oral-ectoderm formation.

The most recent deuterostome nodal gene isolated is from sea urchins, a discovery that pushes the evolutionary emergence of nodal signaling back to the advent of deuterostomy (Fig. 1). Sea urchin nodal, the first nodal gene isolated in a non-chordate deuterostome, is expressed transiently at two stages of development. The first phase of nodal localization appears at the 60-cell stage and decreases significantly at the beginning of gastrulation (Fig. 3K; Duboc et al.,2004). During this time, nodal expression appears in about a third of the embryo, with transcripts localized in the presumptive ectoderm (Duboc et al.,2004; Flowers et al.,2004). At the prism and pluteus stages of gastrulation, nodal expression increases, although levels remain lower than those seen throughout the blastula stages. Nodal expression is restricted to the presumptive oral ectoderm, which marks the future oral side of the larva (Fig. 3L; Duboc et al.,2004; Flowers et al.,2004). The archenteron, which is guided by mesenchyme cells, then bends towards the prospective oral region in response to the signals (Wray,1997). Importantly, over-expression of nodal promotes the formation of oral ectoderm, whereas blocking nodal translation using morpholinos leads to a lack of both oral and aboral ectoderm (Duboc et al.,2004). Interestingly, it has been discovered that the oral-aboral polarity of the embryo may be specified by the asymmetric distribution of mitochondria in the unfertilized egg (Coffman et al.,2004). The side of the egg with the highest density of mitochondria is strongly biased towards developing into the oral pole of the embryo (Coffman et al.,2004). These experiments also suggest that the oxidizing state created by mitochondrial respiration is required for the successful activation of nodal (Coffman et al.,2004). To date, the specific effect of nodal on mesoderm induction during early gastrulation has yet to be examined. However, experiments aimed at determining its role in oral-aboral axis formation failed to indicate any significant mesodermal abnormalities as a result of nodal over-expression or deficiency. The current results indicate that nodal signaling is essential for setting up the oral-aboral axis in sea urchins, just as it is essential for setting up the left-right axes in vertebrates.

CONCLUSIONS

When nodal expression within the deuterostomes is examined, several conserved features emerge. First, although nodal expression is extremely dynamic and is localized in different tissue layers in different animals, it is consistently expressed in the organizer in vertebrates and lancelets, which share a common ancestor (Zeng and Swalla,2005). This conserved expression suggests that nodal signaling played a critical role in organizer development and maintenance in the ancestral vertebrate. Additionally, nodal is asymmetrically expressed on the left side in all chordates suggesting that the chordate ancestor had left-right asymmetry, determined by nodal signaling. In chordates, this asymmetrical expression is necessary for setting up the left-right axis since the A-P and D-V axes are already elaborated. Examination of nodal expression in sea urchin embryos indicates that nodal may have had an ancestral role in determining oral-aboral polarity. This is further supported by expression data from lancelet and ascidian embryos, in which nodal and/or Pitx are found to be localized in the presumptive oral ectoderm, even though expression of nodal has not been examined during formation of the mouth in vertebrates. It is known that in vertebrates, Pitx is expressed in the oral ectoderm, which forms the mouth as well as parts of the pituitary. The idea that the ancestral function of nodal is to specify the region of ectoderm where the mouth, or oral siphon in ascidians, will form contrasts with Palmer's suggestion that nodal's ancestral role was to regulate brain asymmetry (Palmer,2004). In addition, only zebrafish embryos have been shown so far to display asymmetric nodal expression in the brain during development (Rebagliati et al.,1998; Sampath et al.,1998; Gamse et al.2003). However, it would be interesting to look for expression of nodal during mouth formation in vertebrates and solitary ascidians to see if this is the ancestral role of nodal in the deuterostomes.

Despite the conserved left-sided expression of nodal between the different phyla, the germ layer(s) in which nodal is expressed varies in different embryos. This difference in germ layer expression probably represents a divergence from an initial ancestral state, which according to the expression data obtained from the invertebrate deuterostomes, is ectodermal. The appearance of nodal in all three germ layers in lancelets and possibly ascidians, as indicated by Pitx expression, may have been an evolutionary advance that led to the conserved localization of nodal in the lateral plate mesoderm of vertebrate embryos. On a cautionary note, it should be emphasized that sea urchins are derived echinoderm larvae and the hypothesis that expression of nodal in ectodermal tissue is a basal trait would be better supported by examining nodal expression in hemichordate embryos, larvae, and adults. Sequencing experiments with the hemichordate species Saccoglossus bromophenolosus indicate that the nodal gene is present, but the spatial and temporal expression of nodal during development is just being studied (Chea and Swalla, unpublished observations). Also, while the role of nodal in inducing mesoderm formation in vertebrates has been confirmed through genetic experiments, the possibility that this function is conserved in all deuterostomes has yet to be established. The early expression of nodal in the dorsal lip of the lancelet, and in the presumptive endoderm of ascidians, hints that nodal may be involved in mesendoderm formation or patterning in chordates, but functional data will be necessary to substantiate this hypothesis.

To date, nodal has only been reported in deuterostomes and there is no nodal found in the ecdysozoa. Drosophila, Anopholes, and C. elegans contain TGF-β homologues, but no distinct nodal subfamily member (Adams et al.,2000; Holt et al.,2002; C. elegans Sequencing Consortium,1998; Herpin et al.2004). However, nodal may be present and play an active role in the development of the third major metazoan group, the lophotrochozoa. For instance, many lophophorates, including mollusks and polychaetes, display morphological asymmetries throughout the larval and adult stages (Halanych,2004). Either nodal signaling is involved in axis determination in this group of protostomes, or this TGF-β superfamily member evolved only in the deuterostomes. As we begin to better study and sequence the genomes of the lophotrochozoa, these questions will be answered. Furthermore, a more detailed examination of nodal in the invertebrate deuterostomes would be very informative. For example, echinoderms, unlike chordates, display radial symmetry as adults, a feature shared only with the cnidarians in the animal kingdom. However, in sea urchin larvae, the adult body plan is laid down in a structure called the imaginal adult rudiment, which develops on the left side of the larva, breaking its bilateral symmetry (Wray,1997). This morphological asymmetry, like the oral-aboral axis, is likely to be regulated by nodal signaling (Duboc et al., 2005). Further analysis of nodal expression and its presumed roles during embryogenesis in deuterostomes, and possibly protostomes, will offer further insight into its ancestral function as well its role in the evolution of gastrulation.

PERSPECTIVES

Nodal is a member of the ancient TGF-β family of signaling molecules that had duplicated and diverged long before the bilaterians split off into the Ecdysozoa, the Lophotrochozoa, and the Deuterostomes (Halanych,2004; Herpin et al.2004). Nodal, and the specific nodal pathway members, such as the nodal receptor Alk-4, have been reported only in the deuterostomes (Palmer,2004). However, the entire conserved nodal pathway is present in both chordates and echinoderms, suggesting that it is present in all deuterostomes. We have examined gastrulation in the deuterostomes and nodal signaling in early specification of germ layers and in later left-right asymmetry, comparing the invertebrate deuterostomes to the vertebrates. Some commonalities have emerged from this exposition, but many more questions remain to be answered by further experiments.

All non-amniote Deuterostomes gastrulate by invagination of cells at or near the vegetal pole of the egg, and the blastopore becomes the anus, thus the term deuterostome or “second mouth” (Zeng and Swalla,2005). In the Ambulacraria (echinoderms and hemichordates) (Zeng and Swalla,2005), the animal-vegetal axis becomes the anterior-posterior axis of the larvae (Fig. 1). In hemichordates, the adult body plan follows the larval body plan, whereas in some echinoderms, the adult axis is shifted from the larval axis. In contrast, in the chordates, the animal-vegetal axis becomes the dorsal-ventral axis, and the anterior-posterior axis forms at right angles to the animal vegetal axis. It is not yet known what maternal or zygotic factors are localized that cause this shift in polarity, but it would be interesting to understand how and why the shift occurs. This axial shift is critical in understanding how the left side of chordates relates to the oral axis in the Ambulacraria.

Nodal was first isolated in mice and gets its name from its role in the mouse node, specifying mesoderm during early gastrulation (Zhou et al.,1993). Although it is not yet clear whether nodal is critical for mesoderm formation in echinoderms, hemichordates, and ascidians, nodal expression has been reported at an early stage in echinoderms (Duboc et al.,2004) and in ascidians (Chea and Swalla, unpublished observations). It will be necessary to examine the role of this early nodal expression to determine if it is necessary and sufficient to specify mesoderm in all deuterostome embryos or is involved in some basal level of embryonic signaling, or regulates other cell fate allocation processes. If nodal is not found in the Ecdysozoa or the Lophotrochozoa, then the gene duplication and subsequent subfunctionalization of nodal from a BMP-like ancestral protein may have been a critical step in the evolution of the Deuterostomes.

A second, and perhaps concomitant ancestral function of nodal may have been the positioning of the mouth, whether symmetric or asymmetric, in relation to the rest of the larval axis. Little has been studied about the mechanism of the positioning and specification of the mouth in deuterostomes, even though it is a critical ancestral function to make a second opening in the gut tube. We suggest that this endodermal-ectodermal interaction would be an important place to study nodal function in tissue interactions. Functional studies are underway in many labs, studying the mechanism of left-right asymmetry in vertebrates and this later function of nodal may have been lost in more derived vertebrates, as the nodal pathway became more canalized during development (Palmer,2004).

In summary, we hope that this review stimulates ideas and discussion about the evolution of gastrulation and the elaboration of larval and adult body plans, especially in the deuterostomes. Our lab is continuing to study the role of nodal in elaborating the larval and adult body plans in tunicates and hemichordates.

Acknowledgements

Rick Elinson is thanked for discussions that led to Figure 2. Adam Keefer, Brenda Schumpert, and Paul Grant are thanked for their work with nodal expression in Molgula oculata. The University of Washington Howard Hughes Biology Program is thanked for their financial support of Helen Chea.

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