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Home , Chordate, Ventral nerve cord

Perspective

Inversion of the body axis: Are there alternatives?

J. Gerhart*

Department of Molecular and Cell , University of California, Berkeley, CA 94720-3200

One major morphological difference between and or is the opposite orientation of the cord and heart. A long-standing proposal is that the chordate axis evolved by inverting the body of an ancestor with the annelid͞arthropod orientation. However, the data can also be explained by a common ancestor with diffuse dorsoventral organization, followed by oppositely directed condensation of the nerve cord and relocation of the heart in the two lines. n 1822 Geoffroy St. Hilaire suggested nificance of this ancestor is great in meta- sally) (12). The common ancestor’s com- Ithat arthropods have the inverse dorso- zoan , wrapped up as it is with plexity is also implied by complex antero- ventral organization of chordates. After the origins of bilateral . Modifica- posterior expression domains (emx, otx, acceptance of Darwin’s descent with mod- tion of a radially symmetric or biradial Hox genes) in the chordate and ification, Dohrn (1) proposed in 1875 that into a bilateral body plan with a nervous systems (8), and by expression the last common ancestor of these groups dorsoventral axis is thought to have in- similarities in , , and was an -like with a volved many changes, including appear- limbs (13, 14). , a dorsal heart, and a ance of and , conver- Furthermore, the embryonic ectoderm with blood flowing an- sion of a closed gut to a through-gut, in flies and the embryonic mesoderm in teriorly in dorsal vessels. This orientation , and centralization of the segregate into two dorsoventral do- was retained by evolving members of the nerve cord from a diffuse net. These mod- mains, inversely related in the two cases 25–30 phyla (such as annelids ifications presumably occurred in steps (3, 10). Cells of one secrete cer- ␤ and arthropods) whereas, within the deu- between the time of the first simple bilat- tain type transforming growth factor terostomes (chordates, , eral animal, variously called the ‘‘urbilat- signals (bone morphogenetic proteins 2, 4, and ), an ancestor of chor- eria’’ (3) or ‘‘bilaterogastrea’’ (4), and the and 7 in chordates; Dpp in Drosophila),

dates inverted its body, sideways over, but time of the last common ancestor of mod- and cells of the other do not but do secrete PERSPECTIVE retained the same relative organ place- ern bilateral animals. Because the antagonists of these signals (Chordin in ments. The nerve cord was now dorsal, record and modern phylogenies give few chordates; Sog in Drosophila). These the heart ventral, and the blood flowed clues (see below) about the ancestor, it genes seem particularly significant be- anteriorly in ventral vessels (Fig. 1). In would be significant if comparative mo- cause their domains may serve as dorso- chordates, axial muscle blocks were dor- lecular and anatomical data could illumi- ventral compartments of the body axis solateral and visceral mesoderm was ven- nate its character. (like Hox compartments of the anteropos- trolateral whereas had the op- terior axis). Hence, they might reveal posite arrangement. In the chordate line, Recent Evidence for Inversion. Modern pro- global body organization rather than spe- SPECIAL FEATURE the mouth eventually formed on the new ponents of inversion, who have taken gene cific cell type differentiation or local organ ventral side and vanished from the old lo- expression data as evidence, include development. The molecular data support cation. Thus, in the amended inversion hy- Arendt and Nu¨bler-Jung (5–9), De Rob- the assertions from morphology that nerve cords, hearts, and dorsoventral or- pothesis, the last common ancestor already ertis and Sasai (2), and Holley and Fer- ganization of all modern bilateral animals had a complex differentiated dorsoventral guson (10). Data are striking on the sim- derive from organs and organization of a axis. After protostomes and ilar but inverted domains of expression of complex common ancestor, with body diverged, members of each group would several orthologous genes in embryos of inversion in one of the two lines. have added organs along this axis. In chor- chordates (mostly frogs and mice) and When did inversion occur? Deutero- dates, for example, besides a dorsal hollow protostomes (mostly Drosophila). In the stomes currently include chordates, hemi- nerve cord and ventral heart, there evolved nerve cord, three genes, vnd͞Nk2, ind͞ ͞ chordates, and echinoderms (15, 16). Be- a dorsal , ventrolateral slits, Gsh1,2, and Msh Msx1,3, are expressed in cause the body axis of echinoderms is a ventral in the , and a three columns of nerve cells aligned me- greatly (and intriguingly) modified, only dorsal postanal tail. None of these is found dial to lateral (11). Also, the netrin gene(s) the -chordate comparison in protostomes, which would have added is expressed in the medial floor plate of has been pursued. The worm-like entero- other organs. the chordate nerve cord and the ventral pneust hemichordates differ from chor- After denunciation of it by Cuvier in midline mesectoderm of Drosophila. Be- dates in having a ventral nerve cord, a 1830, the hypothesis was reasserted and cause the similarities seem too complex to protostome-like direction of blood flow, a rejected every 50 to the present (1). have arisen by evolutionary convergence, -like organ protruding dorsally from Recent data on regional gene expression the data imply a complex nerve cord in the the gut (rather than ventrally as in chor- in embryos of fruit flies, frogs, and mice last common ancestor. Results with the dates), a notochord-like pygocord located ͞ have brought new credibility to the hy- heart are also striking. The tinman csk ventrally and posteriorly, a ventral post- pothesis and to deductions about the com- (also called Nkx2-5) gene is expressed very plexity of the last common ancestor of early in the prospective heart region in protostomes and deuterostomes. The sig- chordates (ventrally) and arthropods (dor- *E-mail: [email protected].

PNAS ͉ April 25, 2000 ͉ vol. 97 ͉ no. 9 ͉ 4445–4448 Downloaded by guest on September 30, 2021 new ventral nerve cord in an animal al- ready having a mouth (ventral by defini- tion). Then, inversion occurred later in the chordate line to make it a dorsal cord, and the mouth relocated to the opposite side. Lacalli (21) suggested that a larval ances- formed the nerve cord from ciliary rows at a time when it had only one terminal gut opening, and, hence, its dor- soventral axis was ambiguous. Descen- dents on the branch perfo- rated a mouth on the side opposite the nerve cord, making it dorsal. Those on the Fig. 1. The inversion hypothesis. (A) An annelid worm, side view. The mouth (m) and nerve cord (dark shading) are ventral. The gut (light shading) is midlevel. Arrows indicate the direction of blood flow. protostome branch formed a mouth on the Inverted, it is a chordate, with the nerve cord dorsal, the gut ventral, and the blood flowing in the opposite same side as the nerve cord, making it direction. A new mouth (stomadeum) and (proctodeum) evolve in the chordate. Modified from ref. ventral. Inversion did not really occur 2. (B) The dorsoventral axis in cross section, trunk level. because the mouth arose after the nerve cord. In a related idea (22), a ctenophore- like ancestor with a concentrated nerve anal tail, and a dorsal endostyle-like organ have a mouth separate from the anus, by cord had two opposing openings (anal (Table 1). From these differences, several which to assign dorsoventral orientation? pores) in addition to a terminal gut open- authors (9, 17–19) have concluded that In the absence of intermediate phyla, the ing, also precluding a definition of the enteropneusts have protostome-like orga- possibility of a complex, well organized dorsoventral axis. One of these pores was nization and that inversion must have ancestor cannot be excluded, but it gains retained as a mouth by protostomes and occurred in the chordate line after they no additional support. the other by deuterostomes. These hy- branched off. As noted later, other fea- Critics of inversion have usually favored potheses have in common that the dorso- tures can be used to argue for non- a less complex common ancestor and a ventral axis of the last common ancestor of inversion. different path of chordate evolution. modern bilateral animals was relatively Some of these hypotheses can accommo- undifferentiated and ambiguous, and, Alternatives to Inversion. Recent molecular date the morphological and molecular hence, inversion from one orientation to phylogenies (18S RNA sequences) have data as well as does the inversion hypoth- another is not an issue. As discussed later, deepened the uncertainty about the com- esis. In the Auricularia Hypothesis (20), these alternatives must still rationalize the plexity of the last common ancestor (15, hemichordates and chordates evolved striking inverted gene expression patterns. 16). Bilateral metazoa now fall into three from a bilateral larval ancestor. Through a Related hypotheses involving an ancestor large groups of phyla: the , lo- series of intermediates, bilateral ciliary such as a bilaterogastrea (4) or a -like photrochozoa, and deuterostomes. Deu- rows and the associated moved bilateral micrometazoan (23) as the plat- terostomes are the sister group of the dorsally, fused at the midline, and sank form for the diversification of bilateral other two protostome groups. In this phy- inside to form a new dorsal cord, without metazoa address early steps of evolving logeny, few, if any, intermediate phyla inversion (paedomorphosis followed). To bilaterality more so than they do questions exist between radially or biradially sym- include inversion, Nielsen (19) suggested of inversion and the complexity of the last metric animals (coelenterates, cteno- that protostomes and hemichordates common ancestor. phores) and bilateral animals, to reveal evolved from a ciliated larva by a ventral von Salvini-Plawen (24) raises another steps of the evolution of the dorsoventral convergence of ciliary rows to generate a kind of alternative, that of a bilateral axis (15). In previous morphology-based phylogenies, , nemertines, and Table 1. Evidence for and against inversion: The dorsoventral location of anatomical occupied intermediate posi- elements and gene expression domains in protostomes, hemichordates, and chordates tions, implying intermediate Location of element and associated gene expression such as ones with mesoderm but no coe- Anatomical lom or with a blind gut and diffuse ner- element/gene Protostomes Hemichordates Chordates vous system. Development had protos- tome-like traits. However, flatworms and Mouth Ventral Ventral Ventral nemertines now fall within lophotrocho- Nerve cord Ventral Dorsal (hollow) Dorsal (hollow) zoa, and nematodes fall within ecdysozoa. Ventral (solid) They are probably secondarily simplified. Neural genes vnd/ind/msh, netrin ?? Nkx2/Gsh/Msx, Netrin Deuterostomes are now basal (and some sog/chordin domain Ventrolateral ?? Dorsal deuterostome-like groups such as dpp/bmp domain Dorsal ?? Ventrolateral chaetognaths scatter among protostomes). Heart Dorsal Anterior, dorsal Ventral With regard to the of the Heart genes tinman ?? Csx/Nkx2-5 last common ancestor of modern bilateral Notochord Absent Dorsal stomacord Dorsal notochord animals, we are left with a wide range of Ventral pygocord Post-anal tail Absent Posterior, ventral to anus Posterior, dorsal to possibilities. Did it have a diffuse nerve anus net or multiple nerve bundles with no Liver Absent Dorsal Ventral dorsoventral differentiation, as do mod- Endostyle Absent Dorsal? ventral? Ventral ern radial and biradial animals, or did it Gill slits Absent Lateral Ventrolateral have a single concentrated cord, situated Left-right Absent? Left pore of anterior Left dominant Nodal, at a pole of the dorsoventral axis, as do asymmetry Shh, Pitx many modern bilateral animals? Did it

4446 ͉ www.pnas.org Gerhart Downloaded by guest on September 30, 2021 ancestor with a two-part nervous system: cause they are at the posterior or anterior body organization of the last common one part a plexus of the apical organ and terminus, respectively. The tail is ventral ancestor. The ancestor may have had dif- the other a diffuse nerve net in the body. to the anus, but a small displacement of fuse organization, and then centralization Descendents in the protostome line may the blastopore in development would suf- and placement of organs on the dorsoven- have condensed a nerve cord from the fice to make the tail dorsal, without body tral axis occurred independently in the apical organ complex whereas those of the inversion. In the chordate gastrula, the two lines. Gene expression patterns of deuterostome line may have done so from blastopore͞anus indeed moves ventrally modern organs could still come out the the diffuse net. Important to his argument through posterior mesoderm until the tail same but inversely related in the two lines, (also see ref. 25) is the fact that hemichor- rudiment is dorsal to it (30). The hemi- without body inversion (Fig. 3). dates actually have two nerve cords (Fig. chordate heart is located in the anterior As noted above, the most compelling 2), perhaps reflecting an intermediate coelom of the , anterior to the evidence for inversion may be the com- step in the condensation of nerve tracts mouth and slightly dorsal. In chordate plementary gene expression domains of from the ancestor’s diffuse net. The dorsal evolution, the anterior and middle coe- bmp and chordin in chordates and dpp and cord is hollow and subepithelial in the loms have shrunk greatly, and the heart sog in Drosophila because they seem to animal’s mesosomal (collar) region (27, has come to lie in a ventral position pos- define dorsoventral compartments of the 28). The collar portion of the cord devel- terior to the mouth (31). This displace- body plan (3, 10). In both chordates and ops by a chordate-like process ment might not require inversion but only Drosophila, the entire ectoderm possesses in some species: that is, by inrolling of a local changes of archenteron morphogen- a potential for neural development, which sheet of ectoderm cells. The cord extends esis in the spherical gastrula. The ventral is repressed via a type ␤ transforming anteriorly into the proboscis and posteri- midline of the hemichordate pharynx was growth factor signal [bone morphogenetic orly to the anus. In contrast, the larger previously thought from light microscopy protein (BMP) or Dpp]. The antagonists ventral cord is epithelial and solid. It to resemble the ventral endostyle of chor- (Chordin or Sog) are then released locally, extends from the collar to the anus, but dates, but, from recent micros- and neural development is derepressed at ͞ not into the proboscis. At the pharynx copy (32), the dorsal midline appears that site (35). Although the last common collar boundary, it bends to meet the more endostyle-like, implying inversion. ancestor probably had this default mode dorsal cord. It develops separately from However, incorporation was not of neural development, we cannot con- the dorsal cord. Neither cord resembles a done to localize the endostyle by function. clude that the antagonist was released at ganglion or brain (29). von Salvini-Plawen As a speculation against inversion, per- only one place on the dorsoventral axis. proposes that the ventral cord eventually haps the endostyle is double or circumfer- What determines nowadays the location ential in the hemichordate pharynx and disappeared in the chordate line whereas of the Sog͞Chordin compartment? Dro- has been narrowed down to a single ven- the collar cord was retained in a series of sophila uses a gradient of activated Dorsal tral endostyle in chordates. evolutionary intermediates, as its neuru- transcription factor to locate two bilateral Finally, is the ventral pygocord of hemi- lating mode of formation extended poste- domains of sog expression in the ectoderm chordates (of the ptychoderids) the riorly to the blastopore, becoming the whereas frogs use a quite different means, homolog of the dorsal notochord of chor- chordate neural plate. Hence, inversion a combination of ␤-catenin and nodal- PERSPECTIVE dates (implying inversion)? The alterna- never took place because, with two nerve related proteins to activate chordin ex- cords, the ancestor was dorsoventrally am- tive is the dorsal stomacord (no inversion). biguous. The mouth was already ventral. Both homolog candidates contain vacuo- As noted above, Nu¨bler-Jung and Arendt lated cells like the notochord (9, 33). The (9) instead propose that the ventral cord stomacord does not express the brachyury was preserved and the dorsal cord became gene, which the notochord does, and bra is the chordate brain, with inversion and expressed posteriorly and ventrally in mouth relocation. hemichordates, perhaps including the py- What about the inversion evidence cited gocord (34). This would favor inversion SPECIAL FEATURE above: namely, the locations of the hemi- (9). However, the role of bra expression in chordate tail, heart, endostyle, and pygo- notochord evolution is unknown. It may cord? The tail and heart may also be not signify notochord differentiation but, ambiguous on the dorsoventral axis be- rather, convergent extension morphogen- esis. Perhaps such morphogenesis was only added late in notochord evolution: hence, its absence from the hemichor- date’s short stomacord. But, then, maybe neither candidate is the homolog, which might have arisen in the chordate line as an independent endoderm modification.

What Do the Gene Expression Patterns Re- veal? The strikingly similar but inverted gene expression patterns of protostomes Fig. 3. An alternative to inversion. The hypothet- and deuterostomes provide strong evi- ical ancestor (Left, cross section) has little dorso- dence that the common ancestor pos- ventral differentiation except for the mouth on the sessed certain organs (heart, nerve cord) ventral side. Anterior is toward the reader. The in which certain genes were expressed, body has multiple nerve cords and a centrally lo- cated anterior heart. In the protostome line (Upper Fig. 2. Two nerve cords of an enteropneust hemi- and from which homologous organs of Right), the cords coalesce toward the mouth side chordate, Saccoglossus cambrensis. The animal is modern bilateral animals are derived, ex- (ventral) and the heart shifts dorsally whereas, in shown in semitransparent view. and nerve pressing modern orthologs. Although this the deuterostome line (Lower Right), the opposite bundles, drawn in black, have been silver-stained. may be true, expression patterns in mod- occurs. Two body plans thus arise with inverse dor- Modified from ref. 26. ern animals need not reveal details of the soventral organization, without inversion.

Gerhart PNAS ͉ April 25, 2000 ͉ vol. 97 ͉ no. 9 ͉ 4447 Downloaded by guest on September 30, 2021 pression in midline mesoderm. Secreted contiguous domain in the separate proto- axis. However, the data may also be ex- Chordin protein then antagonizes BMP of stome and deuterostome lines. Presum- plained by alternatives in which the last the mesoderm and ectoderm (3, 10, 36). ably, a hemichordate with two cords has common ancestor of protostomes and deu- These different means for localizing the two locations in the embryo in which terostomes had ambiguous dorsoventral or- Sog͞Chordin compartment may indicate Sog͞Chordin releases neural develop- ganization, such as a diffuse nerve net or separate evolutionary paths from a less ment. And, presumably, both nerve cords multiple nerve cords, and perhaps a heart at localized ancestor. For example, perhaps express a full array of neural genes. an anterior location. From this ancestor, the default mechanism predates bilateral- In conclusion, the hypothesis of body protostome descendents may have further ity and was used in radial or biradial inversion has been vitalized recently by data differentiated the dorsoventral axis in one animals to generate their 4–8 nerve tracts on inverted patterns of orthologous gene direction, and deuterostome descendents in (regions receiving Chordin͞Sog) spaced expression in and . Re- the opposite direction, generating inversely between epidermal zones (regions not re- sults are consistent with the hypothesis and related gene expression patterns and coa- ceiving Chordin͞Sog). Perhaps chordin͞ with the existence of a complex ancestor lesced organs without an actual inversion of sog expression was only unified into a with a highly differentiated dorsoventral the body.

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