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Home , Cathaymyrus, Haikouella, Head, Myllokunmingia, Pikaia, Vertebrate

Perspective

Vertebrate innovations

Sebastian M. Shimeld* and Peter W. H. Holland*

School of and Microbial Sciences, University of Reading, Whiteknights, Reading RG6 6AJ, United

Vertebrate innovations include cells and their derivatives, neurogenic placodes, an elaborate segmented , endoskeleton, and an increase in the number of in the genome. Comparative molecular and developmental data give new insights into the evolutionary origins of these characteristics and the complexity of the vertebrate . ll , at some stage in their of both amphioxus (5) and ascidian (6) minded specifically disrupts an early mi- Acycle, possess a hollow embryos, although the expression pattern grating cell population that contributes to dorsal to a , plus lateral muscle differs in detail between taxa. Lateral spinal ganglia, without affecting later- blocks. These characters unite (in- neural plate cells in amphioxus embryos migrating neural crest cells (13). Further- cluding ascidians), amphioxus, and verte- express members of the Msx (7), more, this mutant also affects Rohon- brates in the Chordata. Compara- slug͞snail (8), and Distalless (9) Beard cells; these are not considered to be tive molecular and developmental analyses families whereas the latter gene is also neural crest derivatives but are a dorsal have refined this anatomical picture, sug- expressed in adjacent to (and neural population of cells involved in gesting that the neural tube of the common overgrowing) the neural plate. In ascid- mechanosensory reception (and also ancestor of chordates had three major sub- ians, Msx (10) and slug͞snail (11) homo- present in amphioxus). This genetic link divisions along the anterior-posterior axis logues are expressed in cells contributing between early-migrating neural crest and (1), was patterned along the dorsoventral to the lateral neural plate (and in some Rohon-Beard cells suggests a possible axis by hedgehog and Bmp signaling (2), and other tissues); the Pax-3͞7 gene is ex- common origin. An early step in the evo- was probably segmented (3). These data pressed in the immediately adjacent ecto- lution of neural crest, therefore, may have suggest considerable complexity of the com- derm, after earlier expression in cells been the origin of a specific dorsal neural mon ancestor of chordates. are fated to contribute to neural plate (3). cell population contributing to sensory more complex still, both morphologically These studies demonstrate that the rela- processing; this would predate the diver- and genetically, and are characterized by a tive spatial expression patterns of many gence of the amphioxus and vertebrate considerable number of derived features genes involved in neural crest induction lineages. The of cell migration (see Fig. 1). Here we review recent molec- were already present before the evolution

was probably of later evolutionary origin, PERSPECTIVE ular and developmental data that give in- of vertebrates. Evolutionary changes to as was the origin of neural crest cell lin- sight into the evolutionary origin of these the regulation of these genes, therefore, eages with primarily non-neural fates, vertebrate innovations, notably the neural have probably not contributed directly to such as melanocytes and connective crest, placodes, complex brain, , and neural crest origins. The homologues of tissue. additional genes. several other genes involved in neural crest induction have not yet been exam- Placodes. Placodes are paired ectodermal The Neural Crest. Neural crest cells are a key ined in amphioxus or embryos; it thickenings that contribute to the formation vertebrate character. They form at the will be particularly informative if any of of a number of specialized structures of the boundary between neural plate and sur- these show significant expression differ- vertebrate . They can be divided into SPECIAL FEATURE face ectoderm and migrate to contribute ences to vertebrates. the ‘‘sensory placodes,’’ which contribute to to many of the structures considered to be It is probably inappropriate to consider the , , lateral line, and olfactory or- vertebrate novelties, including the cra- the evolution of neural crest cells as a gans, and the ‘‘neurogenic placodes,’’ which nium, branchial skeleton, and sensory single evolutionary step or a single verte- contribute sensory neurons to cranial gan- ganglia. Experimentally, trunk neural brate character. First, head and trunk glia. It is probable that the two types of crest cells can be induced from chick neural crest have different developmental placode have separate evolutionary origins. embryonic neural plate by application of fates and potentials, and it is not yet clear Sensory placodes form a variety of cell Bmp-4 or -7 (4), the genes en- whether they evolved as a single cell pop- types, including neuroendocrine cells, sen- coding which are expressed by ectodermal ulation or at different times in evolution. sory neurons, ciliated sensory receptors, and cells. Neural crest cells themselves are The existence of head neural crest in early glia. They may also not be confined to marked by expression of a number of vertebrate such as vertebrates. Amphioxus has a putative ho- genes, including members of the Msx, (Fig. 2) is inferred by the presence of a mologue of the olfactory placode in the slug͞snail, Zic, Pax-3͞7, and Distalless complex branchial skeleton (12). Interest- corpuscles of de Quatrefages, a specialized gene families. Lateral neural plate cells ingly, no evidence of trunk neural crest group of anterior ectoderm cells. These cells also express these genes in vertebrate em- derivatives is seen in these fossils (al- bryos, suggesting some continuity of char- though this is not strong evidence for send axonal projections to the central ner- acter between these two cell populations. absence because preservation of such vous system along the most anterior nerve Homologues of several putative neural structures may be poor). Second, neural and are marked by expression of the ho- crest cell inducers and markers have been crest cells at one axial level may them- meobox gene AmphiMsx (7). This putative cloned from amphioxus and͞or tunicates, selves be a diverse population, with cells to olfactory placodes could be and their expression patterns have been that migrate at different times having dif- compared with vertebrates. These studies ferent developmental properties and pos- ͞ *To whom reprint requests should be addressed. E-mail: have revealed that the single BMP-2 4 sibly distinct evolutionary origins. For [email protected] and [email protected] gene is expressed in non-neural ectoderm example, the mutant narrow- ac.uk.

PNAS ͉ April 25, 2000 ͉ vol. 97 ͉ no. 9 ͉ 4449–4452 Downloaded by guest on September 27, 2021 novelty, therefore, but the concentration of neurogenesis to discrete focal regions of ectoderm. Experiments show that the chick trigeminal placode is induced by signals from the neural plate (18) whereas pharyngeal endoderm is the source of the inductive signal (Bmp-7) for epibranchial placodes (16). In both cases, competence to respond to the inductive signal is wide- spread in cranial ectoderm, but absent from trunk ectoderm (16, 19). These data suggest that the focused neurogenesis of vertebrate epibranchial and dorsolateral placodes is achieved by a combination of localized inductive signals and restricted ectodermal competence. In summary, the collective term ‘‘pla- codes’’ refers to some rather different structures, probably with different evolu- tionary origins. Some sensory placodes (at least the otic and olfactory) may have homologues in chordates. Even if this is so, it is apparent that they were elaborated considerably during early ver- tebrate evolution. Epibranchial and dor- solateral placodes appear to be new; we infer that their origin depended on the evolution of specific inductive signals.

Elaboration of the Vertebrate Brain. A com- plex brain with specialized fore-, mid-, and hindbrain regions is characteristic of all ver- Fig. 1. What is a vertebrate? Phylogeny showing the relationship between living members of the Phylum tebrates. Other chordates also possess a Chordata (in bold, at top), plus the fossils Myllokunmingia (12) (a ) and (30) distinct structure at the rostral end of the (a basal ). Some putative chordates, including and (possibly related to amphioxus) (31, 32) and the Euconodonts (25) (possible vertebrates) are omitted, as their precise rela- neural tube, called the cerebral vesicle (am- tionships are less clear. Systematics reserves the Vertebrata for those possessing vertebrae: phioxus) or sensory vesicle (ascidian larvae). that is, lampreys and jawed vertebrates, with hagfish excluded as a sister group. These three taxa are The extent of homology between these united by the term Craniata. Some molecular analyses, however, support of lampreys and structures has long been controversial, but is hagfish (comprising the ) (33), which would make the Vertebrata paraphyletic. Here we central to discerning which aspects of brain depict the node at the base of hagfish, lampreys, and jawed vertebrates as unresolved to reflect this organization are vertebrate innovations. conflict between molecular and morphological data. We view the evolution of vertebrates as the Gene expression patterns, together with fine acquisition of characters along the chordate stem lineage. These characters, established by comparison to structural studies, have clarified the picture outgroups (i.e., amphioxus and tunicates), include (i) neural crest cells and their derivatives; (ii) elaboration of placode-derived structures; (iii) elaboration of the brain, including rhombomeric ; (iv) considerably. These studies reveal that em- (and possibly mineralization); (v) the and the head skeleton; and (vi) a large bryos of vertebrates, amphioxus, and ascidia increase in total number of genes in the genome. Other characters present only in jawed vertebrates, each have a distinct rostral of the including paired , hinged , an adaptive , and specialization of the axial neural tube, marked by Otx gene expression, skeleton along the anterior-posterior axis, were probably acquired later, on the jawed vertebrate stem separated from a more caudal region in lineage, and will not be considered here. which Hox genes are active. Between these two domains in vertebrates lies the isthmo- cerebellar region, including rhombomere 1 investigated further by functional studies on between the atrial primordia of ascidia and of the hindbrain and the -hindbrain the amphioxus organs and by examination the otic placodes of vertebrates. boundary (MHB). This region is marked by of gene expression in Neurogenic placodes contribute sen- expression of all three Pax 2͞5͞8 genes of amphioxus. Adult ascidians have sensory sory neurons to cranial ganglia and may vertebrates, both En genes, Wnt-1, and hair cells grouped into gelatinous cupular themselves be divided into two groups. FGF-8. Strikingly, the single ascidian Pax organs, located in the atrium of the adult; The dorsolateral placodes (trigeminal and 2͞5͞8 homologue is also expressed in cells these cells have striking structural similari- vestibular) develop from ectoderm lateral between the Otx and Hox domains of Halo- ties to the sensory hair cells of the vertebrate to the brain whereas the epibranchial pla- cynthia (15). These comparisons led Wada et acousticolateralis system (14). Further- codes (geniculate, petrosal, and nodose) al. to propose a universal tripartite organi- more, the ascidian atrium develops from a lie more ventrally, close to the pharyngeal zation for chordate neural tubes, comprising pair of ectodermal invaginations, topo- pouches (16). Although epibranchial and Otx, Pax 2͞5͞8, and Hox domains; these graphically similar to sensory placodes; dorsolateral have no identified homo- correspond to the fore-͞midbrain, isthmo- these atrial primordia, like vertebrate otic logues in amphioxus or tunicates, the gen- cerebellar͞MHB, and hindbrain͞spinal placodes, express members of the Pax- eration of individual neurons from ecto- cord of vertebrates (15). The amphioxus Pax 2͞5͞8 gene (15). Thus, cellular or- derm is a character common to many 2͞5͞8 gene is not expressed in the central ganization, embryological origin, and gene animal taxa (17). It is not the neurogenic domain, however, implying secondary mod- expression all point to probable homology potential of ectoderm that is a vertebrate ification in (or convergent

4450 ͉ www.pnas.org Shimeld and Holland Downloaded by guest on September 27, 2021 changes downstream of these signals. The co-option of Pax gene expression into the is likely to be one of several such changes.

Conclusions: Toward a Causal Understanding of Vertebrate Origins. In the above sections, we have highlighted developmental differ- ences that underlie some of the key anatom- ical distinctions between vertebrates and their closest relatives. Because all evolution- ary change must be based on genetic change, we have asked whether it is possible to identify candidate molecular changes that cause each developmental difference. In some cases (e.g., neural crest origins), the candidate molecular changes are not evi- dent, despite extensive comparative work. Fig. 2. Reconstruction of the early Cambrian craniate Myllokunmingia (12). [Reproduced with permis- In other cases, evidence points to changes in sion from John Sibbick (Copyright 1999 John Sibbick).] the regulation of specific genes (e.g., Hox, Krox, FGF-8, Pax-1͞9).Ifwewishtoprobe evolution between tunicates and verte- notochord. The two earliest craniate fos- brates) (20). sils, Myllokunmingia (Fig. 2) and Haik- Despite the underlying homologies, the ouichthys from the early Cambrian, have a vertebrate brain is massively more com- cartilaginous branchial skeleton but show plex. The fore-͞midbrain region has been no evidence of (12). greatly expanded, for example, and shows Late Cambrian fossils, the Euconodonts, evidence of compartmentalization not yet show extensive biomineralization (in the described in other chordates. The second -like elements), a probable subdivision includes the MHB in verte- cartilaginous head skeleton, but still no brates: a structure with important pattern- evidence of an axial skeleton (25). These ing roles, probably mediated by FGF8 observations suggest that cartilage pre- (21). It is unclear whether this role of the dates biomineralization, and that a cranial MHB is a vertebrate innovation. Two ob- skeleton evolved before the axial skeleton. servations combine to suggest it might be: There are few insights into the molecular PERSPECTIVE FGF8 inhibits vertebrate Hox gene ex- changes underlying the origins of cartilage pression in rhomobomere 1 of the hind- or biomineralization. The origins of the ax- brain (21) whereas Hox-1 gene expression ial skeleton seem more tractable because extends more rostrally in ascidia then in comparisons can be made to mesoderm vertebrates (15). A more definitive state- development in outgroup taxa. In am- ment on the origins of MHB signaling phioxus, the entire mesoderm is segmented. awaits cloning of FGF8 homologues from In lampreys and jawed vertebrates, only the

amphioxus and tunicates. The hindbrain paraxial and is fully SPECIAL FEATURE region is also more complex in vertebrates segmented, although the lateral plate meso- than other chordates, notably through its derm of embryos shows traces of stereotyped subdivision into rhom- shallow segmental grooves (M. J. Cohn, bomeres with distinct developmental personal communication). This suggests a properties. Genes such as Krox-20 and progressive dorsal restriction of mesoder- kreisler play key roles in hindbrain segmen- mal segmentation in vertebrate evolution, tation, and in the control of rhombomere which may be linked to increasing complex- fate through modulation of Hox gene ex- ity of mesodermal patterning. Within the pression (22, 23). Current data suggest somite, it is the ventromedial region (the Fig. 3. Mesodermal subdivision in amphioxus (A that this elaboration of segmentation is a sclerotome) that produces the axial skeleton and B) compared with a schematic vertebrate (C). A vertebrate innovation because amphioxus of vertebrates. The of amphioxus and B show progressive stages in the development of amphioxus mesoderm. A ventrolateral zone of am- and ascidian Hox genes do not show a are also subdivided into distinct regions, phioxus mesoderm (red) grows down to surround similar pattern of modulation (15, 24), and although there is no histological evidence the gut. Homology of this zone to the lateral plate amphioxus homologues of Krox-20 and for sclerotome (Fig. 3). Furthermore, Am- mesoderm of vertebrates (red in C) is supported by kreisler are not expressed in appropriate phiPax-1͞9, a gene orthologous to the an- site of origin and fate. Medial amphioxus somite cells patterns in the neural tube (R. D. Knight, cestor of the vertebrate sclerotome markers form myotome (yellow) whereas dorsolateral cells W. Jackman, personal communication). Pax-1 and Pax-9, is not expressed in am- (orange) eventually move to surround the noto- phioxus somites (26). Vertebrate scle- chord. On the basis of position of origin, these may be Cartilage, , and Teeth. Vertebrate car- rotome is induced by hedgehog (and possi- homologous to the myotome and dermatome of vertebrates (yellow and orange, respectively, in C), tilage and are used for bly ) signaling from the notochord although these cells do not contribute to a dermis in protection, , and endoskeletal competing with Bmp signaling from more amphioxus. The vertebrate sclerotome (gray in C) has support. There are no similar tissues in lateral cells. Because at least two of these no equivalent in amphioxus and is a novelty linked amphioxus or tunicates, where endoskel- signals are present in amphioxus (2, 5), the with the evolution of the axial skeleton. A and B are etal support is provided by a vacuolated evolution of sclerotome probably involved adapted from ref. 34.

Shimeld and Holland PNAS ͉ April 25, 2000 ͉ vol. 97 ͉ no. 9 ͉ 4451 Downloaded by guest on September 27, 2021 deeper into vertebrate origins, therefore, it by which this duplication occurred is con- between daughter genes (e.g., by differen- is important to consider how gene regula- troversial (, multiple gene du- tial silencing of enhancers) (29). Never- tion evolves, and how genes can acquire new plications, or both), but its prevalence can theless, the widespread duplication of developmental roles. hardly be disputed. Indeed, increased ge- developmental genes in early vertebrate In the context of vertebrate evolution, it netic complexity can be validly viewed as evolution could conceivably be one of the is now very clear that numerous gene a vertebrate innovation. key events underlying the evolution of families expanded by gene duplication on One long-standing hypothesis is that vertebrate innovations, linking an increase the vertebrate stem lineage (27). These gene duplications promote the evolution in morphological complexity with an in- include gene families encoding transcrip- of new functions because duplicate genes crease in genetic complexity via the inter- tion factors (Hox, ParaHox, En, Otx, Msx, are freed from the constraining effects of mediary of developmental control. Pax, Dlx, HNF3, bHLH), signaling mole- natural selection by redundancy (28). cules (hh, IGF, BMP), and others (dystro- This, however, has proven hard to test We thank A. Graham and I. Mason for helpful phin, cholinesterase, actin, ). In because there are many examples of genes discussions, and John and Bridgette Sibbick for permission to reproduce the reconstruction of each case, vertebrates possess more copies evolving multiple roles without gene du- Myllokunmingia. The authors’ work is funded by than did the common ancestor of chor- plication, and a fundamentally different the Medical Research Council and the Biotech- dates, as inferred by comparison with am- route of increasing gene number by dupli- nology and Biological Sciences Research phioxus and͞or ascidia. The mechanism cation is if these roles become partitioned Council.

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