Nicholas D. Holland

Axial patterning in cephalochordates and the evolution of the organizer.

The organizer of the vertebrate gastrula is an important signalling centre that induces and patterns dorsal axial structures. Although a topic of long-standing interest, the evolutionary origin of the organizer remains unclear. Here we show that the gastrula of the cephalochordate amphioxus expresses dorsal/ventral (D/V) patterning genes (for example, bone morphogenetic proteins (BMPs), Nodal and their antagonists) in patterns reminiscent of those of their vertebrate orthlogues, and that amphioxus embryos, like those of vertebrates, are ventralized by exogenous BMP protein. In addition, Wnt-antagonists (for example, Dkks and sFRP2-like) are expressed anteriorly, whereas Wnt genes themselves are expressed posteriorly, consistent with a role for Wnt signalling in anterior/posterior (A/P) patterning. These results suggest evolutionary conservation of the mechanisms for both D/V and A/P patterning of the early gastrula. In light of recent phylogenetic analyses placing cephalochordates basally in the chordate lineage, we propose that separate signalling centres for patterning the D/V and A/P axes may be an ancestral chordate character.

Early central nervous system evolution: an era of skin brains?

The central nervous system (CNS) in different animals develops under the control of genes that are often conserved both structurally and functionally. The question of whether such conservation means that the common ancestor of all bilateral animals had an anatomically complex CNS is controversial. Recently, a hemichordate was found to express homologues of many of these genes in a rostrocaudal order resembling that in Drosophila and vertebrates. Surprisingly, however, the hemichordate genes are expressed not in a localized CNS, but in widespread epidermal domains pervaded by a basiepidermal nerve net. Here, I discuss the implications of this discovery for CNS evolution.

Origin and early evolution of the vertebrates: new insights from advances in molecular biology, anatomy, and palaeontology

Recent advances in molecular biology and microanatomy have supported homologies of body parts between vertebrates and extant invertebrate chordates, thus providing insights into the body plan of the proximate ancestor of the vertebrates. For example, this ancestor probably had a relatively complex brain and a precursor of definitive neural crest. Additional insights into early vertebrate evolution have come from recent discoveries of Lower Cambrian soft body fossils of Haikouichthys and Myllokunmingia (almost certainly vertebrates, possibly related to modern lampreys) and Yunnanozoon and Haikouella (evidently stem‐group vertebrates). The earliest vertebrates had an unequivocally marine origin, probably evolved mineralised pharyngeal denticles before the dermal skeleton, and evidently utilised elastic recoil of the visceral arch skeleton for suction feeding. Moreover, the new data emphasise that the advent of definitive neural crest was supremely important for the evolutionary origin of the vertebrates. BioEssays 23:142–151, 2001.

Evolution of neural crest and placodes: amphioxus as a model for the ancestral vertebrate?

Recent studies of protochordates (ascidian tunicates and amphioxus) have given insights into possible ancestors of 2 of the characteristic features of the vertebrate head: neural crest and placodes. The neural crest probably evolved from cells on either side of the neural plate–epidermis boundary in a protochordate ancestral to the vertebrates. In amphioxus, homologues of several vertebrate neural crest marker genes (BMP2/4, Pax3/7, Msx, Dll and Snail) are expressed at the edges of the neural plate and/or adjacent nonneural ectoderm. Some of these markers are also similarly expressed in tunicates. In protochordates, however, these cells, unlike vertebrate neural crest, neither migrate as individuals through embryonic tissues nor differentiate into a wide spectrum of cell types. Therefore, while the protochordate ancestor of the vertebrates probably had the beginnings of a genetic programme for neural crest formation, this programme was augmented in the earliest vertebrates to attain definitive neural crest. Clear homologues of vertebrate placodes are lacking in protochordates. However, both amphioxus and tunicates have ectodermal sensory cells. In tunicates these are all primary neurons, sending axons to the central nervous system, while in amphioxus, the ectodermal sensory cells include both primary neurons and secondary neurons lacking axons. Comparisons of developmental gene expression data suggest that the anterior ectoderm in amphioxus may be homologous to the vertebrate olfactory placode, the only vertebrate placode with primary, not secondary, neurons. Similarly, biochemical, morphological and gene expression suggest that amphioxus and tunicates also have homologues of the adenohypophysis, one of the few vertebrate structures derived from nonneurogenic placodes. In contrast, the origin of the other vertebrate placodes is very uncertain.

Evolution of Genetic Networks Underlying the Emergence of Thymopoiesis in Vertebrates

About 500 million years ago, a new type of adaptive immune defense emerged in basal jawed vertebrates, accompanied by morphological innovations, including the thymus. Did these evolutionary novelties arise de novo or from elaboration of ancient genetic networks? We reconstructed the genetic changes underlying thymopoiesis by comparative genome and expression analyses in chordates and basal vertebrates. The derived models of genetic networks were experimentally verified in bony fishes. Ancestral networks defining circumscribed regions of the pharyngeal epithelium of jawless vertebrates expanded in cartilaginous fishes to incorporate novel genes, notably those encoding chemokines. Correspondingly, novel networks evolved in lymphocytes of jawed vertebrates to control the expression of additional chemokine receptors. These complementary changes enabled unprecedented Delta/Notch signaling between pharyngeal epithelium and lymphoid cells that was exploited for specification to the T cell lineage. Our results provide a framework elucidating the evolution of key features of the adaptive immune system in jawed vertebrates.

Chordate origins of the vertebrate central nervous system.

Fine structural, computerized three-dimensional (3D) mapping of cell connectivity in the amphioxus nervous system and comparative molecular genetic studies of amphioxus and tunicates have provided recent insights into the phylogenetic origin of the vertebrate nervous system. The results suggest that several of the genetic mechanisms for establishing and patterning the vertebrate nervous system already operated in the ancestral chordate and that the nerve cord of the proximate invertebrate ancestor of the vertebrates included a diencephalon, midbrain, hindbrain, and spinal cord. In contrast, the telencephalon, a midbrain-hindbrain boundary region with organizer properties, and the definitive neural crest appear to be vertebrate innovations.

AmphiPax3/7, an amphioxus paired box gene: insights into chordate myogenesis, neurogenesis, and the possible evolutionary precursor of definitive vertebrate neural crest.

SUMMARY Amphioxus probably has only a single gene (AmphiPax3/7 ) in the Pax3/7 subfamily. Like its vertebrate homologs (Pax3 and Pax7 ), amphioxus AmphiPax3/7 is probably involved in specifying the axial musculature and muscularized notochord. During nervous system development, AmphiPax3/7 is first expressed in bilateral anteroposterior stripes along the edges of the neural plate. This early neural expression may be comparable to the transcription of Pax3 and Pax7 in some of the anterior neural crest cells of vertebrates. Previous studies by others and ourselves have demonstrated that several genes homologous to genetic markers for vertebrate neural crest are expressed along the neural plate–epidermis boundary in embryos of tunicates and amphioxus. Taken together, the early neural expression patterns of AmphiPax3/7 and other neural crest markers of amphioxus and tunicates suggest that cell populations that eventually gave rise to definitive vertebrate neural crest may have been present in ancestral invertebrate chordates. During later neurogenesis in amphioxus, AmphiPax3/7, like its vertebrate homologs, is expressed dorsally and dorsolaterally in the neural tube and may be involved in dorsoventral patterning. However, unlike its vertebrate homologs, AmphiPax3/7 is expressed only at the anterior end of the central nervous system instead of along much of the neuraxis; this amphioxus pattern may represent the loss of a primitive chordate character.

Amphioxus Postembryonic Development Reveals the Homology of Chordate Metamorphosis

Most studies in evolution are centered on how homologous genes, structures, and/or processes appeared and diverged. Although historical homology is well defined as a concept, in practice its establishment can be problematic, especially for some morphological traits or developmental processes. Metamorphosis in chordates is such an enigmatic character. Defined as a spectacular postembryonic larva-to-adult transition, it shows a wide morphological diversity between the different chordate lineages, suggesting that it might have appeared several times independently. In vertebrates, metamorphosis is triggered by binding of the thyroid hormones (THs) T(4) and T(3) to thyroid-hormone receptors (TRs). Here we show that a TH derivative, triiodothyroacetic acid (TRIAC), induces metamorphosis in the cephalochordate amphioxus. The amphioxus TR (amphiTR) mediates spontaneous and TRIAC-induced metamorphosis because it strongly binds to TRIAC, and a specific TR antagonist, NH3, inhibits both spontaneous and TRIAC-induced metamorphosis. Moreover, as in amphibians, amphiTR expression levels increase around metamorphosis and are enhanced by THs. Therefore, TH-regulated metamorphosis, mediated by TR, is an ancestral feature of all chordates. This conservation of a regulatory network supports the homology of metamorphosis in the chordate lineage.

AN AUTORADIOGRAPHIC INVESTIGATION OF THE GONADS OF THE PURPLE SEA URCHIN (STRONGYLOCENTROTUS PURPURATUS)

1. The DNA-synthesizing cells in the gonads of the purple sea urchin were labeled with tritiated thymidine and detected with autoradiography.2. Some fibroblasts in the connective tissue-muscle layer and some cells of the visceral peritoneum covering the gonads synthesize DNA.3. Some of the non-germinal cells (the nutritive phagocytes) of the germinal layer proliferate during the spring and early summer, but proliferation ceases in the late summer as the nutritive phagocytes begin to accumulate cytoplasmic globules of reserve. Nutritive phagocytes labeled with tritiated thymidine in their deglobulated phase in the spring are still labeled up to several months later, after they have acquired cytoplasmic globules.4. In the testes of male urchins at the beginning of the reproductive season, not only relict spermatozoa, but also some newly-formed spermatozoa are ingested by the nutritive phagocytes. The phagocytosis of newly-formed spermatozoa ceases later in the reproductive season, at approximately the same ...

Retinoic acid signaling acts via Hox1 to establish the posterior limit of the pharynx in the chordate amphioxus.

In the invertebrate chordate amphioxus, as in vertebrates, retinoic acid (RA) specifies position along the anterior/posterior axis with elevated RA signaling in the middle third of the endoderm setting the posterior limit of the pharynx. Here we show that AmphiHox1 is also expressed in the middle third of the developing amphioxus endoderm and is activated by RA signaling. Knockdown of AmphiHox1 function with an antisense morpholino oligonucleotide shows that AmphiHox1 mediates the role of RA signaling in setting the posterior limit of the pharynx by repressing expression of pharyngeal markers in the posterior foregut/midgut endoderm. The spatiotemporal expression of these endodermal genes in embryos treated with RA or the RA antagonist BMS009 indicates that Pax1/9, Pitx and Notch are probably more upstream than Otx and Nodal in the hierarchy of genes repressed by RA signaling. This work highlights the potential of amphioxus, a genomically simple, vertebrate-like invertebrate chordate, as a paradigm for understanding gene hierarchies similar to the more complex ones of vertebrates.