Molecular evidence from Ciona intestinalis for the evolutionary origin of vertebrate sensory placodes

Cranial sensory placodes are focused areas of the head ectoderm of vertebrates that contribute to the development of the cranial sense organs and their associated ganglia. Placodes have long been considered a key character of vertebrates, and their evolution is proposed to have been essential for the evolution of an active predatory lifestyle by early vertebrates. Despite their importance for understanding vertebrate origins, the evolutionary origin of placodes has remained obscure. Here, we use a panel of molecular markers from the Six, Eya, Pax, Dach, FoxI, COE and POUIV gene families to examine the tunicate Ciona intestinalis for evidence of structures homologous to vertebrate placodes. Our results identify two domains of Ciona ectoderm that are marked by the genetic cascade that regulates vertebrate placode formation. The first is just anterior to the brain, and we suggest this territory is equivalent to the olfactory/adenohypophyseal placodes of vertebrates. The second is a bilateral domain adjacent to the posterior brain and includes cells fated to form the atrium and atrial siphon of adult Ciona. We show this bares most similarity to placodes fated to form the vertebrate acoustico-lateralis system. We interpret these data as support for the hypothesis that sensory placodes did not arise de novo in vertebrates, but evolved from pre-existing specialised areas of ectoderm that contributed to sensory organs in the common ancestor of vertebrates and tunicates.     

Chordate betagamma-crystallins and the evolutionary developmental biology of the vertebrate lens

Several animal lineages, including the vertebrates, have evolved sophisticated eyes with lenses that refract light to generate an image. The nearest invertebrate relatives of the vertebrates, such as the ascidians (sea squirts) and amphioxus, have only basic light detecting organs, leading to the widely-held view that the vertebrate lens is an innovation that evolved in early vertebrates. From an embryological perspective the lens is different from the rest of the eye, in that the eye is primarily of neural origin while the lens derives from a non-neural ectodermal placode which invaginates into the developing eye. How such an organ could have evolved has attracted much speculation. Recently, however, molecular developmental studies of sea squirts have started to suggest a possible evolutionary origin for the lens. First, studies of the Pax, Six, Eya and other gene families have indicated that sea squirts have areas of non-neural ectoderm homologous to placodes, suggesting an origin for the embryological characteristics of the lens. Second, the evolution and regulation of the betagamma-crystallins has been studied. These form one of the key crystallin gene families responsible for the transparency of the lens, and regulatory conservation between the betagamma-crystallin gene in the sea squirt Ciona intestinalis and the vertebrate visual system has been experimentally demonstrated. These data, together with knowledge of the morphological, physiological and gene expression similarities between the C. intestinalis ocellus and vertebrate retina, have led us to propose a hypothesis for the evolution of the vertebrate lens and integrated vertebrate eye via the co-option and combination of ancient gene regulatory networks; one controlling morphogenetic aspects of lens development and one controlling the expression of a gene family responsible for the biophysical properties of the lens, with the components of the retina having evolved from an ancestral photoreceptive organ derived from the anterior central nervous system.     

Activation of Six1 target genes is required for sensory placode formation

In vertebrates, cranial placodes form crucial parts of the sensory nervous system in the head. All cranial placodes arise from a common territory, the preplacodal region, and are identified by the expression of Six1/4 and Eya1/2 genes, which control different aspects of sensory development in invertebrates as well as vertebrates. While So and Eya can induce ectopic eyes in Drosophila, the ability of their vertebrate homologues to induce placodes in non-placodal ectoderm has not been explored. Here we show that Six1 and Eya2 are involved in ectodermal patterning and cooperate to induce preplacodal gene expression, while repressing neural plate and neural crest fates. However, they are not sufficient to induce ectopic sensory placodes in future epidermis. Activation of Six1 target genes is required for expression of preplacodal genes, for normal placode morphology and for placode-specific Pax protein expression. These findings suggest that unlike in the fly where the Pax6 homologue Eyeless acts upstream of Six and Eya, the regulatory relationships between these genes are reversed in early vertebrate placode development.     

The evolutionary history of vertebrate cranial placodes – I: Cell type evolution

Vertebrate cranial placodes are crucial contributors to the vertebrate cranial sensory apparatus. Their evolutionary origin has attracted much attention from evolutionary and developmental biologists, yielding speculation and hypotheses concerning their putative homologues in other lineages and the developmental and genetic innovations that might have underlain their origin and diversification. In this article we first briefly review our current understanding of placode development and the cell types and structures they form. We next summarise previous hypotheses of placode evolution, discussing their strengths and caveats, before considering the evolutionary history of the various cell types that develop from placodes. In an accompanying review, we also further consider the evolution of ectodermal patterning. Drawing on data from vertebrates, tunicates, amphioxus, other bilaterians and cnidarians, we build these strands into a scenario of placode evolutionary history and of the genes, cells and developmental processes that underlie placode evolution and development.     

Increased complexity of gene structure and base composition in vertebrates

How the structure and base composition of genes changed with the evolution of vertebrates remains a puzzling question. Here we analyzed 895 orthologous protein-coding genes in six multicellular animals: human, chicken, zebrafish, sea squirt, fruit fly, and worm. Our analyses reveal that many gene regions, particularly intron and 3' UTR, gradually expanded throughout the evolution of vertebrates from their invertebrate ancestors, and that the number of exons per gene increased. Studies based on all protein-coding genes in each genome provide consistent results.We also find that GC-content increased in many gene regions (especially 5' UTR) in the evolution of endotherms, except in coding-exons.Analysis of individual genomes shows that 3′ UTR demonstrated stronger length and CC-content correlation with intron than 5' UTR, and gene with large intron in all six species demonstrated relatively similar GC-content. Our data indicates a great increase in complexity in vertebrate genes and we propose that the requirement for morphological and functional changes is probably the driving force behind the evolution of structure and base composition complexity in multicellular animal genes.     

The amphioxus SoxB family: implications for the evolution of vertebrate placodes

Cranial placodes are regions of thickened ectoderm that give rise to sense organs and ganglia in the vertebrate head. Homologous structures are proposed to exist in urochordates, but have not been found in cephalochordates, suggesting the first chordates lacked placodes. SoxB genes are expressed in discrete subsets of vertebrate placodes. To investigate how placodes arose and diversified in the vertebrate lineage we isolated the complete set of SoxB genes from amphioxus and analyzed their expression in embryos and larvae. We find that while amphioxus possesses a single SoxB2 gene, it has three SoxB1 paralogs. Like vertebrate SoxB1 genes, one of these paralogs is expressed in non-neural ectoderm destined to give rise to sensory cells. When considered in the context of other amphioxus placode marker orthologs, amphioxus SoxB1 expression suggests a diversity of sensory cell types utilizing distinct placode-type gene programs was present in the first chordates. Our data supports a model for placode evolution and diversification whereby the full complement of vertebrate placodes evolved by serial recruitment of distinct sensory cell specification programs to anterior pre-placodal ectoderm.     

Vertebrate cranial evolution: Contributions and conflict from the fossil record

In modern vertebrates, the craniofacial skeleton is complex, comprising cartilage and bone of the neurocranium, dermatocranium and splanchnocranium (and their derivatives), housing a range of sensory structures such as eyes, nasal and vestibulo-acoustic capsules, with the splanchnocranium including branchial arches, used in respiration and feeding. It is well understood that the skeleton derives from neural crest and mesoderm, while the sensory elements derive from ectodermal thickenings known as placodes. Recent research demonstrates that neural crest and placodes have an evolutionary history outside of vertebrates, while the vertebrate fossil record allows the sequence of the evolution of these various features to be understood. Stem-group vertebrates such as Metaspriggina walcotti (Burgess Shale, Middle Cambrian) possess eyes, paired nasal capsules and well-developed branchial arches, the latter derived from cranial neural crest in extant vertebrates, indicating that placodes and neural crest evolved over 500 million years ago. Since that time the vertebrate craniofacial skeleton has evolved, including different types of bone, of potential neural crest or mesodermal origin. One problematic part of the craniofacial skeleton concerns the evolution of the nasal organs, with evidence for both paired and unpaired nasal sacs being the primitive state for vertebrates.     

Expression of AmphiCoe, an amphioxus COE/EBF gene, in the developing central nervous system and epidermal sensory neurons

The COE/EBF gene family marks a subset of prospective neurons in the vertebrate central and peripheral nervous system, including neurons deriving from some ectodermal placodes. Since placodes are often considered unique to vertebrates, we have characterised an amphioxus COE/EBF gene with the aim of using it as a marker to examine the timing and location of peripheral neuron differentiation. A single COE/EBF family member, AmphiCoe, was isolated from the amphioxus Branchiostoma floridae. AmphiCoe lies basal to the vertebrate COE/EBF genes in molecular phylogenetic analysis, suggesting that the duplications that formed the vertebrate COE/EBF family were specific to the vertebrate lineage. AmphiCoe is expressed in the central nervous system and in a small number of scattered ectodermal cells on the flanks of neurulae stage embryos. These cells become at least largely recessed beneath the ectoderm. Scanning electron microscopy was used to examine embryos in which the ectoderm had been partially peeled away. This revealed that these cells have neuronal morphology, and we infer that they are the precursors of epidermal primary sensory neurons. These characters lead us to suggest that differentiation of some ectodermal cells into sensory neurons with a tendency to sink beneath the embryonic surface represents a primitive feature that has become incorporated into placodes during vertebrate evolution.     

Insights from amphioxus into the evolution of vertebrate cartilage

Central to the story of vertebrate evolution is the origin of the vertebrate head, a problem difficult to approach using paleontology and comparative morphology due to a lack of unambiguous intermediate forms. Embryologically, much of the vertebrate head is derived from two ectodermal tissues, the neural crest and cranial placodes. Recent work in protochordates suggests the first chordates possessed migratory neural tube cells with some features of neural crest cells. However, it is unclear how and when these cells acquired the ability to form cellular cartilage, a cell type unique to vertebrates. It has been variously proposed that the neural crest acquired chondrogenic ability by recruiting proto-chondrogenic gene programs deployed in the neural tube, pharynx, and notochord. To test these hypotheses we examined the expression of 11 amphioxus orthologs of genes involved in neural crest chondrogenesis. Consistent with cellular cartilage as a vertebrate novelty, we find that no single amphioxus tissue co-expresses all or most of these genes. However, most are variously co-expressed in mesodermal derivatives. Our results suggest that neural crest-derived cartilage evolved by serial cooption of genes which functioned primitively in mesoderm.     

Duplication of phospholipase C-delta gene family in fish genomes

Fishes possess more genes than other vertebrates, possibly because of a genome duplication event during the evolution of the teleost (ray-finned) fish lineage. To further explore this idea, we cloned five genes encoding phosphoinositide-specific phospholipase C-delta (PLC-delta), designated respectively PoPLC-deltas, from olive flounder (Paralichthys olivaceus), and we performed phylogenetic analysis and sequence comparison to compare our putative gene products (PoPLC-deltas) with the sequences of known human PLC isoforms. The deduced amino acid sequences shared high sequence identity with human PLC-delta1, -delta3, and -delta4 isozymes and exhibited similar primary structures. In phylogenetic analysis of PoPLC-deltas with PLC-deltas of five teleost fishes (zebrafish, stickleback, medaka, Tetraodon, and Takifugu), three tetrapods (human, chicken, and frog), and two tunicates (sea squirt and pacific sea squirt), whose putative sequences of PLC-delta are available in Ensembl genome browser, the result also indicated that the two paralogous genes corresponding to each PLC-delta isoform originated from fish-specific genome duplication prior to the divergence of teleost fish. Our analyses suggest that an ancestral PLC-delta gene underwent three rounds of genome duplication during the evolution of vertebrates, leading to the six genes of three PLC-delta isoforms in teleost fish.     

Seeing chordate evolution through the <Emphasis Type="Italic">Ciona</Emphasis>genome sequence

A draft sequence of the compact genome of the sea squirt Ciona intestinalis, a non-vertebrate chordate that diverged very early from other chordates, including vertebrates, illuminates how chordates originated and how vertebrate developmental innovations evolved.     

The ectodermal placodes: a dysfunctional family

The ectodermal placodes are focal thickenings of the cranial embryonic ectoderm that contribute extensively to the cranial sensory systems of the vertebrates. The ectodermal placodes have long been thought of as representing a coherent group, which share a developmental and evolutionary history. However, it is now becoming clear that there are substantial differences between the placodes with respect to their early development, their induction and their evolution. Indeed, it is now hard to consider the ectodermal placodes as a single entity. Rather, they fall into a number of distinct classes and it is within each of these that the members share a common development and evolution.     

Impact of gene gains,losses and duplication modes on the origin and diversification of vertebrates

The study of the evolutionary origin of vertebrates has been linked to the study of genome duplications since Susumo Ohno suggested that the successful diversification of vertebrate innovations was facilitated by two rounds of whole-genome duplication (2R-WGD) in the stem vertebrate. Since then, studies on the functional evolution of many genes duplicated in the vertebrate lineage have provided the grounds to support experimentally this link. This article reviews cases of gene duplications derived either from the 2R-WGD or from local gene duplication events in vertebrates, analyzing their impact on the evolution of developmental innovations. We analyze how gene regulatory networks can be rewired by the activity of transposable elements after genome duplications, discuss how different mechanisms of duplication might affect the fate of duplicated genes, and how the loss of gene duplicates might influence the fate of surviving paralogs. We also discuss the evolutionary relationships between gene duplication and alternative splicing, in particular in the vertebrate lineage. Finally, we discuss the role that the 2R-WGD might have played in the evolution of vertebrate developmental gene networks, paying special attention to those related to vertebrate key features such as neural crest cells, placodes, and the complex tripartite brain. In this context, we argue that current evidences points that the 2R-WGD may not be linked to the origin of vertebrate innovations, but to their subsequent diversification in a broad variety of complex structures and functions that facilitated the successful transition from peaceful filter-feeding non-vertebrate ancestors to voracious vertebrate predators.