A conserved role for FGF signaling in chordate otic/atrial placode formation

The widely held view that neurogenic placodes are vertebrate novelties has been challenged by morphological and molecular data from tunicates suggesting that placodes predate the vertebrate divergence. Here, we examine requirements for the development of the tunicate atrial siphon primordium, thought to share homology with the vertebrate otic placode. In vertebrates, FGF signaling is required for otic placode induction and for later events following placode invagination, including elaboration and patterning of the inner ear. We show that results from perturbation of the FGF pathway in the ascidian Ciona support a similar role for this pathway: inhibition with MEK or Fgfr inhibitor at tailbud stages in Ciona results in a larva which fails to form atrial placodes; inhibition during metamorphosis disrupts development of the atrial siphon and gill slits, structures which form where invaginated atrial siphon ectoderm apposes pharyngeal endoderm. We show that laser ablation of atrial primordium ectoderm also results in a failure to form gill slits in the underlying endoderm. Our data suggest interactions required for formation of the atrial siphon and highlight the role of atrial ectoderm during gill slit morphogenesis.     

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.     

Molecular evidence from ascidians for the evolutionary origin of vertebrate cranial sensory placodes

Cranial sensory placodes are specialised areas of the head ectoderm of vertebrate embryos that contribute to the formation of the cranial sense organs and associated ganglia. Placodes are often considered a vertebrate innovation, and their evolution has been hypothesised as one key adaptation underlying the evolution of active predation by primitive vertebrates. Here, we review recent molecular evidence pertinent to understanding the evolutionary origin of placodes. The development of vertebrate placodes is regulated by numerous genes, including members of the Pax, Six, Eya, Fox, Phox, Neurogenin and Pou gene families. In the sea squirt Ciona intestinalis (a basal chordate and close relative of the vertebrates), orthologues of these genes are deployed in the development of the oral and atrial siphons, structures used for filter feeding by the sessile adult. Our interpretation of these findings is that vertebrate placodes and sea squirt siphon primordia have evolved from the same patches of specialised ectoderm present in the common ancestor of the chordates.     

Retinoic acid-driven Hox1 is required in the epidermis for forming the otic/atrial placodes during ascidian metamorphosis

Retinoic acid (RA)-mediated expression of the homeobox gene Hox1 is a hallmark of the chordate central nervous system (CNS). It has been suggested that the RA-Hox1 network also functions in the epidermal ectoderm of chordates. Here, we show that in the urochordate ascidian Ciona intestinalis, RA-Hox1 in the epidermal ectoderm is necessary for formation of the atrial siphon placode (ASP), a structure homologous to the vertebrate otic placode. Loss of Hox1 function resulted in loss of the ASP, which could be rescued by expressing Hox1 in the epidermis. As previous studies showed that RA directly upregulates Hox1 in the epidermis of Ciona larvae, we also examined the role of RA in ASP formation. We showed that abolishment of RA resulted in loss of the ASP, which could be rescued by forced expression of Hox1 in the epidermis. Our results suggest that RA-Hox1 in the epidermal ectoderm played a key role in the acquisition of the otic placode during chordate evolution.     

The evolutionary history of placodes: a molecular genetic investigation of the larvacean urochordate Oikopleura dioica

The evolutionary origin of vertebrate placodes remains controversial because divergent morphologies in urochordates, cephalochordates and vertebrates make it difficult to recognize organs that are clearly homologous to placode-derived features, including the olfactory organ, adenohypophysis, lens, inner ear, lateral line and cranial ganglia. The larvacean urochordate Oikopleura dioica possesses organs that morphologically resemble the vertebrate olfactory organ and adenohypophysis. We tested the hypothesis that orthologs of these vertebrate placodes exist in a larvacean urochordate by analyzing the developmental expression of larvacean homologs of the placode-marking gene families Eya, Pitx and Six. We conclude that extant chordates inherited olfactory and adenohypophyseal placodes from their last common ancestor, but additional independent proliferation and perhaps loss of placode types probably occurred among the three subphyla of Chordata.     

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 preplacodal region: an ectodermal domain with multipotential progenitors that contribute to sense organs and cranial sensory ganglia

The otic primordium belongs to a group of related structures, the sensory placodes that contribute to the paired sense organs - ear, eye and olfactory epithelium - and to the distal parts of the cranial sensory ganglia. Recent evidence suggests that despite their diversity, all placodes share a common developmental origin and a common molecular mechanism which initiates their formation. At the base of placode induction lies the specification of a unique "placode field", termed the preplacodal region and acquisition of this "preplacodal state" is required for ectodermal cells to undergo otic development. Here I review the molecular mechanisms that sequentially subdivide the ectoderm to give rise to the placode territory.     

Ovule is a lateral organ finally differentiated from the terminating floral meristem in rice

Cranial placodes are specialized ectodermal regions in the developing vertebrate head that give rise to both neural and non-neural cell types of the neuroendocrine system and the sense organs of the visual, olfactory and acoustic systems. The cranial placodes develop from a panplacodal region which is specifically marked by genes of the eyes absent/eya and two “six homeobox” family members (sine oculis/six1 and six4). It had been believed that cranial placodes are evolutionary novelties of vertebrates. However, data from non-vertebrate chordates suggest that placode-like structures evolved in the chordate ancestor already. Here, we identify a morphological structure in the embryonic head of the beetle Tribolium castaneum with placode-like features. It is marked by the orthologs of the panplacodal markers Tc-six4, Tc-eya and Tc-sine oculis/six1 (Tc-six1) and expresses several genes known to be involved in adenohypophyseal placode development in vertebrates. Moreover, it contributes to both epidermal and neural tissues. We identify Tc-six4 as a specific marker for this structure that we term the insect head placode. Finally, we reveal the regulatory gene network of the panplacodal genes Tc-six4, Tc-eya and Tc-six1 and identify them as head epidermis patterning genes. Our finding of a placode-like structure in an insect suggests that a placode precursor was already present in the last common ancestor of bilaterian animals.     

Epibranchial and otic placodes are induced by a common Fgf signal, but their subsequent development is independent

The epibranchial placodes are cranial, ectodermal thickenings that give rise to sensory neurons of the peripheral nervous system. Despite their importance in the developing animal, the signals responsible for their induction remain unknown. Using the placodal marker, sox3, we have shown that the same Fgf signaling required for otic vesicle development is required for the development of the epibranchial placodes. Loss of both Fgf3 and Fgf8 is sufficient to block placode development. We further show that epibranchial sox3 expression is unaffected in mutants in which no otic placode forms, where dlx3b and dlx4b are knocked down, or deleted along with sox9a. However, the forkhead factor, Foxi1, is required for both otic and epibranchial placode development. Thus, both the otic and epibranchial placodes form in a common region of ectoderm under the influence of Fgfs, but these two structures subsequently develop independently. Although previous studies have investigated the signals that trigger neurogenesis from the epibranchial placodes, this represents the first demonstration of the signaling events that underlie the formation of the placodes themselves, and therefore, the process that determines which ectodermal cells will adopt a neural fate.     

Neurogenic and non-neurogenic placodes in ascidians

The late differentiation of the ectodermal layer is analysed in the ascidians Ciona intestinalis and Botryllus schlosseri, by means of light and electron microscopy, in order to verify the possible presence of placodal structures. Cranial placodes, ectodermal regions giving rise to nonepidermal cell types, are classically found exclusively in vertebrates; however, data are accumulating to demonstrate that the nonvertebrate chordates possess both the genetic machinery involved in placode differentiation, and ectodermal structures that are possible homologues of vertebrate placodes. Here, the term "placode" is used in a broad sense and defines thickenings of the ectodermal layer that can exhibit an interruption of the basal lamina where cells delaminate, and so are able to acquire a nonepidermal fate. A number of neurogenic placodes, ones capable of producing neurons, have been recognised; their derivatives have been analysed and their possible homologies with vertebrate placodes are discussed. In particular, the stomodeal placode may be considered a multiple placode, being composed of different sorts of placodes: part of it, which differentiates hair cells, is discussed as homologous to the octavo-lateralis placodes, while the remaining portion, giving rise to the ciliated duct of the neural gland, is considered homologous to the adenohypophyseal placode. The neurohypophyseal placode may include the homologues of the hypothalamus and vertebrate olfactory placode; the rostral placode, producing the sensorial papillae, may possibly be homologous to the placodes of the adhesive gland of vertebrates.     

Xenopus Eya1 demarcates all neurogenic placodes as well as migrating hypaxial muscle precursors

We cloned two isoforms of the Xenopus Eya1 orthologue. They show identical patterns of expression that closely resemble the previously described expression of XSix1, but partly differ from the expression of Eya1 in other vertebrates. XEya1 is expressed in the somites and hypaxial muscle precursors, but not in the pronephros. Moreover, all ectodermal placodes except the lens placode strongly express XEya1. At neural plate stages, ectodermal XEya1 expression starts in two domains, the anterior neural folds and a domain lateral to the neural folds. At tailbud stages, XEya1 expression continues in the adenohypophysis, all neurogenic placodes and placodally-derived structures including cranial ganglia, the otic vesicle and lateral line primordia.     

Differential requirements for FGF3, FGF8 and FGF10 during inner ear development

FGF signaling is required during multiple stages of inner ear development in many different vertebrates, where it is involved in induction of the otic placode, in formation and morphogenesis of the otic vesicle as well as for cellular differentiation within the sensory epithelia. In this study we have looked to define the redundant and conserved roles of FGF3, FGF8 and FGF10 during the development of the murine and avian inner ear. In the mouse, hindbrain-derived FGF10 ectopically induces FGF8 and rescues otic vesicle formation in Fgf3 and Fgf10 homozygous double mutants. Conditional inactivation of Fgf8 after induction of the placode does not interfere with otic vesicle formation and morphogenesis but affects cellular differentiation in the inner ear. In contrast, inactivation of Fgf8 during induction of the placode in a homozygous Fgf3 null background leads to a reduced size otic vesicle or the complete absence of otic tissue. This latter phenotype is more severe than the one observed in mutants carrying null mutations for both Fgf3 and Fgf10 that develop microvesicles. However, FGF3 and FGF10 are redundantly required for morphogenesis of the otic vesicle and the formation of semicircular ducts. In the chicken embryo, misexpression of Fgf3 in the hindbrain induces ectopic otic vesicles in vivo. On the other hand, Fgf3 expression in the hindbrain or pharyngeal endoderm is required for formation of the otic vesicle from the otic placode. Together these results provide important insights into how the spatial and temporal expression of various FGFs controls different steps of inner ear formation during vertebrate development.