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.

The conservation of developmental functions exerted by Antpclass homeoproteins in protostomes and deuterostomes suggested that homologs with related functions are present in diploblastic animals. Our phylogenetic analyses showed that Antp-class homeodomains belong either to non-Hox or to Hox͞paraHox families. Among the 13 non-Hox families, 9 have diploblastic homologs, Msx, Emx, Barx, Evx, Tlx, NK-2, and Prh͞Hex, Not, and Dlx, reported here. Among the Hox͞paraHox, poriferan sequences were not found, and the cnidarian sequences formed at least five distinct cnox families. Two are significantly related to the paraHox Gsx (cnox-2) and the mox (cnox-5) sequences, whereas three display some relatedness to the Hox paralog groups 1 (cnox-1), 9͞10 (cnox-3) and the paraHox cdx (cnox-4). Intermediate Hox͞paraHox genes (PG 3 to 8 and lox) did not have clear cnidarian counterparts. In Hydra, cnox-1, cnox-2, and cnox-3 were not found chromosomally linked within a 150-kb range and displayed specific expression patterns in the adult head. During regeneration, cnox-1 was expressed as an early gene whatever the polarity, whereas cnox-2 was up-regulated later during head but not foot regeneration. Finally, cnox-3 expression was reestablished in the adult head once it was fully formed. These results suggest that the Hydra genes related to anterior Hox͞paraHox genes are involved at different stages of apical differentiation. However, the positional information defining the oral͞aboral axis in Hydra cannot be correlated strictly to that characterizing the anterior-posterior axis in vertebrates or arthropods. T he discovery of structural and functional homologies between regulatory genes used by Drosophila and vertebrates during their development led to the hypothesis that animals would share a common set of genes for defining the head, trunk, and posterior regions at early developmental stages (1-6). The proposed genes were homeobox genes belonging either to the Antp class, like empty-spiracle (emx), even-skipped (evx), Hox genes, or to the Prd class, like orthodenticle (Otx), goosecoid. Phylogenetic analyses performed on a vast amount of Hox homeodomain (HD) sequences, including representatives from all classes of homeobox genes from animals, protozoa, fungi, and plants, confirmed the monophyly of the Antp class as well as its position as a sister group to the Paired class (7). Within the Antp class, the Hox gene organization is distinctive and enigmatic: the genes map in clusters, and the order of individual genes within a cluster correlates with their temporospatial expression pattern along the anterior-posterior body axis during development (8). Recently, it was proposed that the common bilaterian ancestor of protostomes and deuterostomes had at least seven Hox genes (9). However, the question of the composition of the ancestral HOX cluster remains open. Analysis of Hox homeobox sequences (10) suggested that the conserved HOX cluster emerged early in the evolution of metazoans from an original cluster harboring three ancestral g...

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A survey against the draft genome sequence and the cDNA/EST database of Ciona intestinalis identified a number of genes encoding transcription factors regulating a variety of processes including development. In the present study, we describe almost complete sets of genes for Fox, ETS-domain transcription factors, nuclear receptors, and NFkappaB as well as other factors regulating NFkappaB activity, with their phylogenetic nature. Vertebrate Fox transcription factors are currently delineated into 17 subfamilies: FoxA to FoxQ. The present survey yielded 29 genes of this family in the Ciona genome, 24 of which were Ciona orthologues of known Fox genes. In addition, we found 15 ETS genes, 17 nuclear receptor genes, and several NFkappaB signaling pathway genes in the Ciona genome. The number of Ciona genes in each family is much smaller than that of vertebrates, which represents a simplified feature of the ascidian genome. For example, humans have two NFkappaB genes, three Rel genes, and five NFAT genes, while Ciona has one gene for each family. The Ciona genome also contains smaller numbers of genes for the NFkappaB regulatory system, i.e. after the split of ascidians/vertebrates, vertebrates evolved a more complex NFkappaB system. The present results therefore provide molecular information for the investigation of complex developmental processes, and an insight into chordate evolution.

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.

Gap junctional conductance is regulated by the number of channels between coupled cells (the balance between formation and loss of these channels) and by the fraction of these channels that are open (gating mechanisms). A variety of treatments are known to affect junction formation. Adenosine 3',5'-cyclic monophosphate (cAMP) is involved in some cases, and protein synthesis may be required but precursor molecules can also exist. Junction removal occurs both by dispersion of particles and by internalization of junctional membrane. Factors promoting removal are not well understood. A variety of gating mechanisms exist. Coupling may be controlled by changes in conductance of nonjunctional membranes. Several kinds of voltage dependence of junctional conductance are known, but rat ventricular junctions at least are electrically linear. Cytoplasmic acidification decreases conductance of most gap junctions. Sensitivity in rat ventricular myocytes allows modulation of coupling by moderate changes near normal internal pH. Increasing intracellular Ca also decreases junctional conductance, but in the better studied cases sensitivity is much lower to Ca than H. A few data support low sensitivity to Ca in cardiac cells, but quantitative studies are lacking. Higher alcohols such as octanol block junctional conductance in a wide range of tissues including rat ventricular myocytes. An antibody to liver gap junctions blocks junctions between rat ventricular myocytes. Cross reactivity indicates at least partial homology between many gap junctions. Although differences among gap junctions are known, a general physiology is being developed, which may have considerable relevance to normal cardiac function and also to conduction disorders of that tissue.

SUMMARY. Freeze fracture and thin section techniques have revealed morphological changes in gap junctions and intercalated discs of adult rat myocytes following enzymatic dissociation. Cell separation leaves behind small vesicular remnants of formerly adjacent cells connected to the intact cell by gap junctions; in contrast, desmosomes cleave at the region of intercellular contact. Apparently, the next step in gap junction breakdown is intemalization of the remnants. In thin section, lanthanum penetration reveals that the cleft of some apparently internalized gap junctions is in contact with the sarcolemma, while that of others is not. In freeze fracture replicas, cytoplasmic gap junctions frequently possess hexagonally packed domains of E-face pits separated by smooth regions that may correspond to separations of membranes of internalized junctions found in thin section. Study of material maintained overnight at 37°C showed no surface junctional remnants; topologies of cytoplasmic gap junctions were generally complex, and concentric membrane vesicles were common. These observations suggest that enzymatic dissociation initiates a progressive, defined sequence of junctional intemalization that begins with attached cell remnants and may have as the last determinable step the separation into single membranes inside the intact cell. (CircRes 56:195-204, 1985)

The endostyle of invertebrate chordates is a pharyngeal organ that is thought to be homologous with the follicular thyroid of vertebrates. Although thyroid-like features such as iodine-concentrating and peroxidase activities are located in the dorsolateral part of both ascidian and amphioxus endostyles, the structural organization and numbers of functional units are different. To estimate phylogenetic relationships of each functional zone with special reference to the evolution of the thyroid, we have investigated, in ascidian and amphioxus, the expression patterns of thyroid-related transcription factors such as TTF-2/FoxE4 and Pax2/5/8, as well as the forkhead transcription factors FoxQ1 and FoxA. Comparative gene expression analyses depicted an overall similarity between ascidians and amphioxus endostyles, while differences in expression patterns of these genes might be specifically related to the addition or elimination of a pair of glandular zones. Expressions of Ci-FoxE and BbFoxE4 suggest that the ancestral FoxE class might have been recruited for the formation of thyroid-like region in a possible common ancestor of chordates. Furthermore, coexpression of FoxE4, Pax2/5/8, and TPO in the dorsolateral part of both ascidian and amphioxus endostyles suggests that genetic basis of the thyroid function was already in place before the vertebrate lineage.