Hee-Chan Seo

Hox cluster disintegration with persistent anteroposterior order of expression in Oikopleura dioica.

Tunicate embryos and larvae have small cell numbers and simple anatomical features in comparison with other chordates, including vertebrates. Although they branch near the base of chordate phylogenetic trees, their degree of divergence from the common chordate ancestor remains difficult to evaluate. Here we show that the tunicate Oikopleura dioica has a complement of nine Hox genes in which all central genes are lacking but a full vertebrate-like set of posterior genes is present. In contrast to all bilaterians studied so far, Hox genes are not clustered in the Oikopleura genome. Their expression occurs mostly in the tail, with some tissue preference, and a strong partition of expression domains in the nerve cord, in the notochord and in the muscle. In each tissue of the tail, the anteroposterior order of Hox gene expression evokes spatial collinearity, with several alterations. We propose a relationship between the Hox cluster breakdown, the separation of Hox expression domains, and a transition to a determinative mode of development.

Expression of two zebrafish homologues of the murine Six3 gene demarcates the initial eye primordia.

The murine homeobox gene Six3 and its Drosophila homologue sine oculis both have regulatory functions in eye development. We report the isolation and characterization of two zebrafish genes, six3 and six6, that are closely related to the murine Six3 gene. Zebrafish six3 may be the structural orthologue, while the six6 gene is more similar with respect to embryonic expression. Transcripts of both zebrafish six genes are first detected in involuting axial mesendoderm and, subsequently, in the overlying anterior neural plate from which the optic vesicles and the forebrain will develop. Direct correspondence between six3/six6 expression boundaries and the optic vesicles indicate essential roles in defining the eye primordia. During later stages only the six6 gene displays similar features of expression in the eyes and rostral brain as reported previously for murine Six3.

Six class homeobox genes in Drosophila belong to three distinct families and are involved in head development

The vertebrate Six genes are homologues of the Drosophila homeobox gene sine oculis (so), which is essential for development of the entire visual system. Here we describe two new Six genes in Drosophila, D-Six3 and D-Six4, which encode proteins with strongest similarity to vertebrate Six3 and Six4, respectively. In addition, we report the partial sequences of 12 Six gene homologues from several lower vertebrates and show that the class of Six proteins can be subdivided into three major families, each including one Drosophila member. Similar to so, both D-Six3 and D-Six4 are initially expressed at the blastoderm stage in narrow regions of the prospective head and during later stages in specific groups of head midline neurectodermal cells. D-Six3 may also be essential for development of the clypeolabrum and several head sensory organs. Thus, the major function of the ancestral Six gene probably involved specification of neural structures in the cephalic region.

The zebrafish Pax3 and Pax7 homologues are highly conserved, encode multiple isoforms and show dynamic segment-like expression in the developing brain.

This study describes the isolation and characterization of zebrafish homologues of the mammalian Pax3 and Pax7 genes. The proteins encoded by both zebrafish genes are highly conserved (>83%) relative to the known mammalian sequences. Also the neural expression patterns during embryogenesis are very similar to the murine homologues. However, observed differences in neural crest and mesodermal expression relative to mammals could reflect some functional divergence in the development of these tissues. For the zebrafish Pax7 protein we report the first full-length amino acid sequences in vertebrates and show the existence of three additional isoforms which have truncations in the homeodomain and/or the C-terminal region. These novel variants provide evidence for additional isoform diversity of vertebrate Pax proteins.

Molecular cloning and embryonic expression of Xenopus Six homeobox genes.

Six genes are vertebrate homologues of the homeobox-containing gene sine oculis, which plays an essential role in controlling Drosophila compound eye development. Here we report the identification and expression patterns of all three subfamilies of Xenopus Six genes. Two Six2 subfamily genes (Six1, Six2) showed very similar expression patterns in cranial ganglia, otic placodes and the eyes. Non-neural expression of Six1 and Six2 was observed with mesodermal head mesenchyme, somites and their derivatives, the muscle anlagen of the embryonic trunk. In addition, Six2 expression was also found with mesenchyme associated with the developing stomach and pronephros. Expression of Six3 subfamily genes (Six3.1, Six3.2, Six6.1, and Six6.2) was restricted to the developing head, where expression was especially observed in derivatives of the forebrain (eyes, optic stalks, the hypothalamus and pituitary gland). Interestingly, expression of all Six3 subfamily members but Six6.2 was also found with the pineal gland primordium and the tegmentum. Expression of Six4 subfamily genes (Six4.1, Six4.2) was present in the developing visceral arches, placodal derivatives (otic vesicle, olfactory system), head mesenchyme and the eye. The observed dynamic expression patterns are largely conserved between lower and higher vertebrates and imply important roles of Six family genes not only in eye formation and myogenesis, but also in the development of the gut, the kidney and of placode-derived structures.

RNA interference: mechanisms and applications.

RNA interference (RNAi) is a phenomenon induced by double-stranded RNA (dsRNA) in which gene expression is inhibited through specific degradation of mRNA. The mechanism involves conversion of dsRNA into short RNAs that direct ribonucleases to homologous mRNA targets. This process is related to normal defence against viruses and mobilisation of transposons. Treatment with dsRNA has become an important method for analysing gene functions in invertebrate organisms. RNAi has also been demonstrated in several vertebrate species but with lower efficiency. Development of procedures for in vivo production of dsRNA may provide efficient tools for tissue- and stage-specific gene targeting.

Isolation and characterization of two teleost melanopsin genes and their differential expression within the inner retina and brain

Melanopsin is a newly discovered photopigment that is believed to be involved in the regulation of circadian rhythms in tetrapods. Here we describe the characterization of the first two teleost melanopsins (opn4a and opn4b) isolated from Atlantic cod (Gadus morhua). These two teleost genes belong to a subgroup of melanopsins that also include members from Xenopus, chicken, and Takifugu. In situ hybridization revealed that opn4a and opn4b are differentially expressed within the retina and brain. In the larval and adult retina, both melanopsins are expressed in a subset of cells in the inner retina, resembling amacrine and ganglion cells. In addition, opn4a is expressed in the horizontal cells, indicating a separate task for this gene. In the brain, the two melanopsins are separately expressed in two major retinal and extraretinal photosensitive integration centers, namely, the suprachiasmatic nucleus (opn4a) and the habenula (opn4b). The expression of opn4a in the suprachiasmatic nucleus in cod is similar to the melanopsin expression found in Xenopus. This suggests a conserved role for this opsin and an involvement in mediation of nonvisual photoreceptive tasks, such as entraining circadian rhythms and/or hypophysiotrophic systems. The differential expression of opn4b in the habenula suggests that this gene plays a role similar to that of opn4a, in that it is also situated in an area that integrates photic inputs from the pineal as well as other brain regions. Thus, the habenula may be an additional region that mediates photic cues in teleosts. J. Comp. Neurol. 456:84–93, 2003.

Differential Evolution of the 13 Atlantic Salmon Hox Clusters

Hox cluster organization represents a valuable marker to study the effects of recent genome duplication in salmonid fish (25-100 Mya). Using polymerase chain reaction amplification of cDNAs, BAC library screening, and genome walking, we reconstructed 13 Hox clusters in the Atlantic salmon containing 118 Hox genes including 8 pseudogenes. Hox paralogs resulting from the genome duplication preceding the radiation of ray-finned fish have been much better preserved in salmon than in other model teleosts. The last genome duplication in the salmon lineage has been followed by the loss of 1 of the 4 HoxA clusters. Four rounds of genome duplication after the vertebrate ancestor salmon Hox clusters display the main organizational features of vertebrate Hox clusters, with Hox genes exclusively that are densely packed in the same orientation. Recently, duplicated Hox clusters have engaged a process of divergence, with several cases of pseudogenization or asymmetrical evolution of Hox gene duplicates, and a marked erosion of identity in noncoding sequences. Strikingly, the level of divergence attained strongly depends on the Hox cluster pairs rather than on the Hox genes within each cluster. It is particularly high between both HoxBb clusters and both HoxDa clusters, whereas both HoxBa clusters remained virtually identical. Positive selection on the Hox protein-coding sequences could not be detected.

Remodelling of the homeobox gene complement in the tunicate Oikopleura dioica

Homeodomain transcription factors are involved in many developmental processes and have been intensely studied in a few model organisms, such as mouse, Drosophila and Caenorhabditis elegans. Homeobox genes fall into 10 classes (ANTP, PRD, POU, LIM, TALE, SIX, Cut, ZFH, HNF1, Prox) and 89 different families/groups, all of which are present in vertebrates. Additional groups may be uncovered by further genome annotation, particularly of complex vertebrate genomes. Eight of these groups have been found only in vertebrates, but not in the genome of the tunicate Ciona intestinalis. The other 81 groups of homeobox gene that have been detected in vertebrates so far probably appeared during the early evolution of bilaterians or earlier, as they are also present outside the chordates. How the homeobox genes evolved during and after the main radiation of the bilaterians remains poorly understood, as only a few animal genomes have been sequenced completely. However, drastic changes have occurred at least in the lineage of C. elegans , such as loss of several Hox genes and Hox cluster fragmentation . Here we report considerable alterations of the homeobox gene complement in the tunicate lineage.

Identification and distribution of nitric oxide synthase in the brain of adult zebrafish.

Nitric oxide (NO) is proposed to be involved in developmental and plastic processes. We investigated the presence and distribution of nitric oxide synthase (NOS) in the zebrafish (Danio rerio) using molecular and histochemical techniques. A partial gene sequence corresponding to the neuronal NOS isoform (nNOS) was identified, and in situ hybridization revealed cellular nNOS mRNA expression throughout the brain of adult zebrafish, distributed in distinct central nuclei and in proliferation zones. NOS immunoreactivity and nicotinamide adenine dinucleotide phosphate diaphorase activity partly coincided with the nNOS mRNA expression, however was present also in additional neuronal and non-neuronal cell types. The results indicate the occurrence of different NOS isoforms in the adult brain, of which nNOS may participate in neurotransmission, and in mechanisms related to the continuous growth and neuronal plasticity of the teleost brain.