magp4 gene may contribute to the diversification of cichlid morphs and their speciation.

Lake Victoria harbors more than 300 species of cichlid fish, which are adapted to a variety of ecological niches with various morphological species-specific features. However, it is believed that these species arose explosively within the last 14,000 years and transcripts among Lake Victoria cichlid species are almost identical in sequence. These data prompted us to develop a DNA chip assay to compare patterns of gene expression among cichlid species. We prepared a DNA chip spotted with 6240 elements derived from cichlid expressed sequence tag (EST) clones and successfully characterized gene expression differences between the cichlid species Haplochromis chilotes and Haplochromis sp. "rockkribensis". We identified 14 transcripts that were differentially expressed between these species at an early developmental stage, 15 days post-fertilization (dpf), and several were further analyzed using quantitative real-time PCR (qPCR). One of these differentially expressed transcripts was a homolog of microfibril-associated glycoprotein 4 (magp4), a putative causative gene for the human inherited disease, Smith-Magenis syndrome (SMS), for which facial defects are among the phenotypic features. Further analysis of magp4 expression showed that magp4 was expressed in the jaw portion of cichlid fry and that expression profiles between Haplochromis chilotes and Haplochromis sp. "rockkribensis" differed during development. These data suggest that the differential expression of a gene associated with human cranial morphogenesis may be involved in the diversification of cichlid jaw morphs.

The expression of neural-specific genes reveals the structural and molecular complexity of the planarian central nervous system.

Planarians are attractive animals in which various questions related to the central nervous system (CNS) can be addressed, such as its origin and evolution, its degree of functional conservation among different organisms, and the plasticity and regenerative capabilities of neural cells and networks. However, it is first necessary to characterize at the gene expression level how this CNS is organized in intact animals. Previous studies have shown that the planarian brain can be divided into at least three distinct domains based on the expression of otd/Otx-related genes. In order to further characterize the planarian brain, we have recently isolated a large number of planarian neural-specific genes through DNA microarrays and ESTs projects. Here, we describe new molecular domains within the brain of intact planarians by the expression of 16 planarian neural-specific genes, including the putative homologues of protein tyrosine phosphatase receptor, synaptotagmin VII, slit, G protein and glutamate and acetylcholine receptors, by in situ hybridization in both whole-mount and transverse sections. Our results indicate that planarian otd/Otx-positive domains can be further subdivided into distinct molecular regions according to the expression of different neural genes. We found differences at the gene expression level between the dorsal and ventral sides of the brain, along its antero-posterior axis and also between the proximal and distal parts of the brain lateral branches. This high level of regionalization in the planarian brain contrasts with its apparent simplicity at the morphological level.

cimp1, a novel astacin family metalloproteinase gene from East African cichlids, is differentially expressed between species during growth.

Lake Victoria cichlid fishes are excellent examples of explosive adaptive radiation. Although Lake Victoria cichlids are believed to have arisen during a short period (approximately 14,000 years), they have various species-specific phenotypes. One important phenotype that distinguishes each species is the shape of the jaw, which has diverged to adapt to the wide variety of trophic habitats present in the lake. Here we demonstrate a new approach to investigate the diversification of cichlid jaw morphology at the genetic level by examining differentially expressed genes. We used a DNA chip to compare gene expression levels between closely related cichlid fishes. This analysis indicated that the expression of some genes differed in the larvae of two cichlid species. One such clone encodes a new astacin family metalloproteinase. The expression level of the isolated gene, named cimp1, was analyzed in more detail by real-time quantitative reverse transcription-polymerase chain reaction. A significant difference in cimp1 expression was observed between two Haplochromis cichlid species during development. Using in situ hybridization, we found that this gene is expressed only in head and gill epithelia. Biochemical analysis showed that cichlid metalloproteinase 1 (CiMP1) has proteolytic activity, a common attribute of all astacin family proteins. Because some astacin family proteins contribute to morphogenesis in animals, CiMP1 is expected to participate in species-specific head morphogenesis in cichlids. This is the first study to demonstrate that differentially expressed genes among cichlids can be identified using a DNA chip.

Search for the evolutionary origin of a brain: planarian brain characterized by microarray.

The origin of the brain remains a challenging problem in evolutionary studies. To understand when and how the structural brain emerged, we analyzed the central nervous system (CNS) of a lower invertebrate, planarian. We conducted a large-scale screening of the head part-specific genes in the planarian by constructing a cDNA microarray. Competitive hybridization of cDNAs between a head portion and the other body portion of planarians revealed 205 genes with head part-specific spikes, including essential genes in the vertebrate nervous system. The expression patterns of the top 30 genes showing the strongest spikes implied that the planarian brain has undergone functional regionalization. We demonstrate the complex cytoarchitecture of the planarian brain, despite its simple superficiality of the morphology.

Origin and evolutionary process of the CNS elucidated by comparative genomics analysis of planarian ESTs.

Among the bilateral animals, a centralized nervous system is found in both the deuterostome and protostome. To address the question of whether the CNS was derived from a common ancestor of deuterostomes and protostomes, it is essential to know kinds of genes existed in the CNS of the putative common ancestor and to trace the evolutionary divergence of genes expressed in the CNS. To answer these questions, we took a comparative approach using different species, particularly focusing on one of the lower bilateral animals, the planarian (Platyhelminthes, Tricladida), which is known to possess a CNS. We determined the nucleotide sequence of ESTs from the head portion of planarians, obtaining 3,101 nonredundant EST clones. As a result of homology searches, we found that 116 clones had significant similarity to known genes related to the nervous system. Here, we compared these 116 planarian EST clones with all ORFs of the complete genome sequences of the human, fruit fly, and nematode, and showed that >95% of these 116 nervous system-related genes, including genes involved in brain or neural morphogenesis, were commonly shared among these organisms, thus providing evidence at the molecular level for the existence of a common ancestral CNS. Interestingly, we found that approximately 30% of planarian nervous system-related genes had homologous sequences in Arabidopsis and yeast, which do not possess a nervous system. This implies that the origin of nervous system-related genes greatly predated the emergence of the nervous system, and that these genes might have been recruited toward the nervous system.

Microarray analysis of embryonic retinoic acid target genes in the ascidian Ciona intestinalis.

Many chordate- and vertebrate-specific characteristics develop depending on retinoic acid (RA). Because the gene encoding the RA receptor exists only in chordates, RA function seems to be involved in chordate evolution. A cDNA microarray analysis of 9287 non-redundant cDNA clones was used to screen for RA target genes in the ascidian Ciona intestinalis. In addition, the spatial expression pattern of 94 candidate RA target genes was examined by in situ hybridization in RA-treated and control embryos. Strong RA-induced upregulation of Hox-1 and Cyp26 was observed, as is the case in vertebrates. In addition, a number of novel candidate target genes was identified. These included transcription factors and signaling molecules, suggesting that various differentiation and/or morphogenetic pathways are modulated by RA. The expression of cell adhesion molecules, cytoskeletal proteins and extracellular matrix components was affected by RA. Changes in the expression pattern of these genes may be a direct cause of abnormal morphogenesis of the anterior neural tissues. RA also affected the expression of genes that seemed to be involved in neuronal functions. Although obvious homeotic transformation has not been observed, the function of various neural cell types seemed to be impaired by RA. The microarray data are reliable and will contribute to comprehensive understanding of RA action in the development and evolution of chordates.

Dissecting planarian central nervous system regeneration by the expression of neural-specific genes.

The planarian central nervous system (CNS) can be used as a model for studying neural regeneration in higher organisms. Despite its simple structure, recent studies have shown that the planarian CNS can be divided into several molecular and functional domains defined by the expression of different neural genes. Remarkably, a whole animal, including the molecularly complex CNS, can regenerate from a small piece of the planarian body. In this study, a collection of neural markers has been used to characterize at the molecular level how the planarian CNS is rebuilt. Planarian CNS is composed of an anterior brain and a pair of ventral nerve cords that are distinct and overlapping structures in the head region. During regeneration, 12 neural markers have been classified as early, mid-regeneration and late expression genes depending on when they are upregulated in the regenerative blastema. Interestingly, the results from this study show that the comparison of the expression patterns of different neural genes supports the view that at day one of regeneration, the new brain appears within the blastema, whereas the pre-existing ventral nerve cords remain in the old tissues. Three stages in planarian CNS regeneration are suggested.

Induction of a noggin-like gene by ectopic DV interaction during planarian regeneration.

In previous studies, we have shown that dorsoventral (DV) interaction evokes not only blastema formation, but also morphogenetic events similar to those that occur in regeneration. However, it is still unclear what kinds of signal molecules are involved in the DV interaction. To investigate the signal systems involved in the DV interaction, we focused on a noggin-like gene (Djnlg) identified by the planarian EST project. Djnlg is the first noggin homologue isolated from an invertebrate. In DjNLG, the positions of nine cysteine residues which may be essential for dimer formation were well conserved, but overall, the amino acid sequence of DjNLG did not show high similarity to the sequences of vertebrate Noggins. Expression of Djnlg was observed only in the proximal region of the branch structures in the brain of intact planarians, suggesting that Djnlg may have a role in pattern formation in the brain. Interestingly, transient strong expression of Djnlg was observed in the amputated region of regenerating planarians. Djnlg-expressing cells were detected beneath the muscle 9 h after amputation and were then detected in the ventral subepidermal region of the blastema. The induction of Djnlg expression by amputation was not affected by X-ray irradiation, even though the stem cells were completely eliminated, implying the existence of signal-producing cells which may provide a positional cue to the stem cells. In DV reversed grafting, expression of Djnlg was strongly induced in the DV boundary between the host and donor. These results suggest that ectopic DV interaction may induce expression of Djnlg in the positional cue-producing cells, and that it might be involved in stimulation of blastema formation as well as DV patterning of the body.

FGFR-related gene nou-darake restricts brain tissues to the head region of planarians.

The study of planarian regeneration may help us to understand how we can rebuild organs and tissues after injury, disease or ageing. The robust regenerative abilities of planarians are based upon a population of totipotent stem cells (neoblasts), and among the organs regenerated by these animals is a well-organized central nervous system. In recent years, methodologies such as whole-mount in situ hybridizations and double-stranded RNA have been extended to planarians with the aim of unravelling the molecular basis of their regenerative capacities. Here we report the identification and characterization of nou-darake (ndk), a gene encoding a fibroblast growth factor receptor (FGFR)-like molecule specifically expressed in the head region of the planarian Dugesia japonica. Loss of function of ndk by RNA interference results in the induction of ectopic brain tissues throughout the body. This ectopic brain formation was suppressed by inhibition of two planarian FGFR homologues (FGFR1 and FGFR2). Additionally, ndk inhibits FGF signalling in Xenopus embryos. The data suggest that ndk may modulate FGF signalling in stem cells to restrict brain tissues to the head region of planarians.