* Center for Information Biology and DNA Data Bank of Japan, National Institute of Genetics, Mishima, Japan
RIKEN, Center for Developmental Biology, Kobe, Japan
Department of Biology, Faculty of Science, Okayama University, Okayama, Japan
Department of Cell and Structural Biology, University of Illinois, Urbana-Champaign
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: central nervous system origin of brain planarian microarray
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We chose a freshwater flatworm, planarian (Platyhelminthes; Dugesia japonica) because this organism shows "cephalization" that has been considered as one of the simplest brains (Keenan, Coss, and Koopowitz 1981). Although the exact phylogenetic position of Platyhelminthes is still under debate, their simple body plan mirror biological features of the groups emerged at the base of Bilateria (Valentine 1994). The planarian CNS is composed of two morphologically distinct structures, the ventral nerve cords (VNCs) and the cephalic ganglion. The latter forms an independent bilobed inverted U-shaped structure located dorsally to the VNCs (Agata et al. 1998). Interestingly, the planarian cephalic ganglion exhibits many morphological features characteristic of the vertebrate CNS, such as multipolar nerve cells and knoblike protrusions along dendrites resembling dendritic spines (Sarnat and Netsky 1985). Three genes homologous to otx are specifically expressed in the planarian cephalic ganglion (Agata et al. 1998; Umesono, Watanabe, and Agata 1997, 1999). These facts categorize the planarian cephalic ganglion as a brain. Thus, it is of particular interest to know whether the contemporary planarian maintains features of an evolutionarily primitive brain that were derived from the common ancestor of the Bilateria.
We identified genes specifically expressed in a planarian brain utilizing a planarian cDNA microarray. Microarrays are powerful tools to examine the expression profile of multiple genes simultaneously, leading to prediction of gene functions and relationships (Duggan et al. 1999). In our EST project, we have sequenced more than 9,000 redundant clones derived from a planarian cDNA library. We randomly selected 1,640 nonredundant genes from these sequenced clones, the upper limits of our spotting capability. To create the cDNA microarray, we overcame several difficulties such as ambiguous reproducibility, inefficient hybridization, and unclearness of quantitative spike criteria by establishing our own experimentation.
We then conducted competitive hybridization experiments of cDNAs between a head portion and the other body portion of planarians in order to screen genes specifically expressed in a planarian brain. In this study, we show extensive evidence that a planarian brain has complex cytoarchitecture characterized by gene expression profiles, implying that the functional regionalization took place in the planarian brain in spite of superficial simplicity of morphology.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Probe Preparation
After homology search in our planarian EST project (Mineta et al. in preparation), 1,640 nonredundant clones from planarian ESTs were spotted. We amplified the clones by PCR in a 100-µl system using vector-specific, amino-linked primers in a 96-well format. After checking by electrophoresis, PCR products were precipitated by centrifugation (4,000 rpm for 1 h with TS-38LB rotor [TOMY]) after incubation with sodium acetate and 2-propanol at -20°C overnight. The luciferase gene (pGL2-Basic Vector [Promega]) was used as an external control.
Fabrication of cDNA Microarray
Microscopic slides coated with poly-L lysine were used as the base of the DNA chip (Matsunami). PCR amplified samples were solved in spotting buffer (DMSO: Polymer [FujiFilm]: TE = 2:1:1). After denaturation at 95°C for 3 min, the samples were spotted onto slide glasses with 32 solid pins at the pitch of 0.5 mm by an SPBIO 2000 arrayer (Hitachi Software Engineering). Spotted glasses were burnt at 80°C for 1 h in an oven, followed by exposure to 60 mJ of UV to immobilize the DNAs. We blocked glass surfaces with 175 mM succinic anhydride in 1-methyl-2-pyrrolidinone and 44 mM sodium borate (pH 8.0).
Target Preparation
As the brain occupies the anterior part of the planarian, we designed experiments to screen brain-related genes as shown in figure 1A. We extracted poly-A tailed messenger RNA (mRNA) separately from the planarian head portion and the body portions. Planarians were starved for at least 1 week before use. Approximately 600 worms were divided into two parts as shown in figure 1A and collected for one experiment. Total RNAs derived from each part were extracted using Isogene (Nippon Gene), and mRNAs were then isolated using OligoTex-dT30 <Super> (Takara).
|
Hybridization and Scanning
Fluoro-labeled targets were adjusted to 5 x SSC and denatured at 95°C for 2 min. After the addition of SDS to a 0.25% final concentration, the target was placed on the microarray under a glass cover slip measuring 22 mm x 45 mm. Hybridization was performed in a humid chamber at 65°C for 24 to 48 h. The slide was then washed in 2 x SSC containing 0.1% SDS, followed by four incubations in 0.2 x SSC and 0.1% SDS at room temperature (RT), at 40°C, and two additional times at RT. The slide was immediately scanned by ScanArray 4000 Version 1.2 software (GSI Lumonics). Analyses were performed using QuantArray Version 1.0.0.0 software (GSI Lumonics).
Whole Mount in Situ Hybridization
For whole mount in situ hybridization, we utilized planarians starved for 1 week. Worms were treated with 2% HCl for 5 min at 4°C and then fixed in Carnoy's solution (ethanol: chloroform: acetic acid in proportions 6:3:1, respectively) for 2 h at 4°C. Hybridization was performed using 20 ng/ml of DIG-labeled riboprobes, as previously described (Umesono, Watanabe, and Agata 1997; Agata et al. 1998).
Analyses of the Data from cDNA Chip Experiments
After the addition of the control samples and the duplicated spots, the planarian cDNA chip comprised a total of 1,728 spots. We used 1,689 spots in the following analysis after excluding those spots with bad forms. The fluorescent intensities of each dye were separately scanned. We calculated the ratio of fluorescent intensities between two dyes on each spot (H/B ratio) according to the following equation: H/B ratio = (fluorescent intensity of a head-part cDNA pool)/(fluorescent intensity of a body-part cDNA pool).
We conducted five independent experiments. As the hybridization efficiency differs among experiments, each H/B ratio was normalized to the median of the data set of each experiment. The average of H/B ratio among five experiments was calculated for each spot. Finally, all the spots were ordered according to the averaged H/B ratio from the highest value to the lowest value.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We calculated an H/B ratio for each spot. An H/B value actually reflects the relative abundance of the spotted gene within the head portion to the abundance of the spotted gene within the other body portion. The histogram of H/B ratios (fig. 1B) demonstrates a tremendous difference between gene groupings, with larger and smaller H/B ratios at approximately 1.45. Thus, we designated an H/B ratio of more than 1.45 as the criterion for head partspecific genes. We then found that 205 genes showed H/B values higher than this criterion.
The 205 genes are separated into four categories as a result of homology search (table 1). Eighty-two genes are homologous to functionally known genes (designated as "Known" in table 1). In particular, 33 genes are homologous to the neural-related genes in other organisms, such as synapsin, synaptotagmin, prohormone convertase 2, and nicotinic acetylcholine receptor. These genes are functionally essential and well conserved throughout CNS of higher organisms. Because of the high similarity at the amino acid level, these homologous genes in the planarian should be basic components in planarian CNS as well as in other organisms. The remainder of the 123 genes, categorized into three additional groups, are functionally unknown (designated as "No match," "Unknown," and "Hypothetical" in table 1). Therefore, we uncovered four times as many unknown head partspecific genes through cDNA microarray analysis as known neural-related genes. The planarian brain contains a large number of functionally unknown genes that are currently beyond our fundamental understanding of the basic neural genes.
|
|
|
Functional Regionalization in Planarian Brain Inferred from the Expression Patterns of Head PartSpecific Genes
We then performed whole mount in situ hybridization for the head partspecific genes, regardless of whether they were known or unknown, in order to know the molecular features of the planarian brain. We detail the top 30 genes having the highest H/B values from the total 205 candidates. We observed clearly distinct expression patterns that are separated into seven categories, designated types A to G (fig. 3).
Type A genes are expressed throughout the CNS. These genes are likely to have basic and fundamental functions in the nervous system of most organisms. Most of the genes in type A show sequence similarities to neural specific genes in other organisms, including 0944_HH/ NCAM (21st), 4307_HH/nicotinic acetylcholine receptor (23rd), and 2814_HH/synapsin (26th). The planarian ESTs 0180_E (10th) and 6698_HH (25th) are also contained within this group, although they are functionally unknown. These genes are indispensable in keeping the general functions of the CNS, as they are ubiquitously expressed.
Compared with type A genes, type B and C genes exhibit the expression patterns in specific regions of the cephalic ganglion. Type B genes are expressed particularly in the leaflike regions on both sides in the bilobed cephalic ganglion. The planarian cephalic ganglion actually contains two leaflike structures connected by a commissure, where neuronal cell bodies accumulate. Type B genes may have a role in processing and transporting the biological signals to the body, as this region is connected to VNCs. One of the type B genes, EST 0107_E (24th), which is an unknown gene, has high sequence similarity with human EST clones, KIAA0513 (E-value: 7.00e-8), derived from human brain (Nagase et al. 1998). This should be a novel brain-related gene conserved from the planarian to the human.
Contrary to type B genes, type C gene expressions are restricted to the branch regions in the cephalic ganglion. These genes are likely to be involved in signal transduction. For example, 1791_HH (11th) homologous to the G-protein alpha subunit (E-value: 5.00e-32) is mainly expressed in the distal part of each branch with reduced levels in other portions. Due to the sequence similarity with G-protein, relating to the signal transduction throughout the species (Simon, Strathmann, and Gautam 1991), 1791_HH may mediate the transduction of signals received in sensory cells locating on the head periphery.
These differing expression patterns imply that functional regionalization occurs within the planarian CNS. Since the branch and leaflike regions are morphologically distinguished, these structurally different regions should possess specialized functions.
The expression patterns of the type E genes, however, demonstrate that the regionalization occurs even inside the leaflike structure, which appears to be morphologically homogenous. EST 1020_HH (8th), 0053_E (18th), and 0639_E (29th) in the type E genes are functionally unknown genes. These expression patterns are characterized by the following three points. First, they show gradient expression patterns within the leaflike region. The posterior portion of the brain stained more strongly than the anterior side, and the interior of the two leaves stained with a greater intensity than the exterior. This gradient pattern suggests that the different areas within the leaflike structure possess different functions, although they appear morphologically homogeneous. Second, 0053_E and 0639_HH are expressed around the eyes, as observed with the type D 0251_HH/beta arrestin homologous gene. Moreover, 0053_E is expressed at the tip of the head. These stained cells may be a kind of a photoreceptor or mechanoreceptor, judging from the cell location. Third, 1020_HH and 0053_E are expressed in dispersed cells along the VNCs. These cells are closely associated to VNCs and could have some kind of supporting role for neurons from the nerve cords.
Type E expression patterns indicate the regionalization in the planarian cephalic ganglion, similar to that observed in the human brain. In fact, it is reported that each branch mediates distinct functions. For example, two eyes are located on the dorsal side of the third branch, with the axon of each eye extending towards the third branch. The sixth to ninth branches extend to the surface of the head region, forming auricles that make a putative sensory organ of taste (Umesono, Watanabe, and Agata 1999). These different sensory neurons should innervate to the specific region of the brain through specific branches, indicating that different signals are processed in distinct areas of the cephalic ganglion. Judging from the expression patterns of type E genes, photosignals may be processed in the posterior/interior region in the planarian cephalic ganglion.
The genes categorized in type F show particular patterns in which expression signals appear not only in cephalic ganglion but also in the entire head region. Based on the results of RNA interference experiments of these genes that are expressed in the anterior side of the planarian body, we presume that they are strongly related to cephalization in planarian (Cebrià et al. 2002).
The last group, type G genes exhibit specific expressions in sensory cells. ESTs 1681_HH (13th) and 0821_HN (14th) are expressed on the lateral ends of the brain branches where sensory organs such as the auricles are located (Umesono, Watanabe, and Agata 1999). 1681_HH, similar to the PKD2 gene that confers autosomal dominant polycystic kidney disease in Homo sapiens (Pennekamp et al. 1998) (E-value: 2e-11), is expressed in two distinct curved regions around the brain. Another gene, unknown 0821_HN, is expressed in a single and inside curved region along periphery of the head. The stained cells by these genes are located in the extended region to the tips of the brain branches, suggesting these cells are related to the CNS.
Collectively, the variety of the expression patterns of the top 30 head partspecific genes demonstrates the highly organized planarian CNS. We designate these 30 genes as the planarian brain-specific genes. Their expressions indicate that planarian CNS is functionally regionalized.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We observed functional regionalization in the planarian CNS. Previous studies suggested that the planarian brain is divided into four regions according to otx homologous gene expressions. The first region characterized by DjotxA shows expression in photoreceptor cells. DjotxB is expressed in the intermediate regions connecting to the VNCs. Djotp is expressed in the brain branches (Umesono, Watanabe, and Agata 1997, 1999). The fourth region is lateral to branches where otx/otp is undetectable. Moreover, we identified at least seven different expression patterns in the planarian CNS, indicating complex cytoarchitecture in the planarian brain as shown in figure 4. The functional regionalization of the planarian brain allows the performance of complex processes, as observed in higher organisms. These data suggest that the planarian brain can be utilized as a model of the CNS in higher organisms.
|
We have discovered brain-specific genes in the planarian, including the genes with unknown functions. It may be because orthologous genes have diverged to a great extent among the species. The possibility remains, however, that these are novel genes not yet identified in other species. Further analysis of these planarian genes may reveal novel brain-specific genes in mammals, as in the case of planarian EST 0107_E, which is homologous to human brain EST. We suggest that evolutionary studies play an important role as new gene finder for higher organisms. Moreover, we would conduct the functional analyses of the candidates obtained as planarian brain-specific genes using knockout methods such as planarian RNA interference (Sánchez Alvarado and Newmark 1999). Competitive hybridization of the expressed genes between knocked out and intact worms on a cDNA chip may reveal the transcriptional pathways of brain-specific genes in the planarian, giving hints for the analyses of genetic networks in mammalian CNS. We suggested that the evolutionary study of brains give profound insights into the understanding of the complex nervous systems in higher organisms such as mammals.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Agata, K., Y. Soejima, K. Kato, C. Kobayashi, Y. Umesono, and K. Watanabe. 1998. Structure of the planarian central nervous system (CNS) revealed by neuronal cell markers. Zool. Sci. 15:433-440.[ISI]
Cebrià, F., C. Kobayashi, and Y. Umesono, et al. (11 co-authors). 2002. FGFR-related gene nou-darake restricts brain tissues to the head region of planarians. Nature 419:620-624.[CrossRef][ISI][Medline]
Duggan, D. J., M. Bittner, Y. Chen, P. Meltzer, and J. M. Trent. 1999. Expression profiling using cDNA microarrays. Nat. Genet. 21:(Suppl.): 10-14.[CrossRef][ISI][Medline]
Edelman, G. M. 1986. Cell adhesion molecules in the regulation of animal form and tissue pattern. Annu. Rev. Cell. Dev. Biol. 2:81-116.[CrossRef][ISI]
Finkelstein, R., D. Smouse, T, M. Capaci, A. C. Spradling, and N. Perrimon. 1990. The orthodenticle gene encodes a novel homeo domain protein involved in the development of the Drosophila nervous system and ocellar visual structures. Genes Dev. 4:1516-1527.[Abstract]
Gerhart, J., and M. Kirschner. 1997. Cells, embryos, and evolution. Blackwell Science, Malden, Mass.
Grenningloh, G., A. J. Bieber, E. J. Rehm, P. M. Snow, Z. R. Traquina, M. Hortsch, N. H. Patel, and C. S. Goodman. 1990. Molecular genetics of neuronal recognition in Drosophila: evolution and function of immunoglobulin superfamily cell adhesion molecules. Cold Spring Harbor Symp. Quant. Biol. 55:327-340.[Medline]
Keenan, L. C., R. Coss, and H. Koopowitz. 1981. Cytoarchitecture of primitive brains: golgi studies in flatworms. J Comp. Neurol. 195:697-716.[ISI][Medline]
Matsuo, I., S. Kuratani, C. Kimura, N. Takeda, and S. Aizawa. 1995. Mouse otx2 functions in the formation and patterning of rostral head. Genes Dev. 9:2646-2658.[Abstract]
Nagase, T., N. Ishikawa, A. Tanaka, H. Kotani, N. Nomura, and O. Ohara. 1998. Prediction of the coding sequences of unidentified human genes. IX. The complete sequences of 100 new cDNA clones from brain which can code for large proteins in vitro. DNA Res. 5:31-39.[Medline]
Pennekamp, P., N. Bogdanova, M. Wilda, A. Markoff, H. Hameister, J. Horst, and B. Dworniczak. 1998. Characterization of the murine polycystic kidney disease (Pkd2) gene. Mamm. Genome 9:749-752.[CrossRef][ISI][Medline]
Sánchez Alvarado, A., and P. A. Newmark. 1999. Double-stranded RNA specifically disrupts gene expression during planarian regeneration. Proc. Natl. Acad. Sci. USA 96:5049-5054.
Sarnat, H. B., and M. G. Netsky. 1985. The brain of the planarian as the ancestor of the human brain. Can. J. Neurol. Sci. 12:296-302.[ISI][Medline]
Shinohara, T., T. Kikuchi, M. Tsuda, and K. Yamaki. 1992. A family of retinal s-antigens (arrestins) and their genes: comparative analysis of human, mouse, rat, bovine and Drosophila. Comp. Biochem. Physiol. 103B:505-509.[CrossRef][ISI]
Simon, M. I., M. P. Strathmann, and N. Gautam. 1991. Diversity of G protein in signal transduction. Science 252:802-808.[ISI][Medline]
Umesono, Y., K. Watanabe, and K. Agata. 1997. A planarian orthopedia homolog is specifically expressed in the branch region of both the mature and regenerating brain. Dev. Growth Differ. 39:723-727.[ISI][Medline]
Umesono, Y., K. Watanabe, and K. Agata. 1999. Distinct structural domains in the planarian brain defined by the expression of evolutionarily conserved homeobox genes. Dev. Genes Evol. 209:31-39.[CrossRef][ISI][Medline]
Valentine, J. W. 1994. Late Precambrian bilaterians: grades and clades. Proc. Natl. Acad. Sci. USA 91:6751-6757.[Abstract]