Kazuhiro W. Makabe

The ANISEED database: Digital representation, formalization, and elucidation of a chordate developmental program

Developmental biology aims to understand how the dynamics of embryonic shapes and organ functions are encoded in linear DNA molecules. Thanks to recent progress in genomics and imaging technologies, systemic approaches are now used in parallel with small-scale studies to establish links between genomic information and phenotypes, often described at the subcellular level. Current model organism databases, however, do not integrate heterogeneous data sets at different scales into a global view of the developmental program. Here, we present a novel, generic digital system, NISEED, and its implementation, ANISEED, to ascidians, which are invertebrate chordates suitable for developmental systems biology approaches. ANISEED hosts an unprecedented combination of anatomical and molecular data on ascidian development. This includes the first detailed anatomical ontologies for these embryos, and quantitative geometrical descriptions of developing cells obtained from reconstructed three-dimensional (3D) embryos up to the gastrula stages. Fully annotated gene model sets are linked to 30,000 high-resolution spatial gene expression patterns in wild-type and experimentally manipulated conditions and to 528 experimentally validated cis-regulatory regions imported from specialized databases or extracted from 160 literature articles. This highly structured data set can be explored via a Developmental Browser, a Genome Browser, and a 3D Virtual Embryo module. We show how integration of heterogeneous data in ANISEED can provide a system-level understanding of the developmental program through the automatic inference of gene regulatory interactions, the identification of inducing signals, and the discovery and explanation of novel asymmetric divisions.

Phylogenetic Relationships between Solitary and Colonial Ascidians, as Inferred from the Sequence of the Central Region of their Respective 18S rDNAs

Ascidians (tunicates) are primitive chordates. In spite of their elevated phylogenetic position in the animal kingdom, ascidians have evolved a varied reproductive repertoire; some of them live as individuals (solitary ascidians), while others form colonies (colonial ascidians). Colonial ascidians propagate asexually by budding and strobilation, and they have an extensive capacity for regeneration. However, the orthodox taxonomic classification of ascidians categorizes them into two major groups (the orders Enterogona and Pleurogona), irrespective of their solitary or colonial life style. To examine whether the orthodox classification of ascidians is substantiated by molecular phylogeny, the complete nucleotide sequence of a region of about 1000 base pairs in the central part of their respective 18S rDNAs was determined, and the sequences were compared among five solitary and three colonial ascidians. The phylogenetic tree deduced from these results suggests that the three species of Enterogona and the five species of Pleurogona examined form discrete and separate groups irrespective of their potential to form colonies. Therefore, a solitary or colonial life style is likely to have developed independently after the divergence of the two major groups of ascidians.

Maternally localized RNA encoding a serine/threonine protein kinase in the ascidian, Halocynthia roretzi.

Maternally localized cytoplasmic determinants play important roles in the embryogenesis of many animals, including ascidians. Cytoplasmic determinants are particularly important in the determination of cell fates, and in the establishment of the embryonic axes. Ascidians, which show mosaic development, are good models for the study of maternal cytoplasmic determinants. Here we report the isolation and characterization of HrPOPK-1 (Halocynthia roretzi posterior protein kinase-1), a putative protein serine/threonine kinase. HrPOPK-1 cDNA was obtained from a Halocynthia roretzi fertilized egg cDNA library by screening for localized RNAs using whole-mount in situ hybridization. HrPOPK-1 mRNA is strongly localized at the posterior pole of embryos. The pattern of HrPOPK-1 mRNA localization during early embryogenesis is identical to that of HrWnt-5 in Halocynthia roretzi, and to those of the posterior end mark (pem) transcripts of Ciona savignyi. In addition, HrPOPK-1 shows zygotic expression in neural tissues at the tailbud stage. These results show that the temporal regulation of HrPOPK-1 transcription is complex.

Temporal and Spatial Expression of a Muscle Actin Gene during Embryogenesis of the Ascidian Halocynthia roretzi

Screening a cDNA library from tailbud embryos of the ascidian Halocynthia roretzi with a Styela plicata mantle actin probe yielded several muscle‐type actin clones. These clones differed from each other in the nucleotide sequences of their 3′non‐coding regions, although the sequences of the coding region were almost identical. One of the clones, HrcMA4, was selected and characterized. HrcMA4 contains an open reading frame of 1137 bp and a 100 bp 3′non‐coding region followed by a poly(A) tail. An antisense probe consisting of a small segment of 3′coding region and a large portion of 3′non‐coding region of the clone was constructed. In situ hybridization analysis with this probe demonstrated that expression of HrMA4 mRNAs was restricted to differentiating muscle cells in tailbud embryos. Signal was not detectable in other regions of the embryo. Northern blot analysis showed that HrMA4 mRNA was undetectable in unfertilized eggs, zygotes and cleavage‐stage embryos. A single band of the HrMA4 transcripts about 1.5 kb in length was first observed in gastrulae. The amount of HrMA4 mRNAs increased rapidly as development progressed. The mRNA was evident in tadpole larvae and newly metamorphosed juveniles. The amount of transcripts, however, decreased after metamorphosis and became undetectable by a week after metamorphosis. Thus, the HrMA4 gene showed strict zygotic expression restricted to the muscle lineage cells.

Introduction and Expression of Recombinant Genes in Ascidian Embryos

In order to examine the expression of exogenous genes introduced into ascidian eggs, two recombinant plasmids pmiwZ and pHrMA4aCAT were microinjected into the cytoplasm of fertilized eggs of Ciona savignyi and Halocynthia roretzi, respectively. The plasmid pmiwZ contains the coding sequence of bacterial β‐galactosidase gene (lac‐Z) fused with animal gene promoters, while pHrMA4aCAT was constructed by fusing about 1.4‐kb long 5′ flanking region of H. roretzi muscle actin gene HrMA4a with bacterial chloramphenicol acetyltransferase gene (CAT). Injection of approximately 160 pl of 10 μg/ml pmiwZ DNA into Ciona eggs did not affect the embryogenesis, although introduction of the same volume of 30 μg/ml pmiwZ DNA resulted in abnormal development of injected eggs. When the expression of lac‐Z was examined by histochemical detection of the enzyme activity, the expression was evident in the early tailbud embryos and later stage embryos, and larvae, irrespective of linear or circular form of the plasmid. The enzyme activity appeared in various cell‐types including epidermis, nervous system, endoderm, mesenchyme, notochord, and muscle. In contrast, when pHrMA4aCAT was introduced into Halocynthia eggs and the appearance of CAT protein was examined later by the anti‐CAT antibody, the CAT expression was restricted to muscle cells. These results indicate that the recombinant genes introduced into ascidian eggs could express during embryogenesis and that the 1.4‐kb long 5′ flanking region of HrMA4a contains regulatory sequences enough for the appropriate spatial and temporal expression of the gene.

Cellular and Molecular Mechanisms of Muscle Cell Differentiation in Ascidian Embryos

Publisher Summary This chapter reviews the current and general information on cellular and molecular mechanisms involved in determination and differentiation of ascidian larval muscle cells, and discusses emerging concepts and future prospects. Ascidians, or sea squirts, are ubiquitous, filter-feeding, sessile marine animals. Most of them attach to rocks, piles, or other substrates in shallow water. Some of them live as individuals, while others form colonies. Ascidians have evolved rich patterns and modes of development; sexual reproduction is common in solitary ascidians, whereas asexual reproduction and regeneration are highly developed in compound ascidians. The fertilized egg of a solitary ascidian shows a determinate cleavage pattern and develops quickly into a tadpole larva that contains either 21 or 18 unicellular striated muscle cells on each side of the tail. Muscle cell differentiation during normal ascidian embryogenesis is examined by various methods, including histochemistry of an enzyme, AChE, immunocytochemistry using a muscle-specific antibody, the ultrastructural examination of myofilaments and myofibrils, and the electrophysiological detection of the membrane properties characteristic of muscle.

Isolation of an early neural maker gene abundantly expressed in the nervous system of the ascidian, Halocynthia roretzi

Abstract. Ascidian tadpole larvae possess a primitive nervous system, which is a prospective prototype of the chordate nervous system. It is composed of relatively few cells but sufficient for complex larval behavior. Here we report on HrETR-1, a gene zygotically expressed in a large proportion of the developing neural cells of the ascidian, Halocynthia roretzi. HrETR-1 is an early neural marker which can be used for analyzing neural differentiation. HrETR-1 expression intensified in most neural cells of genes isolated to date, in both central and peripheral nervous systems including palps as early as the 110-cell stage. Using this gene as a probe, we characterized neural cells in the nervous system as well as confirming their origins. Also, we recognized three types of peripheral epidermal neurons which presumably correlate to the larval neurons previously reported for another ascidian. Among these, five bilateral neurons located in the anterior region of the trunk appeared to be derived from a8.26 blastomeres.

A Large-Scale Whole-Mount in situ Hybridization System: Rapid One-Tube Preparation of DIG-Labeled RNA Probes and High Throughput Hybridization using 96-Well Silent Screen Plates

Abstract Recent progress in multiple and automated-sequencing technology allows large-scale random cDNA sequencing, the so-called EST project, in various fields. In addition to the EST collection, the cDNA project requires analysis of spatiotemporal patterns of gene expression of a large number of clones by whole-mount in situ hybridization (WISH). To facilitate the multiple WISH procedures, we developed a protocol for rapid and uniform synthesis of multiple probes and multi-well based WISH processing. A DIG-labeled RNA probe for WISH was synthesized from a PCR-amplified template which contained an RNA promoter. All reactions of PCR and subsequent RNA synthesis were performed in a single tube by sequential addition of the reagents without phenol extraction or ethanol precipitation steps. An RNA probe was purified and condensed by a centrifugal ultrafilter to achieve high and stable purification efficiency. WISH of 96 samples were performed simultaneously in a 96-well plate attached to silent screen filters that were connected with a vacuum exhausting system. These processes eliminated the labor-intensive steps of WISH and provided opportunities to search for novel genes.

Localization and expression pattern of type I postplasmic mRNAs in embryos of the ascidian Halocynthia roretzi.

The posterior-vegetal cytoplasm (PVC) of fertilized ascidian eggs plays important roles in embryo development. It has been reported that some maternal RNAs are localized to the PVC. We identified four novel type I postplasmic mRNAs that are localized to the PVC through the use of data from a cDNA project of maternal mRNAs in the eggs of Halocynthia roretzi (MAGEST database). The mRNAs are HrGLUT, HrPEN-1, and HrPEM-3, which show similarity to a glucose transporter, a g1-related protein, and Ciona pem-3, respectively; and HrPEN-2, with no similarity. Maternal mRNAs of all four genes were identically localized to the PVC after ooplasmic segregation. During cleavage, they were concentrated in the centrosome-attracting body (CAB) and were then segregated into the small blastomeres located at the posterior pole. This localization pattern is common to all known type I postplasmic mRNAs found so far. HrGLUT, HrPEN-1, and HrPEM-3 were expressed zygotically in various tissues later in embryogenesis: HrGLUT and HrPEM-3 in the mesenchyme and nervous system, and HrPEN-1 in the ectodermal cells.

POPK-1/Sad-1 kinase is required for the proper translocation of maternal mRNAs and putative germ plasm at the posterior pole of the ascidian embryo

Maternal mRNAs localized to specific regions in eggs play important roles in the establishment of embryonic axes and germ layers in various species. Type I postplasmic/PEM mRNAs, which are localized to the posterior-vegetal cortex (PVC) of fertilized ascidian eggs, such as the muscle determinant macho-1 mRNA, play key roles in embryonic development. In the present study, we analyzed the function of the postplasmic/PEM RNA Hr-POPK-1, which encodes a kinase of Halocynthia roretzi. When the function of POPK-1 was suppressed by morpholino antisense oligonucleotides, the resulting malformed larvae did not form muscle or mesenchyme, as in macho-1-deficient embryos. Epistatic analysis indicated that POPK-1 acts upstream of macho-1. When POPK-1 was knocked down, localization of every Type I postplasmic/PEM mRNA examined, including macho-1, was perturbed, showing diffuse early distribution and eventual concentration into a smaller area. This is the probable reason for the macho-1 dysfunction. The postplasmic/PEM mRNAs such as macho-1 and Hr-PEM1 are co-localized with the cortical endoplasmic reticulum (cER) and move with it after fertilization. Eventually they become highly concentrated into a subcellular structure, the centrosome-attracting body (CAB), at the posterior pole of the cleaving embryos. The suppression of POPK-1 function reduced the size of the domain of concentrated cER at the posterior pole, indicating that POPK-1 is involved in the movement of postplasmic/PEM RNAs via relocalization of cER. The CAB also shrank. These results suggest that Hr-POPK-1 plays roles in concentration and positioning of the cER, as well as in the concentration of CAB materials, such as putative germ plasm, in the posterior blastomeres.