Taishin Shimotori

Developmental fates of the first four blastomeres of the chaetognath Paraspadella gotoi: Relationship to protostomes

Experimental analysis of the development of chaetognaths is virtually lacking. To elucidate developmental fates, single blastomeres of the 2‐cell and 4‐cell embryos of Paraspadella gotoi were injected with a lineage‐tracing dye (Fluoro‐Ruby or DiI). The distribution of the labels was observed in the hatchlings. In a previous study, embryos were injected at the 2‐cell stage with Fluoro‐Ruby and two sets of complementary labeling patterns (DL and VR, and DR and VL) were found. The same results were obtained when DiI was used as a tracer dye. The 4‐cell embryo consists of the animal and vegetal cross‐furrow cells in a tetrahedral arrangement and one of the vegetal cross‐furrow cells typically contains the germ plasm. When single cells were injected at the 4‐cell stage, four labeling patterns were observed (D, V, L and R). These four patterns represent subsets of the four patterns observed in the hatchling injected at the 2‐cell stage. The V pattern is probably generated from the blastomere containing the germ plasm. It was found that the positions of the blastomeres at the 4‐cell stage corresponded to the future body axes, similar to classic spiralians and modified spiralians such as crustaceans. Furthermore, it was confirmed that second cleavage occurs in a leiotropic fashion, which is seen in the second cleavage of the classic spiralians. Chaetognaths may have some similarities to protostomes in their developmental program.

Utilization of a particle gun DNA introduction system for the analysis of cis-regulatory elements controlling the spatial expression pattern of the arylsulfatase gene (HpArs) in sea urchin embryos

Abstract. We applied a particle gun method to introduce DNA into fertilized sea urchin eggs for the analysis of cis-regulatory elements responsible for spatial gene expression during development. We introduced HpArs (sea urchin arylsulfatase gene) –GFP and HpArs–LacZ fusion constructs into the fertilized eggs and obtained high expression levels of the fusion genes. Using this assay system, we demonstrated that a fragment of HpArs (–3,484 to +4,636) is sufficient for aboral ectoderm-specific expression, and that the region in the first intron from +406 to +1,993 contains the control elements responsible for the repression of the HpArs promoter activity in secondary mesenchyme cells.

Establishment of Axial Properties in the Arrow Worm Embryo, Paraspadella gotoi (Chaetognatha): Developmental Fate of the First Two Blastomeres

Abstract Chaetognaths are bilateral animals totally symmetrical on both sides of their body. To elucidate the manner in which cell fates are established, single blastomeres of the two-cell stage embryos of the benthic arrow worm, Paraspadella gotoi, were injected with a fluorescent lineage-tracing dye. The distribution of labels was observed in the hatchlings by using a confocal laser scanning microscope. A total of four different labeling patterns was observed: 1) the dorsal epidermis, the right half of the ventral epidermis, and the right half of the dorsal longitudinal muscles (DR pattern); 2) the dorsal epidermis, the left half of the ventral epidermis, and the left half of the dorsal longitudinal muscles (DL pattern); 3) the right half of the ventral epidermis, the ventral longitudinal muscles, the right half of the dorsal body muscles, and the primordial germ cells (PGCs) (VR pattern); and 4) the left half of the ventral epidermis, the ventral longitudinal muscles, the left half of the dorsal body muscles, and the PGCs (VL pattern). Thus, one blastomere of the two-cell stage largely contributes the dorsal epidermis and the other contributes the ventral longitudinal muscles and the PGCs. Bilateral halves of the dorsal longitudinal muscles and the ventral epidermis were labeled as complementary pairs. These labeling patterns indicate that the first cleavage plane runs oblique to the bilateral and dorsoventral axes. In addition, the occurrence of complementary “DL and VR” and “DR and VL” labeling patterns indicates that the first cleavage plane bears one of two different angular relationships relative to the future body axes.

A new G-stretch-DNA-binding protein, Unichrom, displays cell-cycle-dependent expression in sea urchin embryos

We report the identification and characterization of Unichrom, a gene encoding a new G‐stretch‐DNA‐binding protein in the sea urchin embryo. The derived amino acid sequence of Unichrom contains plant homeodomain (PHD) finger and high mobility group (HMG) motifs as well as motifs required for cell‐cycle‐dependent degradation. The expression of a Unichrom‐green fluorescent protein (GFP) fusion protein in sea urchin embryonic cells indicates that Unichrom protein accumulates in nuclei during interphase and disperses into the cytoplasm at mitosis. Overexpression of dominant negative Unichrom, which contains the DNA binding domain lacking the motif for cell‐cycle‐dependent degradation, causes impairment of chromosome segregation. These results suggest that Unichrom binds to genome DNA at G‐stretch and that degradation of Unichrom is required for segregation of chromosomes.

Unichrom, a novel nuclear matrix protein, binds to the Ars insulator and canonical MARs.

Abstract Eukaryotic genomic DNA is organized into loop structures by attachments to the nuclear matrix. These attachments to the nuclear matrix have been supposed to form the boundaries of chromosomal DNA. Insulators or boundary elements are defined by two characteristics: they interrupt promoter-enhancer communications when inserted between them, and they suppress the silencing of transgenes stably integrated into inactive chromosomal domains. We recently identified an insulator element in the upstream region of the sea urchin arylsulfatase (HpArs) gene that shows both enhancer blocking and suppression of position effects. Here, we report that Unichrom, originally identified by its G-stretch DNA binding capability, is a nuclear matrix protein that binds to the Ars insulator and canonical nuclear matrix attachment regions (MARs). We also show that Unichrom recognizes the minor groove of the AT-rich region within the Ars insulator, which may have a base-unpairing property, as well as the G-stretch DNA. Furthermore, Unichrom selectively interacts with poly(dG)•poly(dC), poly(dA)•poly(dT) and poly(dAT)•poly(dAT), but not with poly(dGC)•poly(dGC). Unichrom also shows high affinity for single-stranded G- and C-stretches. We discuss the DNA binding motif of Unichrom and the function of Unichrom in the nuclear matrix.

The Otx binding site is required for the activation of HpOtxL mRNA expression in the sea urchin, Hemicentrotus pulcherrimus

Two distinct types of orthodenticle‐related (HpOtxE and HpOtxL) mRNA are transcribed from a single HpOtx gene by altering the transcription start site and by alternative splicing, and their expressions are differentially regulated during early development of the sea urchin Hemicentrotus pulcherrimus. To understand the mechanism of this regulation, we screened for the enhancer element involved in the stage‐specific expression of HpOtxL mRNA. Different portions of the HpOtx gene, including the 5′‐flanking region and the first intron, were ligated to the minimal HpOtxL promoter driving a luciferase gene, and their constructs were introduced into fertilized eggs using a particle gun. The enhancer element responsible for proper expression consistent with that of the endogenous HpOtxL was found in the first intron of the HpOtx gene. External and internal deletion analyses showed that the 334 bp region (from +8838 bp to +9171 bp) was required for enhancer activity. In addition, deletion of an Otx binding site within the 334 bp region markedly reduced reporter expression. These results suggest that the Otx binding site within the HpOtxL enhancer is required for the activation of HpOtxL mRNA expression. The promoter preference of the HpOtxL enhancer is also discussed.