Mechanisms that regulate the number of cells constituting the body have remained largely elusive. We approached this issue in the ascidian, Halocynthia roretzi, which develops into a tadpole larva with a small number of cells. The embryonic cells divide 11 times on average from fertilization to hatching. The number of cell division rounds varies among tissue types. For example, notochord cells divide 9 times and give rise to large postmitotic cells in the tadpole. The number of cell division rounds in partial embryos derived from tissue-precursor blastomeres isolated at the 64-cell stage also varied between tissues and coincided with their counterparts in the intact whole embryos to some extent, suggesting tissue-autonomous regulation of cell division. Manipulation of cell fates in notochord, nerve cord, muscle, and mesenchyme lineage cells by inhibition or ectopic activation of the inductive FGF signal changed the number of cell divisions according to the altered fate. Knockdown and missexpression of Brachyury (Bra), an FGF-induced notochord-specific key transcription factor for notochord differentiation, indicated that Bra is also responsible for regulation of the number of cell division rounds, suggesting that Bra activates a putative mechanism to halt cell division at a specific stage. The outcome of precocious expression of Bra suggests that the mechanism involves a putative developmental clock that is likely shared in blastomeres other than those of notochord and functions to terminate cell division at three rounds after the 64-cell stage. Precocious expression of Bra has no effect on progression of the developmental clock itself.

The genome of Ciona intestinalis contains eight genes for HSP70 superfamily proteins, 36 genes for J-proteins, a gene for a J-like protein, and three genes for BAG family proteins. To understand the stress responses of genes in the HSP70 chaperone system comprehensively, the transcriptional profiles of these 48 genes under heat stress and endoplasmic reticulum (ER) stress were studied using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Heat stress treatment increased the messenger RNA (mRNA) levels of six HSP70 superfamily genes, eight J-protein family genes, and two BAG family genes. In the cytoplasmic group of the DnaK subfamily of the HSP70 family, Ci-HSPA1/6/7-like was the only heat-inducible gene and Ci-HSPA2/8 was the only constitutively active gene which showed striking simplicity in comparison with other animals that have been examined genome-wide so far. Analyses of the time course and temperature dependency of the heat stress responses showed that the induction of Ci-HSPA1/6/7-like expression rises to a peak after heat stress treatment at 28 degrees C (10 degrees C upshift from control temperature) for 1 h. ER stress treatment with Brefeldin A, a drug that is known to act as ER stress inducer, increased the mRNA levels of four HSP70 superfamily genes and four J-protein family genes. Most stress-inducible genes are conserved between Ciona and vertebrates, as expected from a close evolutionary relationship between them. The present study characterized the stress responses of HSP70 chaperone system genes in Ciona for the first time and provides essential data for comprehensive understanding of the functions of the HSP70 chaperone system.