Molecular chaperones play crucial roles in various aspects of the biogenesis and maintenance of proteins in the cell. The heat shock protein 70 (HSP70) chaperone system, in which HSP70 proteins act as chaperones, is one of the major molecular chaperone systems conserved among a variety of organisms. To shed light on the evolutionary history of the constituents of the chordate HSP70 chaperone system and to identify all of the components of the HSP70 chaperone system in ascidians, we carried out a comprehensive survey for HSP70s and their cochaperones in the genome of Ciona intestinalis. We characterized all members of the Ciona HSP70 superfamily, J-proteins, BAG family, and some other types of cochaperones. The Ciona genome contains 8 members of the HSP70 superfamily, all of which have human and protostome counterparts. Members of the STCH subfamily of the HSP70 family and members of the HSPA14 subfamily of the HSP110 family are conserved between humans and protostomes but were not found in Ciona. The Ciona genome encodes 36 J-proteins, 32 of which belong to groups conserved in humans and protostomes. Three proteins seem to be unique to Ciona. J-proteins of the RBJ group are conserved between humans and Ciona but were not found in protostomes, whereas J-proteins of the DNAJC14, ZCSL3, FLJ13236, and C21orf55 groups are conserved between humans and protostomes but were not found in Ciona. J-proteins of the sacsin group seem to be specific to vertebrates. There is also a J-like protein without a conserved HPD tripeptide motif in the Ciona genome. The Ciona genome encodes 3 types of BAG family proteins, all of which have human and protostome counterparts (BAG1, BAG3, and BAT3). BAG2 group is conserved between humans and protostomes but was not found in Ciona, and BAG4 and BAG5 groups seem to be specific to vertebrates. Members for SIL1, UBQLN, UBADC1, TIMM44, GRPEL, and Magmas groups, which are conserved between humans and protostomes, were also found in Ciona. No Ciona member was retrieved for HSPBP1 group, which is conserved between humans and protostomes. For several groups of the HSP70 superfamily, J-proteins, and other types of cochaperones, multiple members in humans are represented by a single counterpart in Ciona. These results show that genes of the HSP70 chaperone system can be distinguished into groups that are shared by vertebrates, Ciona, and protostomes, ones shared by vertebrates and protostomes, ones shared by vertebrates and Ciona, and ones specific to vertebrates, Ciona, or protostomes. These results also demonstrate that the components of the HSP70 chaperone system in Ciona are similar to but simpler than those in humans and suggest that changes of the genome in the lineage leading to humans after the separation from that leading to Ciona increased the number and diversity of members of the HSP70 chaperone system. Changes of the genome in the lineage leading to Ciona also seem to have made the HSP70 chaperone system in this species slightly simpler than that in the common ancestor of humans and Ciona.

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°C (10°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.

Polycyclic aromatic hydrocarbons (PAHs) are pollutants that exert harmful effects on marine invertebrates; however, the molecular mechanism underlying PAH action remains unclear. We investigated the effect of PAHs on the ascidian Ciona intestinalis type A (Ciona robusta). First, the influence of PAHs on early Ciona development was evaluated. PAHs such as dibenzothiophene, fluorene, and phenanthrene resulted in formation of abnormal larvae. PAH treatment of swimming larva induced malformation in the form of tail regression. Additionally, we observed the Ciona aryl hydrocarbon receptor (Ci-AhR) mRNA expression in swimming larva, mid body axis rotation, and early juvenile stages. The time correlation between PAH action and AhR mRNA expression suggested that Ci-AhR could be associated with PAH metabolism. Lastly, we analyzed Ci-AhR mRNA localization in Ciona juveniles. Ci-AhR mRNA was localized in the digestive tract, dorsal tubercle, ganglion, and papillae of the branchial sac, suggesting that Ci-AhR is a candidate for an environmental pollutant sensor and performs a neural function. Our results provide basic knowledge on the biological function of Ci-AhR and PAH activity in marine invertebrates.

Summary: Temporally controlled induction of gene expression is a useful technique for analyzing gene function. To make such a technique possible in Ciona intestinalis embryos, we employed the cis‐regulatory region of the heat‐shock protein 70 (HSP70) gene Ci‐HSPA1/6/7‐like for heat‐inducible gene expression in C. intestinalis embryos. We showed that Ci‐HSPA1/6/7‐like becomes heat shock‐inducible by the 32‐cell stage during embryogenesis. The 5′‐upstream region of Ci‐HSPA1/6/7‐like, which contains heat‐shock elements indispensable for heat‐inducible gene expression, induces the heat shock‐dependent expression of a reporter gene in the whole embryo from the 32‐cell to the middle gastrula stages and in progressively restricted areas of embryos in subsequent stages. We assessed the effects of heat‐shock treatments in different conditions on the normality of embryos and induction of transgene expression. We evaluated the usefulness of this technique through overexpression experiments on the well‐characterized, developmentally relevant gene, Ci‐Bra, and showed that this technique is applicable for inferring the gene function in C. intestinalis. genesis 53:170–182, 2015.