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First published online June 22, 2006
doi: 10.1242/10.1242/dev.02446

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Dynamic redistribution of vasa homolog and exclusion of somatic cell determinants during germ cell specification in Ciona intestinalis

¹ Laboratory for Germline Development, RIKEN Center for Developmental Biology, Kobe, Hyogo 650-0047, Japan.
² Department of Biology, Konan University, Kobe, Hyogo 658-8501, Japan.
³ Department of Marine Biotechnology, Fukuyama University, Fukuyama, Hiroshima 729-0292, Japan.
⁴ Laboratory of Cellular Biochemistry, RIKEN, Wako, Saitama 351-0198, Japan.

View larger version (41K): [in a new window]   Fig. 1. Expression patterns of CiVH and Ci-PEM RNAs during embryogenesis. (A-G) CiVH RNA expression; (H-N) Ci-PEM RNA expression. Although both Ci-PEM and CiVH RNA localize to the postplasm and to the middle of the tail at the tailbud stage (arrowheads), the CiVH RNA shows a different specific distribution in the posterior region of the tail in tailbud embryos (arrows).

View larger version (56K): [in a new window]   Fig. 2. CiVH protein is localized to two discrete regions through the B7.6 cell division. Embryos were stained for CiVH protein (green) and for F-actin (magenta) to visualize the cell boundary. A-C, dorsal views; E-G, lateral views. Anterior is towards the left in all panels. (A,B) CiVH protein (green) accumulated at the posterior cortex of the B7.6 cells with an F-actin layer (A',B', arrowheads). (A'',B'') Phalloidin staining alone highlights a thick layer of cortical F-actin in the CAB region (arrowheads). (C,C') At the mid-gastrula stage, while some CiVH protein remained associated with an F-actin mass (C', arrowhead), some was diffused in the cytoplasm of the B7.6 cells (C', arrows). Diagrams show the position of the B7.6 cells in above embryos (upper row; outlined in red) and distribution of CiVH protein within the B7.6 cells (lower row; red). (D) A mid-gastrula embryo showing the pH3-positive chromosomes in one of the B7.6 cells (D', yellow). pH3-positive chromosomes are aligned, indicating that this pH3-positive B7.6 cell was in metaphase (arrow). (E-G) In the B8.11 cells (E',F',G', arrowheads), the CiVH signals were faint. By contrast, the CiVH protein was upregulated in the B8.12 cells (E,F, arrows), forming CiVH granules in the cytoplasm (E',F' arrows). (G) The B8.12 cells divided to form four CiVH-positive cells at the late-tailbud stage (arrows in G,G'''). These cells formed perinuclear CiVH granules (arrows in G'''). (G'') Phalloidin staining alone highlighting the F-actin aggregates in the B8.11 cells (arrowhead). Diagrams show the positions of CiVH-positive B7.6 descendants in above embryos.

View larger version (141K): [in a new window]   Fig. 3. CiVH-positive cells in the tail region become germ cells. (A,B) An early tadpole larva. Eight CiVH-positive cells were localized to the distal region of the tail (arrowheads in B). CiVH signals were also detected in the trunk region (arrow). (C) A late tadpole-stage larva. CiVH signals in the trunk were remarkably reduced (arrow). (D) A larva during tail resorption. CiVH-positive cells were found in the tail (arrowhead), and the CiVH signals in the trunk were undetectable. (E) A stage 3a juvenile. CiVH-positive cells were among those in the tail debris (arrowhead). (F,F') A stage 3b juvenile. CiVH-positive cells (arrowheads) remained among the cells in the tail debris (outlined). (G,G') A stage 4 juvenile. CiVH-positive cells were aligned within the tail debris, which was almost completely resorbed (outlined). (H,H') A stage 5 juvenile. CiVH-positive cells (arrowheads) were aligned within the tube-like structure. (I,I') A stage 6 juvenile. CiVH-positive cells (arrowhead) changed position within the tube to form a drop-like cluster. (J,J') A stage 7 juvenile. The CiVH-positive cells (arrowheads) were entirely surrounded by somatic tissue. The number of CiVH-positive cells in the gonad increased.

View larger version (118K): [in a new window]   Fig. 4. B8.12 descendants coalesce into the gonad. (A) A gastrula embryo with DiI-labeled B7.6 cells (arrowhead). (B) A tailbud embryo with DiI signals in the B8.11 (arrowhead) and B8.12 (arrow) cells. (C) A stage 6 juvenile, in which the B7.6 cells were DiI labeled, were stained for CiVH (green). The arrowhead indicates the primitive gonads. Five DiI-labeled cells (arrowheads) that were also CiVH positive coalesced into the gonads (inset). (D) A larva in which B7.6 blastomeres were DiI-labeled and the distal part of the tail containing the B8.12 descendants was cut off. The DiI signals remained in the B8.11 cells of the tail (arrowhead). (E) A stage 5 tail-cut juvenile. The DiI-positive B8.11 cells were present on the gut wall (arrowhead). Owing to the capture of a faint DiI signal, the auto-fluorescence from the tail debris was relatively prominent (arrow). No CiVH-positive cells were detected in this juvenile (data not shown).

View larger version (88K): [in a new window]   Fig. 5. CiVH RNA and protein are inherited by the B8.12 cells through their release from the CAB. Embryos were stained for CiVH protein (magenta) and CiVH RNA (green), and their nuclei were labeled (blue in J,O). (A-E) A 32-cell stage embryo; (F-J) a gastrula embryo; (K-O) a mid-tailbud embryo. (B-E,G-J,L-O) Enlarged views of A,F,K. (A,B,F,G,K,L) Nomarski optics. (C-E) CiVH protein and RNA concentrated to the CAB during cleavage stages (arrowheads). (H-J) Some CiVH protein (white arrows) and RNA (yellow arrows) diffused together from the CAB (arrowheads) in gastrula stage embryos. (M-O) In tailbud embryos, although CiVH protein and RNA colocalized in the B8.11 cells (arrowheads), they were separated in the cytoplasm within the B8.12 cells (white and yellow arrows, respectively).

View larger version (86K): [in a new window]   Fig. 6. Ci-PEM RNA and CiVH protein show different distributions after the gastrula stage. Embryos were stained for CiVH protein (magenta) and for Ci-PEM RNA (green). Nuclei were labeled in blue (J,O). (A-E) A 32-cell stage embryo; (F-J) a gastrula embryo; (K-O) a tailbud embryo. (B-E,G-J,L-O) Enlarged views of A,F,K. (A,B,F,G,K,L) Nomarski optics. Although the CiVH protein was released from the CAB and diffused into the cytoplasm (H,J, arrows), the Ci-PEM RNA remained in the anterior-most region in the B7.6 cells at the gastrula stage (I,J, arrowheads), resulting in its inheritance only by the B8.11 cells at the tailbud stage (N,O, arrowheads).

View larger version (74K): [in a new window]   Fig. 7. CiYB1 protein is inherited only by the B8.11 cells, along with Ci-PEM RNA. Embryos were stained for CiYB1 protein (magenta) and Ci-REM RNA (green). (A-E) A 32-cell embryo; (F-J) a gastrula embryo; (K-O) a mid-tailbud embryo. (B-E,G-J, L-O) Enlarged views of A,F,K. (A,B,F,G,K,L) Nomarski optics. The CiYB1 protein tightly colocalized with Ci-PEM RNA and segregated only into the B8.11 cells (E,J,O).

View larger version (73K): [in a new window]   Fig. 8. CiVH protein production in the B8.12 cells depends on the translation of maternally inherited RNA. (A,A') Untreated mid-tailbud-stage embryos, showing CiVH signals in the B8.11 (arrowhead) and B8.12 (arrow) cells. (B,B') AcD-treated embryos. Although the treatment severely affected embryogenesis, strong CiVH signals were detected in the presumptive B8.11 (arrowhead) and B8.12 (arrow) cells. (C,C') Puromycin-treated embryos, in which CiVH signals in the presumptive B8.12 cells were almost undetectable. Owing to the capture of a faint CiVH signals (C' arrow), background signals in the cortical rim were relatively prominent. (A'',B'',C'') Histochemical staining for AP activity under each condition. (D) Expression of Ci-AP, CiVH and ß-tubulin RNAs, analyzed by RT-PCR. A severe reduction in the expression of Ci-AP mRNA in the AcD-treated embryos was confirmed by using two different primer sets (AP-1, AP-2). The negative controls without reverse transcriptase (RT) are shown in the lanes (-) to the left of each RT-positive lane (+).