In the normal sea urchin embryo, Vasa expression is exclusively enriched only in the small micromere lineage during gastrula and early larval stages. The four small micromere descendants undergo only 1–2 cell cycles during gastrulation, resulting typically into 8–10 cells and translocate into the coelomic pouches where the adult rudiment is formed on the left side of the larval body during the late larval stage (Tanaka and Dan, 1990; Yajima and Wessel, 2012). In S. purpuratus, typically five out of the eight small micromere descendants transfer into the left coelomic pouch and are thought to resume cell cycling in the late larva during formation of the adult rudiment, whereas the remaining three small micromere descendants in the right coelomic pouch disappear and are thought to undergo apoptosis by late larval stage (Luo and Su, 2012; Campanale and Hamdoun, 2012). In Seawi knockdown embryos, however, we found an over-proliferation of Vasa positive cells in the right coelomic pouch at prism and early larval stages compared with controls (Left:Right = 4.75 ± 0.5:13.3 ± 4.79, n = 10, respectively) (Fig. 4A), and Vasa protein expression was also increased in these larvae (Fig. 4B). To test if Seawi/Piwi-like1 knockdowns affected PGC-related transcripts, we assayed nanos, vasa, and gustavus mRNA abundance by transcript accumulation. We found no significant change in the Seawi-knockdown embryos compared with the controls (Fig. 4C), suggesting that a posttranscriptional mechanism regulates Vasa over-expression. These over-proliferated Vasa-positive cells were then rapidly decreased in number (Left:Right = 5 ± 0.8:4 ± 1.8, n = 10, respectively) by late larval stage (Fig. 4A,L pluteus). To test if these Vasa-positive cells were derived from the small micromere lineage or from another lineage, Nanos2 MO was co-injected with Seawi MO. We previously identified Nanos2 knockdown results in a loss of small micromere descendants by late gastrula stage (Juliano et al., 2010), and thus Nanos2 MO can be used as a molecular tool to specifically remove the small micromere lineage. The double knockdown of Nanos2 and Seawi resulted in a reduced number of Vasa positive cells at Prism stage (Fig. 4D), suggesting the over-proliferated Vasa-positive cells in the Seawi MO embryo was likely caused by excess proliferation of small micromere descendants. In these Seawi-knockdown embryos, activated Caspase-3, an indicator of apoptosis, was detected in the coelomic pouch area as well in the mesenchyme cells (Fig. 4E, Seawi MO). The apoptotic signal was, however, reduced following addition of Z-VAD-fmk, a pan-caspase inhibitor (Fig. 4E, MOCK and Seawi MO [Higher dose]) (Fujii et al., 2009), suggesting that the Seawi knockdown led to apoptotic cell death broadly in the embryo. Taken all together, the cell cycle control of the small micromere lineage appears to be based on a Nanos-dependent mechanism that trumps Vasa over-function, and Seawi appears to be involved in this regulation together as well as functioning not just in the germ line but also in somatic lineages during embryogenesis, potentially by altering the Vasa and Gus functions (Yajima and Wessel, 2011b) (Fig. 4F).
Figure 4. Seawi knockdown induced the proliferation of Vasa positive cells in the right coelomic pouch at Prism stage. A: Left panels are control (MOCK) larvae immunostained with anti-Vasa antibody. Vasa signal was restricted into the coelomic pouches with less enriched in the right coelomic pouch (arrows) compared with the left coelomic pouch (arrowhead). Right panels are Seawi MO-injected larvae. Vasa-positive cells were more accumulated in the right coelomic pouch (arrows) compared with the left side (arrowheads) during prism and early pluteus (Day 3, E pluteus) stages yet shrunk down by the late pluteus (Day 4, L pluteus) stage. The pictures of the Prism embryo are the same embryo with a different focus. Scale bars = 50 μm. B: Anti-Vasa Immunoblotting. Vasa signal (arrow, approximately 85 kDa) was increased in the MO-injected embryos. A total of 50 embryos were loaded per lane. A graph indicates the signal value of each immunoblot band. Each value was calculated by Image J and normalized to the value of MOCK. C: Expression profiles of germ line determinants in the Seawi knockdown embryos by the quantitative RT-PCR analysis. A slight increase of the seawi transcripts was observed yet little difference was observed in other gene expressions. Each 1/Ct value was first normalized to that of Ubiquitin, and then the resultant value was standardized to that of the control embryos. D: 0.5 mM stock each of Seawi and Nanos2 MO was injected into the fertilized eggs, and the resultant embryos were reduced in number of Vasa-positive cells at Prism stage (Nanos MO +Seawi MO). Arrow indicates a right coelomic pouch area. A graph demonstrates the average number of Vasa-positive cells per embryo injected with MOCK, Seawi MO, or Seawi + Nanos MO. E: the Caspase-3 signal (red) was detected in the right coelomic pouch of the Seawi MO embryos (arrow), and increased further in the gut area with the higher dose (1mM) of Seawi-MO (dashed circle) yet the signal was reduced by Z-VAD-fmk, a pan-Caspase inhibitor. A graph indicates the Capase-3 signal value of gut (circled) region in each picture. Each value of Seawi MO injected larvae was calculated by Image J and normalized to the value of Z-VAD-fmk treated larvae (n = 3). F: A summary diagram of potential functions of Piwi in the soma and germ line. Piwi is involved in the degradation of Vasa in the somatic lineage during early embryogenesis, whereas Nanos specifically represses Vasa function and cell cycling in the PGC lineage and Piwi appears to be involved in this process together.
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