Supplementary MaterialsSupplementary information develop-145-155663-s1. this animal. We learned that Nodal inhibits germ cell factor accumulation in three ways including: inhibition of specific transcription, degradation of specific mRNAs and inhibition of tissue morphogenesis. These results document a signaling mechanism required for the sequential restriction of germ cell factors, which causes a specific set of embryonic cells to become the primordial germ cells. imaging capabilities. Here, we show that Nodal signaling is required for the sequential restriction of Nanos and Vasa mRNAs in early development. Although the function of Nanos and Vasa remains to be tested in PF-06855800 the germ line of sea stars, we strongly suggest that they are required for germ cell specification because: (1) these factors are usually found together in the germ cell lineage (Juliano et al., 2010); (2) these factors are required for germ cell specification in many animals (Juliano et al., 2010); and (3) these factors accumulate in the posterior enterocoel (PE), a structure that has previously been shown to contribute to primordial germ cells (Inoue et al., 1992). Although we are not able to test Vasa function specifically in the germ line by conventional means (knockdown of Vasa expression in early embryos leads to aborted development, as it does in the sea urchin; data not shown), we propose that the sequential restriction of germ cell factors is a significant mechanism involved in germ cell specification: i.e. germ cell factors are present broadly in cells during early development and embryonic signals reduce the field of cells to the future germ line. RESULTS Germ cell factors are sequentially restricted during early development We noticed in previous studies in that the mRNA of the germ cell factors Vasa, Nanos and Piwi are present broadly in early development but then become restricted to the posterior enterocoel (PE) (Fresques et al., 2014, 2016). The restriction of Vasa and Nanos mRNA in particular shows a similar restriction pattern during two stages of embryonic development: i.e. Vasa and Nanos accumulate in a vegetal ring at the mid-gastrula stage and, subsequently, by the late-gastrula stage, these two factors are eliminated from cells in the ventral part of the developing gut (Fig.?1Ci-vi). Then, in the transition from late-gastrula to early larva, these same germ cell factors are eliminated from cells in the right side of the developing gut, and the cells with the remaining mRNA on the left side form the posterior enterocoel (Fig.?1Cix-xiv). In order to test whether germ factor mRNAs are decreasing or just shifting during this dynamic period, we performed qPCR. Our results show that during the dorsal and left phases of restriction, Vasa and Nanos mRNA levels decrease significantly (Fig.?1Cxvii-xviii). This suggests that Vasa and Nanos mRNA is lost from cells in the ventral and right part of the developing gut. As a result, Vasa and Nanos mRNA is specifically retained in cells in the dorsal and left side of the gut. Nodal is required for the restriction of germ cell factors We next sought to determine what embryonic signal(s) could be involved in the dorsal and left restriction of Vasa and Nanos. Previous research in a closely related animal, the sea urchin, PF-06855800 shows that Nodal is required for the patterning of the dorsal/ventral and left/right axes (Duboc et al., 2004, 2005). In order to test whether Nodal is relevant for restriction of PF-06855800 germline factor mRNAs in the sea star, we first determined where Nodal mRNA was localized during sea star development (Fresques et al., 2014). We found that Nodal is expressed in the domain opposite to germ cell factors: in the ventral side of the embryo during the blastula stage and then in the right side of PF-06855800 the embryo during the late gastrula stage (Fig.?1Cvii,xv; Fig.?S1). These data suggest that Nodal expression counteracts Rabbit Polyclonal to CYC1 the retention of germ cell factor mRNA’s (Fig.?1Ci,ii,ix,x,.