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Abstract
Specification of the germ cell lineage is required for sexual reproduction in animals. The mechanism of germ cell specification varies among animals but roughly clusters into either inherited or inductive mechanisms. The inductive mechanism, the use of cell-cell interactions for germ cell specification, appears to be the ancestral mechanism in animal phylogeny, yet the pathways responsible for this process are only recently surfacing. Here, we show that germ cell factors in the sea star initially are present broadly, then become restricted dorsally and then in the left side of the embryo where the germ cells form a posterior enterocoel. We find that Nodal signaling is required for the restriction of two germ cell factors, Nanos and Vasa, during the early development of 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.
Germ cell factors are restricted dorsally and on the left during embryogenesis. (A) Primordial germ cells can be specified by either inherited mechanisms or inductive mechanisms. The inherited mechanism requires asymmetric localization of germ cell factors in the egg and whichever embryonic cells acquire these factors will become the germ cell lineage. In the inductive mechanism, germ cell factors are either not present or are present ubiquitously in the egg, and embryonic signals instruct which cells will accumulate germ cell factors and will eventually become the germ cell lineage. (B) Dorsal/ventral asymmetry in sea star development can be seen by two morphological events best visualized in the left side view. The mouth forms on the ventral side of the outer cell layer, while a posterior pouch begins to form on the dorsal side of the developing gut (purple). Left/right asymmetry in sea star development can be seen by one morphological event best visualized in the dorsal side view. The posterior pouch is present only in the left side of the larva (purple). (Ci-viii) Germ cell factors Vasa and Nanos are present in a vegetal ring at the mid-gastrula stage (purple). By the late gastrula stage, Vasa and Nanos accumulate only in the dorsal side of the developing gut (purple). During the dorsal restriction of germ cell factors, Nodal is present in the ventral side of the embryo (red). Areas outlined with a dotted circle lose the presence of germ cell factors. Scale bar: 100 μm. (Cix-xvi) Germ cell factors Vasa and Nanos are present in a dorsal patch at the late gastrula stage (purple). By the early larva stage, Vasa and Nanos accumulate only in the left side of the developing gut (purple). During the left restriction of germ cell factors, Nodal is present in the right side of the embryo (red). Areas outlined with a dotted circle lose the presence of germ cell factors. (Cxvii,xviii) Nanos and Vasa RNA levels sequentially decrease during early embryogenesis when examined using qPCR analysis.
Nodal restricts the germ cell factors Vasa and Nanos. (A) Experimental design. Oocytes were injected with Nodal morpholino or control morpholino and the resulting embryos were fixed at the gastrula stage. Germ cell markers (purple) and dorsal/ventral markers (red and blue) were visualized using RNA in situ hybridization. (B) Our results support the model that Nodal expression (red) in the ventral side of the embryo is required for the loss of germ cell factors (purple) in the ventral side of the embryo. Nodal expression in the ventral side of the embryo is also required for the later restriction of itself and Tbx2/3 to the dorsal side of the embryo. (C,D) Vasa and Nanos accumulate in a dorsal patch on the gut in 95% and 98% of embryos injected with control morpholino. In contrast, Vasa and Nanos are not restricted and instead accumulate in a ring around the gut in 94% and 97% of embryos injected with a Nodal morpholino. P=0.00002, Vasa; P=0.0007, Nanos. (E,F) Nodal accumulates in the dorsal-right side of the developing gut in 100% of embryos injected with control morpholino. Tbx2/3 mRNA accumulates in the dorsal side of the developing gut in 99% of embryos injected with control morpholino. In contrast, Nodal and Tbx2/3 mRNAs are not restricted and instead become radialized in 100% and 86% of embryos injected with a Nodal morpholino. P=2.5E-13, Nodal; P=0.0002, Tbx2/3. Scale bars: 100 μm.
Nodal restricts transcription of germ cell factors and causes their degradation. (A) Experimental design: EU RNA nucleotide analog was added to embryos at the mid-gastrula stage. RNA was extracted from larva stage embryos and ‘New RNA’ (EU+) was separated from ‘Old RNA’ (EU−) by covalently attaching biotin to EU and using magnetic streptavidin beads. (B) qPCR analysis shows that germ cell factor RNAs (Vasa and Nanos) produced prior to EU labeling are significantly increased in embryos injected with Nodal morpholino (green) compared with embryos injected with control morpholino (blue). As a control for inhibition of the Nodal signaling pathway, Lefty RNA levels are significantly decreased in embryos injected with Nodal morpholino (green) compared with embryos injected with control morpholino (blue). (C) qPCR analysis shows that levels of germ cell factor RNAs (Vasa and Nanos) produced during EU labeling are significantly increased in embryos injected with Nodal morpholino (green) compared with embryos injected with control morpholino (blue). As a control for inhibition of the Nodal signaling pathway, Lefty RNA levels are significantly decreased in embryos injected with Nodal morpholino (green) compared with embryos injected with control morpholino (blue).
Nodal restricts cells from bulging out of the mid-gut. (A) When oocytes are injected with control morpholino, a patch of cells bulges out of the dorsal side of the gut during the late gastrula stage. By the early larva and larva stage, cells only bulge out from the left side of the gut and only one pouch begins to form. (B) When oocytes are injected with Nodal morpholino, cells bulge out in a ring around the gut during the late gastrula stage and early larva stage. By the larva stage, multiple discrete pouches begin to form radially all around the gut.
Nodal inhibits the apical constriction of mid-gut cells. (A) DIC and GFP images of an embryo injected with a control morpholino and PH domain of PLCγ-GFP. The first column represents a section in which the endodermal cells are in cross-section. The second column represents a section in which the posterior enterocoel cells are in cross-section. Cells are outlined in pink and apical and basal sides of the cells are labeled A and B, respectively. (B) DIC and GFP images of an embryo injected with Nodal morpholino and PH domain of PLCγ-GFP. In these images, the posterior enterocoel cells are in cross-section. Cells are outlined in pink and apical and basal sides of the cells are labeled A and B, respectively. (C) The cell volume of endodermal cells and posterior enterocoel cells is similar in embryos injected with control morpholino. (D) In embryos injected with control morpholino, the ratio of basal:apical cell length is significantly increased in posterior enterocoel cells (∼4) compared with endodermal cells (∼1). In embryos injected with Nodal morpholino, the ratio of basal:apical cell length in multiple posterior enterocoels (∼4 and ∼3) is similar to that of the posterior enterocoel in embryos injected with control morpholino (∼4). Scale bars: 25 μm.
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