Elements of divergence in germline determination in closely related species

Abstract

Evolutionary transitions are particularly important in development of the germ line, cells which directly impact sexual reproduction. Differences in the primordial germ cells (PGCs) of two sea urchin species were examined here by stage-matched, integrated, single cell RNA-seq (scRNA-seq) datasets. Even though both species rely on inherited mechanisms to specify their germ line, this analysis revealed a variety of differences in germline gene expression, including a broader expression of the germline factor Nanos2 (Nan2) in Lytechinus variegatus (Lv) compared to Strongylocentrotus purpuratus (Sp). In Sp, Nan2 mRNA expression is highly restricted to the PGCs by a lability element in its 3'UTR, which is lacking in the mRNA of Lv-Nan2, thus explaining the difference. We discovered that the Lv-Nan2 3'UTR instead leads to its specific translation in the PGCs. The results emphasize that regulatory mechanisms resulting in germline specification rely greatly on post-transcriptional restrictions of key gene products.

Keywords: Cell biology; Evolutionary biology.

Graphical abstract

Figure 1

Identification of 23 cell-populations in Lytechinus variegatus embryos (A) Diagram of sea urchin embryogenesis. We collected embryos at eight developmental time points from 2 h postfertilization (hpf) to 18 hpf. The diagrams of embryos at 2 hpf (2H), 4 hpf (4H), 10 hpf (EB), 12 hpf (MB), 14 hpf (EG) and 18 hpf (LG) are illustrated. Undifferentiated cells (gray), ectodermal cells (blue), endodermal cells (green), mesodermal cells (purple) and germline cells (red) are shown. This figure was created with BioRender.com (https://app.biorender.com/). (B) Identification of 23 cell-populations using UMAP visualization for integrated dataset of 8 developmental time points. (C) Dot plot represents marker gene expression characterizing cell types in the Lv dataset. Dot size and dot color indicate the percentage of cells expressing the gene and the average expression level, respectively.

Figure 2

Transcriptional profile of Nan2-expressing cells in Lv embryos (A–H) Violin plots showing Lv-Nan2 expression levels of each cluster (Lv1–Lv23) in 2H (A), 4H (B), 6H (C), 8H (D), EB (E), MB (F), EG (G) and LG (H) embryo. Dots represent expression level of Lv-Nan2 in individual cells. (I–K) Venn diagrams showing the number of shared marker gene between germline cells (Lv23) and undifferentiated cells (I; Lv1–Lv3), ectodermal cells (J; Lv10 and Lv11) and endo/mesodermal cells (K; Lv18 and Lv21).

Figure 3

Gene expression changes during germline development in Lv embryos (A and A′) UMAP plot generated by subclustering of the germ cells (Lv23) extracted from the Lv-dataset (A) and UMAP plots split by developmental stages (A′) are presented. (B) The graph shows Germline_Lv1 (purple), Germline_Lv2 (blue) and Germline_Lv3 (brown) cells as a percentage of total cell number analyzed for each developmental time point. (C) Dot plot showing marker gene expression in the germline subpopulations (Germline_Lv1–Lv3). Dot size and dot color indicate the percentage of cells expressing the gene and the average expression level, respectively.

Figure 4

Marker gene expression in the Lv-Sp datasets (A–H) UMAP plots (A, C, E and G) and dot plots (B, D, F and H) performed using Lv-Sp datasets. EB (A and B), MB (C and D), EG (E and F) and LG (G and H) embryos are analyzed individually. Dot plot represents marker gene expression in Lv-derived cells (green) and Sp-derived cells (purple) separately. Dot size and dot color indicate the percentage of cells expressing the gene and the average expression level, respectively.

Figure 5

Germline subpopulations in the Lv-Sp dataset (A) UMAP plots for Nan2-positive cells extracted from the Lv-Sp datasets. UMAP plots of Lv-derived cells (B) and Sp-derived cells (C) in each developmental time point (EB–LG) are presented. (D) Dot plot showing marker gene expression in Nan2-positive cells (Nan2pos_1–7).

Figure 6

NPDE-mediated protein degradation in Sp embryos (A) NPDE located at the N-terminal side of Sp-Nan2 ORF (gray box) was divided into eight fragments containing 14–16 amino acids (aa) (NPDE1–5) and 22–23 aa (NPDE6–8). Amino acid position within the NPDE is shown at both side of each fragment. The fragments are color-coded by the percentage of amino acid residue conserved between Sp-Nan2 and Lv-Nan2 (red:＞60%; orange: 30–60%; yellow: ＜30%). (B–K and B′–K′) Sp embryos were injected with Control GFP or GFP-NPDE fusion mRNAs and mCherrymRNA. Fluorescence of GFP (B–K; green) and mCherry (B′–K′; red) were observed in the EB stage of Sp embryos. Scale bar: 20 μm. (L) Relative signal intensity against the Control GFP is presented. GFP signal intensity was normalized with mCherry signal intensity within the same embryo. The normalized GFP signal intensities of GFP-NPDE fusion proteins were compared with that of Control GFP. Graph is color-coded by the percentage of amino acid residue conserved between Sp-Nan2 and Lv-Nan2 as described above. At least 50–100 embryos were injected in each experiment, and at least 20 embryos were used for each quantification. Significance was calculated between Control GFP and GFP-NPDE fusion proteins (NPDEFL and NPDE1–8) by Student’s t test (∗: p < 0.05). Error bars indicate standard errors of samples. The number of embryos (n) examined are shown in parentheses.

Figure 7

Lv-Nan2 3′UTR regulates its protein expression Lv embryos injected with Control GFP mRNA (A), Lv-Lv mRNA (B), Lv-Sp mRNA (C), Sp-Lv mRNA (D) and Sp-Sp mRNA (E) were labeled by whole mount in situ hybridization (magenta; A–E) and immunostaining for GFP protein (green; A′–E′). DNA staining (blue) merged with in situ hybridization and immunostaining are presented (A′′–E′′). White arrows in B′ and D′ indicate the specific translation of the constructs in the germ cells. Scale bar: 20 μm.