Research ArticleDEVELOPMENTAL BIOLOGY

Hedgehog signaling controls segmentation dynamics and diversity via msx1 in a spider embryo

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Science Advances  09 Sep 2020:
Vol. 6, no. 37, eaba7261
DOI: 10.1126/sciadv.aba7261
  • Fig. 1 Early patterning in the spider embryo.

    (A) Polarity phenotypes of late stage 5 gfppRNAi, Pt-hhpRNAi, and Pt-ptcpRNAi germ discs stained by FISH for Pt-Delta (green), Pt-otd (red), and Pt-opa (blue) transcripts. (B) Stripe pattern development during the germ disc to germ band transition. Left: Semi-flat preparations of sibling embryos stained for Pt-hh (blue), Pt-Delta (red), and Pt-noto1 (green) transcripts and DNA (shown only in right panels). Approximate times AEL (top left) and surface cell counts (bottom right). Right: Signal intensity profiles in the surface cell layer along the midline (white line, left; averaged across 20-μm width); extracted images (80-μm wide) are shown together. Yellow lines refer to the center of the germ disc and the likely corresponding position (39 to 45 hours) and the posterior limit of the Pt-Delta L4 stripe (45 to 51 hours). Blue arrows indicate the anterior Pt-hh stripe(s) that undergo traveling and splitting (45 to 48 hours), green dots indicate Pt-noto1 stripes that appear simultaneously in the thorax (48 and 51 hours), and white arrows mark Pt-hh stripes generated through oscillations for L4 and O1 segments (45 to 51 hours). Scale bars, 200 μm.

  • Fig. 2 Genome-wide identification and categorization of genes regulated by Hh signaling.

    (A) Strategy for identifying DEGs between untreated and pRNAi-treated embryos. BLAT, Blast-like alignment tool. (B) Venn diagram showing the numbers of identified DEGs. DEGs identified in both Pt-hh and Pt-ptc pRNAi experiments at late stage 5 (yellow) were deduced into 98 nonredundant genes. (C) The heatmap showing fold changes (log2, the color code at the bottom) of expression levels of DEGs from stage 5 experiments, calculated from three comparisons (1) to (3). The column to the immediate right of the heatmap shows spatial expression ranges along the radius of the late stage 5 germ disc with color-coded horizontal bars and triangles. Gray, unclear staining; n.d., not determined. In the right, four categories of genes, their representative expression pattern in the germ disc, and their regulation by Pt-hh and Pt-ptc are schematically represented. (D) Explanation of the expression color code and expression areas shown in (C). Spatial expression ranges observed in WT, Pt-ptcpRNAi, and Pt-hhpRNAi germ discs are illustrated in purple, orange, and green, respectively. (E) Expression of some of the DEGs in late stage 5 pRNAi germ discs. These photos are the same as in data file S5. (F) Varied expression profiles of DEGs and Pt-hh in late stage 5 WT germ discs. Data and images are shown as in Fig. 1B, except that extracted images are 40-μm wide and along the radius. The numbers in parentheses indicate the classes of the genes. Germ discs are oriented so that the CM cells are to the right. Scale bars, 200 μm (E and F). Photo credit: Yasuko Akiyama-Oda, JT Biohistory Research Hall.

  • Fig. 3 Complimentary striped expression of Pt-msx1and Pt-hh.

    (A) WT sibling embryos serially fixed at 2.5-hour intervals starting from early stage 7 and stained for Pt-hh and Pt-msx1 transcripts. Brackets mark the opisthosomal region. (B and C) WT sibling embryos stained for Pt-msx1 (red) and Pt-hh (green) transcripts and DNA (blue). Semi-flat preparations (B, top), the extracted surface image of the opisthosomal region of the 45-hour embryo (B, bottom), and the germ band of a 54-hour embryo (200-μm wide) (C). Signal intensity profiles in the surface cell layer along the midline (white line; averaged across 20 μm width) is shown together (C). White arrows indicate cells expressing Pt-msx1 (B). Scale bars, 50 (B, bottom) and 200 μm (others).

  • Fig. 4 Effects of Pt-msx1 pRNAi on periodic stripe formation.

    (A) Developmental time course of gfppRNAi and Pt-msx1pRNAi embryos. These images are related to movie S1. Approximate times AEL and developmental stages are shown. The Pt-msx1pRNAi embryo does not transform into a germ band, remaining in a fan-like shape. (B) gfppRNAi and Pt-msx1pRNAi embryos at stage 7 stained for DNA. The boxed caudal regions are magnified in bottom panels, which show the images at three different depths: 0 μm, surface epithelial cell layer; 8 μm, mesodermal cell layer; 22 μm, yolk layer (gfppRNAi) or interior cells, some of which appear to be dying (msx1pRNAi). The white dotted lines indicate the boundary between embryonic and extraembryonic (ex) area. (C to E) gfppRNAi and Pt-msx1pRNAi embryos at late stage 5 (C), stage 7 (D), and stages indicated at the bottom right (E) stained for transcripts indicated. The gfppRNAi germ discs stained for Pt-otd and Pt-AP2A are the same as in data file S5. Arrows, the stripes in the head; white brackets, the thoracic region; black brackets, the opisthosomal region (D). The Pt-msx1pRNAi embryos do not show repetitive striped expressions of genes (D and E). Anterior is to the left. Scale bars, 50 (B, bottom) and 200 μm (others). Photo credit: Yasuko Akiyama-Oda, JT Biohistory Research Hall.

  • Fig. 5 Striped and oscillatory expression of DEGs identified in Pt-msx1 pRNAi and RNA-seq experiments.

    (A) Summary of expression of 40 selected DEGs: (a) nine DEGs that were also identified in Pt-hh and Pt-ptc pRNAi experiments; (b) other 31 DEGs. Expression areas in the epithelium of WT germ discs (a) and germ bands (b), numbers of DEGs (N), and expression in Pt-msx1pRNAi embryos, where “–” indicates expression domains diminish, “+” indicates expression domains enlarge, and “+/−” indicates expression domains not clearly different from WT. T/O boundary, thorax-opisthosoma boundary. (B to D) gfppRNAi and Pt-msx1pRNAi embryos stained for transcripts of genes that are listed in Aa (B) and Ab (C and D) (“not clear” genes were omitted). Embryos were at late stage 5 (B), stage 7 (C and D), or stages as indicated. The numbers in parentheses indicate the classes of the genes (B). Arrows (C) (aug3.g3963) point to the boundary between the thorax and opisthosoma, where very faint signals are observed. (E) Relative times of onsets of segmentation clock gene expression during the oscillation cycle at the posterior pole of the germ band. Anterior is to the left (stage 7). Scale bars, 200 μm. Photo credit: Yasuko Akiyama-Oda, JT Biohistory Research Hall.

  • Fig. 6 Dynamics of the primary wave of Pt-msx1 expression.

    (A to D) Sibling embryos at the indicated time points stained for transcripts of Pt-msx1 (red), Pt-AP2-A (green), and DNA (blue). (A) Semi-flat preparations. (B) Extracted images (80-μm wide) along the midline (white line, A). At the position of nuclei, white spots are placed, from where the quantitative signal values were extracted. Bar graphs show numbers of cells expressing substantial transcript levels (color-coded, inset) in each subdivided area (20 μm by 80 μm). (C) Dot plots showing relationships between cell positions and signal intensities of Pt-msx1 (red) and Pt-AP2-A (green). (D) Dot plots showing relationships between signal intensities of Pt-msx1 and Pt-AP2-A in individual cells. Gray spots (45 to 51 hours) (D) were derived from cells at the AP position range that are indicated by gray bars in (C), which mark Pt-msx1 expression for the O1 segment. Yellow lines refer to the center of the germ disc and the likely corresponding position (33 to 45 hours) and the posterior limit of the Pt-msx1 L4 stripe (45 to 51 hours). Gray dots, three Pt-msx1 stripes (L2 to L4) resulting from tri-splitting of the primary Pt-msx1 wave; blue arrows, Pt-msx1 stripes at the bi-splitting sites (A and C). Anterior is to the left (36 to 51 hours). Germ disc orientation is arbitrary at 33 hours. Scale bar, 200 μm.

  • Fig. 7 Propagation and attenuation dynamics of the Pt-msx1 primary wave.

    (A) Initiation and propagation of the Pt-msx1 wave independent of the central part of the early germ disc. An embryo was laser-irradiated to extirpate the region surrounding the embryonic pole (arrow) at 24 hours AEL and was photographed at 27 and 38.5 hours AEL together with an untreated sibling. Both embryos were fixed at 39.5 hours and were stained for Pt-msx1 (red) and Pt-otd (green) transcripts and DNA (blue). (B) Early regulation of Pt-msx1 expression by Hh signaling. Pt-msx1 transcript staining of gfppRNAi, Pt-hhpRNAi, and Pt-ptcpRNAi germ discs at early stage 5. The expression already expanded to the periphery in the hhpRNAi germ disc. (C) Pt-msx1 expression dynamics in a static field. A live embryo with a labeled cell clone (yellow) was photographed at 33.5 and 41.5 hours AEL and was then stained as indicated. Arrows indicate the clone position, which remained largely unchanged, and the expression of Pt-msx1 was detected in the more peripheral region. (D) Effects of Pt-ci knockdown on the Pt-msx1 expression. gfppRNAi and Pt-cipRNAi embryos at stage 7 were stained for Pt-msx1 transcripts. Pt-msx1 continued to be expressed in the corresponding caudal region in the Pt-cipRNAi embryo. Anterior is to the left (D). Scale bars, 200 μm. Photo credit: Yasuko Akiyama-Oda, JT Biohistory Research Hall.

  • Fig. 8 Tri-splitting dynamics of the Pt-msx1 expression.

    (A to D) Sibling embryos at the indicated time points stained for Pt-msx1 (red) and Pt-noto1 (green) transcripts and DNA (blue). Data and images are shown as in Fig. 6. In (D), intensity values were extracted from cells at the position range indicated by blue bars in (B). Yellow lines refer to the posterior limit of the Pt-msx1 L4 stripe. Gray dots, three Pt-msx1 stripes (L2 to L4) resulting from tri-splitting of the primary Pt-msx1 wave; gray arrows, Pt-msx1 expression peaks or stripes for O1 and O2 segments generated through oscillations; blue arrows, Pt-msx1 stripes at the bi-splitting sites; white circles, the Pt-msx1 L1 stripe appearing later (A and C). (E) Phenotypes of Pt-msx1eRNAi and gfpeRNAi cell clones during tri-splitting. The samples were stained for Pt-msx1 (magenta) and Pt-noto1 (green) transcripts, eRNAi cell clones (red), and DNA (blue). Dot plots show relationships between positions of cells (spots) along the AP axis and signal intensities of Pt-msx1 (red) and Pt-noto1 (green) in the eRNAi cell clones (outlined). White arrows indicate disordered patterns of Pt-noto1 expression in the Pt-msx1eRNAi cell clone. Anterior is to the left. Scale bars, 200 μm (A) and 100 μm (E).

  • Fig. 9 Pt-msx1–mediated distinct segmentation dynamics in different body regions.

    (A and B) Initiation of clock gene expression. Sibling embryos at 39 and 42 hours AEL were stained for the indicated transcripts and DNA (blue). Extracted images (50-μm wide) along the line (left) are shown with bar graphs that show numbers of cells (spots) expressing substantial transcript levels (color-coded, inset) in each subdivided area (20 μm by 50 μm). (C) Phenotypes of Pt-msx1eRNAi and gfpeRNAi cell clones in the opisthosomal region at early stage 6. The samples stained for Pt-ci and Pt-krü-1 transcripts, eRNAi cell clones, and DNA are shown as indicated. Dot plots (insets) show signal intensities of Pt-ci and Pt-krü-1 in individual cells (spots) in the cell clones. Yellow arrows, the extraembryonic (ex) region; yellow dotted lines, the boundary between embryonic and ex areas (Pt-msx1eRNAi). The Pt-msx1eRNAi embryo is slightly older than the gfpeRNAi embryo, but most cells in the Pt-msx1eRNAi cell clone (white arrows) remained expressing Pt-ci and not Pt-krü-1. (D) Insertion of Pt-msx1 expression between the splitting Pt-hh stripes in the head region. The head region of sibling embryos stained for Pt-msx1 (red) and Pt-hh (green) transcripts and DNA (blue). The boxed 80-μm-wide regions were extracted, and bar graphs were produced as in (A) and (B), except that each subdivided area is 10 μm by 80 μm. (E) Phenotypes of Pt-msx1eRNAi and gfpeRNAi cell clones during bi-splitting. The samples stained for Pt-hh and Pt-msx1 transcripts, eRNAi cell clones, and DNA are shown as indicated. White arrows mark unseparated Pt-hh stripes. Anterior is to the top (A to C) and to the left (D and E). Scale bars, 100 μm.

  • Fig. 10 Schematic diagram of early patterning in the spider embryo.

    Germ-disc patterning and varied stripe-forming events were controlled by Hh signaling along the radius of the germ disc or along the AP axis of the germ band. Expressions of class I, II, and III genes in the germ disc are regulated by Pt-hh and Pt-ptc. The class II gene Pt-msx1 is involved in generating distinct gene expression dynamics in the three body regions. See the main text for details.

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