A cellular and molecular analysis of SoxB-driven neurogenesis in a cnidarian
- Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Ireland
- Centre for Microscopy and Imaging, Discipline of Anatomy, National University of Ireland, Galway, Ireland
- National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland
- Carbohydrate Signalling Group, Microbiology, School of Natural Sciences, National University of Ireland Galway, Ireland
- Whitney Laboratory for Marine Bioscience, University of Florida, United States
- Department of Biology, University of Florida, United States
- Computational and Statistical Genomics Branch, Division of Intramural Research, National Human Genome Research Institute, National Institutes of Health, United States
Figures
Figure 1 with 4 supplements
Piwi1 and Soxb1 are co-expressed in i-cells and germ cells.
(A–D) Analysis of sexual polyps’ mid (A) and lower (B) body column, and early (C) and late (D) oocytes. (E–G) Analysis of feeding polyps’ head (E), mid-(F), and lower (G) body column. (A–D) were …
Figure 1—figure supplement 1
The animal model Hydractinia.
(A) Simplified cladogram showing the position of various cnidarian clades relative to bilaterians. (B) The life cycle of Hydractinia. (B) Schematic representation of Hydractinia’s body wall and the …
Figure 1—figure supplement 2
i-Cell location during different stages in ontogeny, marked by Piwi1.
(A) During embryogenesis and in the larval stage, i-cells are exclusively present in the endoderm; they migrate to the epidermis during metamorphosis. (B) Schematic representation.
Figure 1—figure supplement 3
The structure of the nervous system in embryogenesis and larvae.
(A) GLWamide+ neurons. (B) RFamide+ neurons. (C) Nematocyst capsules, representing mature nematocytes, visualized by lectin staining. (D) Acetylated tubulin immunostaining. (E) Ncol3+ nematoblasts. …
Figure 1—figure supplement 4
The structure of the nervous system in adult feeding and sexual polyps.
(A) GLWamide+ neurons. (B) RFamide+ neurons. (C) Ncol1+ and Ncol3+ nematoblasts in feeding polyps. (D) Ncol1+ and Ncol3+ nematoblasts in sexual polyps. (E) Acetylated tubulin immunostaining in …
Figure 2
Partial overlap in the expression of Soxb1 and Soxb2, and Soxb2 and Soxb3 in feeding polyps.
(A–C) Single-molecule fluorescence in situ hybridization (FISH) with probes against Soxb1 and Soxb2 showing upper-mid (A), lower-mid (B), and lower (C) body column. (D–F) Single-molecule FISH with …
Figure 3
Soxb1, Soxb2, and Rfamide transgenic reporter animals.
(A–E) Soxb1::tdTomato reporter animal. Animals were fixed and stained with an anti-dsRed antibody. (A) Whole feeding polyp. (B) Lower polyp body column. (B’) Higher magnification of tdTomato+ …
Figure 4 with 1 supplement
Sequential expression of Soxb1 and Soxb2.
(A) Immunofluorescence of Soxb1::tdTomato and Soxb2::GFP double positive cells that resemble differentiating neurons by morphology. (B) In vivo time lapse imaging of a Soxb1::tdTomato+ cells (shown …
Figure 4—figure supplement 1
In vivo time lapse imaging of a Soxb1::tdTomato/Soxb2::GFP double transgenic reporter animal.
(A, B) Two cases of gradual upregulation of the Soxb2 reporter in Soxb1-expressing cells over an 8 hr time frame.
Figure 5 with 3 supplements
Analysis of dissociated Hydractinia cells by conventional and imaging flow cytometry.
(A) Gating strategy of i-cells, nematocytes, mixed progenitors, and GFP- cells from a Piwi1 reporter animal. (B) Gating of putative neural progenitors from a Soxb2 reporter animal. (C) Gating two …
Figure 5—figure supplement 1
Representative flow cytometry density plots identifying subpopulations as defined by internal complexity (side scatter [SSC]) and level of GFP expression of Hydractinia transgenic reporter animals and matched wild type animals (A–I) and imaging flow cytometry (J).
(A–C) Characterization of feeding polyps, (D–F) male and (G–I) female sexual polyps. The nature of the reporter transgene is indicated above each density plot. (J) Imaging flow cytometric analysis …
Figure 5—figure supplement 2
Typical flow cytometry gating strategy used for cell cycle analysis of transgenic animals and a wild type animal.
(A) Density plot of cells stained with Hoechst 33342 cell permeant dye versus side scatter (SSC-A). The gated population represents Hoechst positive live, nucleated cells. Doublets are subsequently …
Figure 5—figure supplement 3
Transcriptomic analysis of Hydractinia single cells.
(A) UMAP dimensional reduction projection of 7071 Hydractinia symbiolongicarpus cells. (B) Heatmap showing the top 10 markers for each cell cluster across all clusters and within nematoblast …
Figure 6 with 2 supplements
Cellular events during Hydractinia head regeneration in an Rfamide::GFP transgenic reporter animal.
(A) Intact head, characterized by few S-phase cells, extensive RFamide+ neuronal network, and no Piwi1high i-cells or their putative Piwi1low progeny. (B) 12 hr post decapitation (hpd) showing many …
Figure 6—figure supplement 1
Piwi2 antibody validation.
Zygotes were injected with an shRNA targeting Piwi2 (shPiwi2) or a control shRNA targeting the GFP sequence (shGFP). At 48 hr post fertilization, animals were fixed and stained with the anti-Piwi2 …
Figure 6—figure supplement 2
In vivo tracing of RFamide-GFP+ neurons during regeneration located in the (A) lower part and (B) upper part of the body column during the first 20 hr post decapitation (hpd), and (C) from 24 hpd until 72 hpd.
No cell proliferation or migration of individual neurons (circled) was observed, indicating no role during this process. Images are single optical slices. Scale bars = 40 μm.
Figure 7 with 2 supplements
Effects of SoxB genes downregulation in embryogenesis.
(A) Short hairpin RNA (shRNA)-mediated knockdown of Soxb1, Soxb2, and Soxb3. (B) Higher magnification of Piwi1+ and S-phase cells showing reduced i-cell numbers following Soxb1 downregulation. Note …
Figure 7—figure supplement 1
Validation of shRNA experiments.
(A) Validation of shSoxB1, shSoxB2, and shSoxB3 knockdowns by single-molecule fluorescence in situ hybridization (SABER FISH) on 3DPF larvae. Larvae injected with shSoxB1 showed lower expression …
Figure 7—figure supplement 2
Effect of Soxb1 downregulation on epidermal ciliation in 3-day old larvae using acetylated tubulin antibody staining and in situ hybridization of Soxb2 in sexual polyps (A) shGFP-injected animal.
(B) shSoxb1-injected animal. (C) Male sexual polyp. (D) Female sexual polyp.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Anti-Piwi1 (rabbit polyclonal) | https://doi.org/10.1126/science.aay6782 | N/A | IF (1:2000) |
| Antibody | Anti-Piwi2 (Guinea pig polyclonal) | In-house | N/A | IF (1:500) |
| Antibody | Anti-acetylated tubulin (mouse monoclonal) | Sigma-Aldrich | Ca# T7451 | IF (1:1000) |
| Antibody | Anti-RFamide (mouse polyclonal) | https://doi.org/10.1007/s004270050181 | IF (1:1000) | |
| Antibody | Anti-GLWamide (rabbit polyclonal) | https://doi.org/10.1007/s004270050181 | IF (1:1000) | |
| Antibody | Anti-Ncol1 (rabbit polyclonal) | https://doi.org/10.1371/journal.pone.0022725.g001 | IF (1:500) | |
| Antibody | Anti-Ncol3 (guinea pig polyclonal) | https://doi.org/10.1371/journal.pone.0022725.g001 | IF (1:500) | |
| Antibody | Anti-GFP (rabbit polyclonal) | Santa Cruz | Ca# 8334 | IF (1:1000) |
| Antibody | Anti-RFP (rat polyclonal) | Chromotek | Ca# 5F8 | IF (1:1000) |
| Strain, strain background (Hydractinia symbiolongicarpus) | 293-10 wild type animals | This paper | N/A | Materials and methods |
| Strain, strain background (Hydractinia symbiolongicarpus) | SoxB1::tdTomato line | This paper | N/A | Materials and methods |
| Strain, strain background (Hydractinia symbiolongicarpus) | SoxB2::GFP line | This paper | N/A | Materials and methods |
| Strain, strain background (Hydractinia symbiolongicarpus) | RFamide::GFP line | This paper | N/A | Materials and methods |
| Strain, strain background (Hydractinia symbiolongicarpus) | RFamide::SoxB1::GFP | This paper | N/A | Materials and methods |
Additional files
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Supplementary file 1
Combination of cell fractions used for differential gene expression analysis.
- https://cdn.elifesciences.org/articles/78793/elife-78793-supp1-v3.docx
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Supplementary file 2
Differentially expressed gene lists.
- https://cdn.elifesciences.org/articles/78793/elife-78793-supp2-v3.xlsx
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Supplementary file 3
Files pertaining to single-cell transcriptomic analysis.
- https://cdn.elifesciences.org/articles/78793/elife-78793-supp3-v3.zip
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Supplementary file 4
List of SRA accession numbers.
- https://cdn.elifesciences.org/articles/78793/elife-78793-supp4-v3.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/78793/elife-78793-mdarchecklist1-v3.pdf
