Oren M et al. (2019), Individual Sea Urchin Coelomocytes Undergo Soma...

ECB-ART-47286

Front Immunol 2019 Jan 01;10:1298. doi: 10.3389/fimmu.2019.01298.

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Individual Sea Urchin Coelomocytes Undergo Somatic Immune Gene Diversification.

Oren M , Rosental B , Hawley TS , Kim GY , Agronin J , Reynolds CR , Grayfer L , Smith LC .

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The adaptive immune response in jawed vertebrates is marked by the ability to diversify somatically specific immune receptor genes. Somatic recombination and hypermutation of gene segments are used to generate extensive repertoires of T and B cell receptors. In contrast, jawless vertebrates utilize a distinct diversification system based on copy choice to assemble their variable lymphocyte receptors. To date, very little evidence for somatic immune gene diversification has been reported in invertebrate species. Here we show that the SpTransformer (SpTrf ; formerly Sp185/333) immune effector gene family members from individual coelomocytes from purple sea urchins undergo somatic diversification by means of gene deletions, duplications, and acquisitions of single nucleotide polymorphisms. While sperm cells from an individual sea urchin have identical SpTrf gene repertoires, single cells from two distinct coelomocyte subpopulations from the same sea urchin exhibit significant variation in the SpTrf gene repertoires. Moreover, the highly diverse gene sequences derived from single coelomocytes are all in-frame, suggesting that an unknown mechanism(s) driving these somatic changes involve stringent selection or correction processes for expression of productive SpTrf transcripts. Together, our findings infer somatic immune gene diversification strategy in an invertebrate.

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Genes referenced: 185-333 apc LOC100887844 LOC100893907 LOC105441782 LOC115919910
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	Figure 1. The SpTrf genes have a variety of element patterns. Element patterns identified for the genes amplified and sequenced in this study are shown. Elements are presented as rectangles of different colors. Element 10, which has very diverse sequence among genes, defines the gene name (4). This figure is based on data from Buckley and Smith (8).
	Figure 2. The percentages of SpTrf + small phagocytes in the coelomic fluid increase in response to Vibrio challenge. Three adult sea urchins were injected with heat-killed Vibrio diazotrophicus. Coelomic fluid (CF) was collected prior to injection (day 0) and after injection on days 1, 2, and 14. (A–D) Cells were incubated with a mix of three anti-SpTrf (formerly anti-Sp185/333) primary antibodies followed by incubation with a secondary antibody labeled with Alexa Fluor 488 (AF-488) to identified SpTrf+ cells. Nuclear staining with propidium iodine was used to exclude dead cells. Results for sea urchin 1 are shown. Percentages of live SpTrf+ coelomocytes from animal 1 at all time points are shown within the rectangular gate. SpTrf+ coelomocytes increase to the highest recorded level on day 2 post challenge and return to the basal level by day 14. (E) The mean of the percentages of SpTrf+ coelomocytes for the three individual animals at different time points are shown. Differences in the percentages of SpTrf+ cells over time for the individual animals were significant by Anova two factor analysis (p < 0.05). Additionally, the day 2 time point was analyzed compared to days 1 and 14 time points using Anova single factor (*indicates p < 0.05). Vertical bars indicate standard errors.
	Figure 3. The percentages of red spherule cells in the purple sea urchin coelomic fluid does not change in response to Vibrio challenge. Three adult sea urchins were injected with the heat-killed marine bacterial species, Vibrio diazotrophicus. Coelomic fluid was collected before injection (day 0) and post injection at days 1, 2, and 14. Cells were labeled with propidium iodine to exclude dead cells. Red spherule cells were gated by their far red auto-fluorescence (APC channel). (A–D) Percentages of live red spherule cells from animal #1 at all time points (within the rectangular gate) in the coelomic fluid over 14 days post challenge. (E) Percentages of red spherule cells in the total live coelomocyte population of the three animals tested. Differences in the red spherule cell percentages over time were not significant by Anova two factor analysis. Standard deviation is indicated by vertical bars.
	Figure 4. Sorting and isolation of single cells from two coelomocyte sub-populations and from sperm for WGA. Small phagocytes that express SpTrf on their surface and red spherule cells were sorted from animals 1–3 pre-challenge on day 0 as well as on days 1 and 2 post-challenge with Vibrio diazotrophicus. Sperm cells were collected directly from the gonopores of the same sea urchins. (A) Coelomocytes were gated from debris based on forward scatter (FSC) and side scatter (SSC). (B) Live cells were gated for propidium iodide (PI) exclusion and for surface staining of anti-SpTrf and secondary antibody conjugated with Alexa Fluor 488 (AF-488). (C) Live cells incubated with the secondary antibody alone did not show AF-488 staining. (D) Red spherule cells were gated based on their natural far-red auto-fluorescence (allophycocyanin channel; APC). Single cells with high auto-fluorescence were sorted and observed post-sorting by light microscopy (inset). (E) Live cells with high surface SpTrf protein levels were gated based on the AF-488 fluorescence of the secondary antibody. Single cells were sorted and observed post-sorting by fluorescence microscopy (inset). (F) No cells were recorded in the same gate as in (E) for the sample incubated with the secondary antibody only. This demonstrated that all cells within the gate in (E) had SpTrf on the surface and were likely small phagocytes. (G) Sperm cells were obtained by electric shock stimulation (16–20 mA), diluted to 1×105 cells/ml, stained with Hoechst 33342 and serially diluted to 0.25 cells/μl. (H) Single cells of each type were isolated either by FACs or manually into 4 μl of 3.3X PBS in a 384-well plate, observed under epifluorescent microscope, and subjected to WGA using the multiple displacement amplification with the REPLI-g single-cell kit (Qiagen). The scale bars in the insert figures in (D,E) are 10 μm.
	Figure 5. The SpTrf gene family profile is different among single coelomocytes. (A) The standard structure of an SpTrf gene shows the locations of F2 and R9 degenerate primers that were used to amplify most of the second exon. This figure is modified from (6). (B) SpTrf gene amplification profiles from single coelomocytes and single sperm from three sea urchins that were processed for WGA. The upper panel shows the amplicon patterns from individual coelomocytes from three animals. Both single red spherule cells (red numbers) and single small phagocytes (black numbers) show variable sizes of the SpTrf gene amplicons. Circled lane numbers indicate samples for which amplicons were sequenced and include one coelomocyte and one sperm from each animal. Superscript letters associated with lane numbers indicate the time after immune challenge with Vibrio diazotrophicus: A, 1 day post challenge; B, 2 days post challenge. The controls (C) for each of the three sea urchins show amplicons from ~106 coelomocytes that were not processed for WGA. The lower panel shows the amplicon patterns of 10 single sperm cells from each animal. SpGAPDH is a single copy gene that was employed as the positive control for all samples to verify that genomic DNA after the WGA process would support PCR.
	Figure 6. The alignment of single cell derived SpTrf gene amplicon sequences indicate different element patterns and SNPs. All sequences were aligned using Bioedit software (46). The initial alignment was done with ClustalW on the deduced amino acid sequences, which was reverted to nucleotide sequences and optimized manually. The sequence identity level (percent identity) for each position is indicated at the top. Horizontal gray rectangles show regions of matching nucleotides in which SNPs are indicated as vertical black lines. Gaps are shown as black horizontal lines and indicate missing elements. Sequence names indicate source based on sperm (S), coelomocytes (C), or both (M), followed by element pattern, and sequence version number. Detailed alignment is presented in Figure S4.
	Figure 7. Single cell analysis indicates somatic gene deletion, duplication, and sequence diversification in the SpTrf gene family. Amplicons of one coelomocyte and one sperm from each animal (as indicated in Figure 5B) were cloned and sequenced. (A) The Venn diagram illustrates shared and unique gene amplicons (element pattern followed by a sequence variant number after the dash) from single coelomocytes and single sperm from three animals. Underlined element patterns indicate gene sequences that are unique to sperm and are not found in coelomocytes for each animal. (B) qPCR results using primers that amplify the second exon of all SpTrf genes plus primers designed for specific gene sequences are shown as ratios of coelomocyte amplicons relative to sperm amplicons for each animal. Ratios of gene copy number between multiple coelomocyte and multiple sperm samples that were not processed for WGA are indicated in blue. Ratios of gene copy number between single coelomocyte and single sperm from the same animal (same cells as in A) are shown in red. The horizontal line represents a ratio of 1 (identical gene copy numbers between coelomocyte and sperm). Samples in which primers did not generate amplicons by qPCR are indicated with an X. See Table S2 for details.
	Figure 8. Single cell SpTrf gene sequences cluster according to element patterns. The phylogenetic relationships of SpTrf amplicon sequences containing most of the second exon were inferred using maximum parsimony, neighbor joining (results not shown), and maximum likelihood, all of which gave similar results. The tree shown here is based on the maximum likelihood phylogeny with PhyML according to Jukes-Cantor model with 1,000 bootstrap replicates. Bootstrap values for nodes present in >50% of trees are shown. Sequences from sperm (S) or coelomocyte (C) are indicated accordingly and accompanied with animal number (1, 2, or 3). The letter and number after the first dash indicate the element pattern based on previously published nomenclature (7, 8, 16). The last number after the second dash indicates the sequence sub-categories based on SNP variations within an element pattern. Colors indicate the gene element pattern according to Miller et al. (7) and Buckley and Smith (8).

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