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Figure 1. Larval immune cell types in the sea urchin Lytechinus pictus. (a) Larva at 3 days post‐fertilization (dpf). (b) Stellate pigment cell (black arrow, non‐active morphology). (c) Globular cell. (d) Filopodial cell. Black arrow denotes the long filopodia extending from the cell body. White arrow indicates a skeletal rod of the larva. (e) Ameboid cell (black arrow). White dashed line outlines the epithelia of the gut. The “tail” of these typically comma‐shaped cells is out of view. (f) Ovoid cell (black arrow) imaged from a larva infected with Vibrio diazotrophicus for 28 h. Scale for a = 100 μm. Scale for all other panels = 5 μm.
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Figure 2. Basal expression of molecular markers of immune cells in Lytechinus pictus larvae. (a)
PKS1 is localized in pigment cells of the larvae. Signal is evenly distributed in pigment cells embedded in the ectoderm of the animal. (b, c) Localization of srcr142 transcript in the larvae occurs in pigment cells as well as in coelomic pouches of the larva, with variability in the number of srcr142‐positive cells observed in the right pouch (b′–b″ and c′–c″, with and without DAPI overlay). All lower magnification images (b, c) are maximum intensity projections from Z‐stacks of whole fixed larvae at 3 dpf. Insets (b′–b″) and (c′–c″) show individual slices from the Z‐stacks from the same larvae shown in (b) and (c) respectively, at higher magnification. Coelomic pouches are outlined with yellow dashed lines. (d–f) Expression of macpfE2 in the gut is variable, but all macpfE2‐positive cells localize to a ring, or parts of a ring around the stomach. Some larvae have lower numbers of expressing cells (d), while others have enough positive cells to completely encircle the gut (f). Quantification of each type of gene pattern scored is listed above the scale bar (n = 75 larvae combined from three distinct mate pairs). Genes labeled are noted in the lower left of each panel (DAPI – gray, PKS1 – green, srcr142 – magenta and macpfE2 – cyan). Scale = 25 μm for all panels and insets. Detailed quantification of phenotypes is available in Supplementary tables 3 and 4.
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Figure 3. Infection with Vibrio diazotrophicus causes significant differences in pigment cell activation and migration to the gut. (a) Quantification of the migratory pigment cell phenotype pooled from six genetically distinct mate pairs. White circles are control larvae that have not been exposed to Vibrio, red circles are sibling larvae that have been exposed to Vibrio. Statistical labeling: ns, not significant (P > 0.05); ****P < 0.0001. (b, c) Representative micrographs of the phenotypic response to V. diazotrophicus exposure. Uninfected larvae at 3 dpf (b) have little to no activated pigment cells, and instead these cells are localized to the ectoderm (black arrow). The stomach “s” is large with a thin epithelium, and the mouth “m” is annotated. At 24 h post‐exposure (hpe) to Vibrio, the larval stomach (S) has become inflamed and constricted, and pigment cells (black arrow) are clustering at the stomach (c).
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Figure 4. Immune gene markers respond variably to Vibrio diazotrophicus infection. (a)
srcr142 transcripts are localized to pigment cells throughout the larval body and within cells of the left and right coelomic pouch. (b–d)
srcr142 transcripts are evident in several cells in the left CP as well as within 1–2 cells in the right pouch at 6 h post‐exposure (hpe; b–b″), increasing in the number of cells in both coelomic pouches through 24 hpe (c–d″). Boxed insets highlight the localization of srcr142‐positive cells within the coelomic pouches (outlined in yellow dashed lines), both with DAPI overlay (a′–d′) and without (a″–d″). (e–h) (e) Prior to bacterial exposure, pigment cells marked by PKS1 are evenly distributed throughout the ectoderm; (f) cells begin to round and migrate towards and surround the midgut by 6 hpe; (g) tightly surrounding the midgut at 12 hpe; and (h) beginning to migrate away at 24 hpe. The box insets highlight the localization of pigment cells around the midgut within each figure, both with DAPI overlay (e′–h′) and without (e″–h″). (i–l). (i)
IL17‐1a transcription is absent in larvae prior to exposure; (j, k) At 6 and 12 hpe, transcription of IL17‐1a is localized to the midgut, with a slight decrease in abundance at hour 12; (l) By 24 hpe, transcription of IL17‐1a is generally present in about 1–3 cells near the midgut. The box insets highlight the localization of IL17‐1a‐positive cells near the midgut within each figure, both with DAPI overlay (i′–l′) and without (i″–l″). (m–p)
MacpfE2 transcription is localized to cells surrounding the apical portion of the midgut. Little variation is observed throughout exposure (n–p); however, the number of cells transcribing macpfE2 varies between mate pairs (see Supplementary table 4). The box insets highlight the localization of macpfE2‐positive cells in the midgut within each figure, both with DAPI overlay (m'–p') and without (m''–p''). Quantification of the phenotype is shown in the lower right of each panel (n = # of larvae out of the total scored from three mate pairs). Scale for all panels = 50 μm.
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Figure 5. Quantification of immune gene expression territories in response to bacterial exposure. (a) Patterning of srcr142; (b)
PKS; (c)
IL17‐1a; and (d)
macpfE. Scoring for all genes is pooled from 25 larvae each from three mate pairs (n = 75 total). Above each stacked bar, “−” indicates control, unexposed larvae and “+” indicates larvae exposed to Vibrio diazotrophicus. Larvae were assessed at 0, 6, 12 or 24 h post‐exposure (hpe).
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Figure 6. The intensity of srcr142 signal increases in the coelomic pouches during bacterial exposure. (a) Distribution of net fluorescence intensity of srcr142 signal. Control larvae without exposure to bacteria at T0 (black), control larvae at T24 (light pink), and larvae exposed to Vibrio at T24 (magenta). (b) Distribution of DAPI signal of control larvae without exposure to bacteria at T0 (black), control larvae at T24 (light blue) and larvae exposed to Vibrio at T24 (cyan). Measurements are pooled from five larvae each from three mate pairs (n = 15 total). Asterisks denote statistical significance for comparisons between treatments within an anatomical region as determined by two‐way ANOVA. Comparisons that are not significantly different are not marked. *P < 0.03; **0.0021 < P < 0.03; *** = 0.0059.
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Figure 7. Three‐dimensional rendering of srcr142 expression in the coelomic pouches of bacteria‐exposed larvae. (a, a″) Representative larva at T0, with DAPI displayed in white with transparency. Cells that are srcr142‐positive are filled with light pink, and cells that are srcr142‐positive and located at the coelomic pouch are filled in with magenta. Yellow box indicates the region for coelomic pouch‐centered insets showing only srcr142‐positive cells (DAPI removed; a′) or coelomic pouches rendered with srcr142‐positive cells in magenta and DAPI in white with transparency (a″). (b, b″) Representative larva at T24 of Vibrio exposure. Color and inset schemes are the same as in Panel A. Scale for panel (a) and (b) = 100 μm. Scale for a′, a″ and b′, b″ = 50 μm. Additional views of the 3D renderings are provided in Supplementary videos 5 and 6.
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