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FIGURE 1. Histological observation reveals the intestinal regeneration process of Apostichopus japonicus. (A) Schematic diagram of the internal anatomy of A. japonicus in normal and in different regenerative stages (from 2 to 28 dpe). Sea cucumbers were dissected along the body length, at the right side of the mid‐dorsal, and flattened. Red solid line: the intestine breaks at its anterior end from the esophagus and at the posterior end from the cloaca. Red broken line: the edge of the mesentery; yellow area: newly formed intestinal lumen; (B) transverse tissue sections of the mesentery and regenerating intestine at normal, 2, 7, 12, 20, and 28 dpe were stained with hematoxylin–eosin (a–f). The free edge of the mesentery is marked with a red arrow. At 2 dpe, the epithelium covers the tip of the mesentery and the tear edge of the mesentery gradually recovered (b). At 7 dpe, a small enlargement can be observed at the mesenterial tip (c). At 12 dpe, the intestinal rudiment has increased considerably in size (d). At 20 dpe, the lumen has formed, but the epithelial fold has not been fully formed (e). At 28 dpe, the intestinal rudiment continued to grow in size and all tissue layers of the mature intestine can be found within the rudiment (f). a1–3–f1–3 are the high magnification images of the black boxes in a–f. The muscular layer is marked by a blue arrow. Bar (a–f) = 100 μm, bar (a1–3–f1–3) = 10 μm
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FIGURE 2. Cell proliferation in Apostichopus japonicus during intestinal regeneration. (A) Schematic reconstruction of the sequence of events during regeneration of the intestinal system in A. japonicus. The diagram presents only the organs involved in this process. Other organs, such as the right respiratory tree, have been removed. a1–3–f1–3 marked in the schematic correspond to the sampling sites in (B), respectively. (B) EdU assay was performed to detect the cell proliferation levels in normal and different regenerative stages (2–28 dpe). The sampling locations of dorsal, ventral, and lateral mesentery at each stage of intestinal regeneration were stained. Blue color indicates Hoechst‐stained nuclei, and green indicates EdU‐stained proliferating cells. Bar = 200 μm. (C) EdU‐based flow cytometry was used to analysis the changes in cell proliferation levels of normal and different regenerative stages (2–28 dpe). (D) The number of EdU‐labeled cells in the normal group and five regeneration groups were comparatively analyzed and represented by a histogram. Results are the mean ± SD from three independent experiments. In each experiment, the cells of three A. japonicus were analyzed. Per sample, 10,000 events were acquired on an FACS flow cytometer. “ns” indicates no significant difference versus the control. *p < 0.05; **p < 0.01; ****p < 0.0001 versus the control
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FIGURE 3. AjFGF4 is identified as a target to regulate cell proliferation during intestinal regeneration. (A) A Venn diagram showed overlapping genes between the normal group and different regenerative groups. (B) Expression changes mean normalized expression behavior of highlighted clusters from the intersection of four regeneration groups (7 dpe vs. normal, 12 dpe vs. normal, 20 dpe vs. normal, 28 dpe vs. normal). (C) Gene ontology enrichment analysis of DEGs of upregulated clusters (Clusters 1, 3, 4). Ontology domains comprise biological process (BP), cellular component (CC), and molecular function (MF). (D) The protein expression level of AjFGF4 in the regenerating mesentery and intestine in the normal and different regenerative groups. (E) Sea cucumbers were transfected with siNC or siAjFGF4 for six times in every 2 days post‐evisceration treatment. The protein levels of AjFGF4 in the regenerating mesentery and intestine at 2, 7, and 12 dpe were determined by western blotting with anti‐AjFGF4 Ab and anti‐Ajβ‐Tubulin Ab as the internal reference. (F) EdU assay detection of cell proliferation and the size of the regenerative tissues in the groups of siRNA‐NC, siAjFGF4, BSA, and rAjFGF4 groups at 2, 7, and 12 dpe. The regeneration tissues in each group were taken from the same location in the anterior end (next to the esophagus) of the sea cucumber. Blue color indicates Hoechst‐stained nuclei, and green indicates EdU‐stained proliferating cells. Bar = 200 μm. (G) Flow cytometry analysis for detecting EdU incorporation into cells. (H) Comparison of the EdU‐labeled cells in siRNA‐NC, siAjFGF4, BSA and rAjFGF4 groups at 2, 7, and 12 dpe. Results are the mean ± SD of three independent experiments. In each experiment, the cells of three Apostichopus. japonicus were analyzed. Per sample, 10,000 events were acquired on a FACS flow cytometer. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
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FIGURE 4. AjFGF4 interacts with AjFGFR2. (A) The binding capacity of AjFGF4 and AjFGFR2 was detected by the co‐IP assay in vitro. HEK293T cells were transfected with 5 μg of empty vector or AjFGF4‐flag and AjFGFR2‐EGFP plasmids, respectively. At 48 h posttransfection, cell lysates were immunoprecipitated with protein A/G magnetic beads which were bound with anti‐flag or anti‐GFP antibodies and immunoblotted with anti‐flag and anti‐GFP Abs by western blotting. (B) Colocalization of AjFGF4 and AjFGFR2 in single‐cell suspensions of regenerating tissues at 12 dpe was analyzed by laser confocal microscopy. Red color represents Cy3‐labeled AjFGF4, green represents FITC‐labeled AjFGFR2, and blue represents DAPI‐stained nuclei. The last row indicates the images in the first three panels as a digital overlay to visualize colocalization. Single‐cell suspensions from the first to the fourth rows were, respectively, incubated with the rabbit anti‐AjFGF4 Ab and mouse anti‐AjFGFR2 Ab, the mouse anti‐AjFGFR2 Ab and the pre‐immune rabbit sera, the rabbit anti‐AjFGF4 Ab and the pre‐immune mouse sera, and the pre‐immune rabbit sera and the pre‐immune mouse sera as primary Abs, and Cy3‐conjugated goat anti‐rabbit IgG and FITC‐conjugated goat anti‐mouse IgG were served as secondary Abs. Scale bar = 5 μm.
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FIGURE 5. AjFGF4 regulates Apostichopus japonicus cell proliferation depending on the recognition of AjFGFR2 during intestinal regeneration. (A) Sea cucumbers were transfected with siRNA‐NC or siAjFGFR2 for six times in every 2 days postevisceration treatment. The protein levels of AjFGF4 in the regenerating mesentery and intestine at 2, 7, and 12 dpe were determined by western blotting with anti‐AjFGFR2 Abs and anti‐Ajβ‐Tubulin Ab as the internal reference. (B) EdU assay detection of cell proliferation and the size of the regenerative primordium in the siRNA‐NC group and siAjFGFR2 group at 2, 7, and 12 dpe. The regeneration tissues in each group were taken from the same location in the anterior end (next to the esophagus) of the sea cucumber. Blue color indicates Hoechst‐stained nuclei, and green indicates EdU‐stained proliferating cells. Bar = 200 μm. (C) Flow cytometry analysis for detecting EdU incorporation into cells at 2, 7, and 12 dpe in the siAjFGFR2 group and siRNA‐NC group. (D) Comparison of the number of EdU‐labeled cells at 2, 7, and 12 dpe between the siAjFGFR2 and siRNA‐NC groups. (E) EdU assay detection of cell proliferation and the size of regenerative tissues in the groups of siRNA‐NC + BSA, siAjFGFR2 + BSA, siAjFGFR2 + rAjFGF4 at 2, 7, and 12 dpe. Blue color indicates Hoechst‐stained nuclei, and green indicates EdU‐stained proliferating cells. Bar = 200 μm. (F) Comparison of the numbers of EdU‐labeled cells in the siRNA‐NC + BSA, siAjFGFR2 + BSA, siAjFGFR2 + rAjFGF4 group. All data are presented as the mean ± SD of three independent experiments. In each experiment, the cells of three A. japonicus were analyzed. Per sample, 10,000 events were acquired on an FACS flow cytometer. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
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FIGURE 6. AjFGF4/AjFGFR2‐mediated cell proliferation is depending on ERK–MAPK pathway during intestinal regeneration. (A) The protein or phosphorylation level of AjFGFR2, ERK, p38, and JNK in the single‐cell suspension of the mesentery at the stage of 12 dpe were detected at 48 and 72 h post‐siAjFGFR2 treatment by western blotting using anti‐p38 Ab and anti‐p‐p38 Ab, anti‐JNK Ab and anti‐p‐JNK Ab, anti‐ERK1/2 Ab, anti‐p‐ERK1/2 Ab, and anti‐Ajβ‐Tubulin Ab as the internal reference, respectively. (B) The protein and phosphorylation levels of ERK in the single‐cell suspension of the mesentery at the stage of 12 dpe were detected at 48 and 72 h after being treated with FR180204 (ERK inhibitor, 5 μM) by western blotting using anti‐ERK1/2 Ab, anti‐p‐ERK Ab, and anti‐Ajβ‐Tubulin Ab as the internal reference. (C) EdU assay detection of cell proliferation in the mesentery single‐cell suspension at the stage of 12 dpe after being treated with BSA, DMSO, DMSO + BSA, DMSO + rAjFGF4, FR180204 + BSA, and FR180204 + rAjFGF4. Blue color indicates Hoechst‐stained nuclei, and green indicates EdU‐stained proliferating cells. The last row shows the images in the first two panels as a digital overlay to visualize colocalization by laser confocal microscopy. Scale bar = 10 μm. In each experiment, the sample of six Apostichopus japonicus was analyzed and each experiment was repeated three times.
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FIGURE 7. AjFGF4/AjFGFR2‐ERK–MAPK induced the expression of cell cycle‐related proteins (CDK2, Cyclin A, and Cyclin B) during intestine regeneration in Apostichopus japonicus. The protein expression levels of CDK2, Cyclin A, and Cyclin B in the regenerating mesentery and intestine at the stage of 2, 7, and 12 dpe were determined after siAjFGF4 (A), siAjFGFR2 (B), FR180204 (C), or FR180204 + rAjFGF4 (E) were analyzed by western blotting using anti‐CDK2 Ab, anti‐Cyclin B Ab, anti‐Cyclin A Ab, and anti‐Ajβ‐Tubulin Ab as the internal reference. Sea cucumbers were transfected with siRNA‐NC, siAjFGF4, siAjFGFR2, FR180204, DMSO, or FR180204 + rAjFGF4 in eviscerated‐sea cucumbers for six times in every 2 days. (D) EdU assay detection of cell proliferation and the size of regenerative tissues in the groups of FR180204 + BSA, FR180204 + rAjFGF4, and DMSO +BSA at 2, 7, and 12 dpe. The regeneration tissues in each group were taken from the same location in the anterior end (next to the esophagus) of the sea cucumber. Blue color indicates Hoechst‐stained nuclei, and green indicates EdU‐stained proliferating cells. Bar = 200 μm. In each experiment, the sample of six A. japonicus were analyzed and each experiment was repeated at least three times.
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FIGURE 8. Scheme depicting how mesentery gradually thickened to form a new intestine and established a close relationship with cell proliferation. Furthermore, it illustrates how AjFGF4 induces cell proliferation in the process of intestinal regeneration. AjFGF4 associates with AjFGFR2 to stimulate cell proliferation through ERK activation, and then activated the expression of cell cycle‐related proteins, cyclins, and CDKs, to increase cell cycle progression, which finally promotes cell proliferation.
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