Live imaging reveals differing roles of macrophages and neutrophils during zebrafish tail fin regeneration.

Macrophages and neutrophils are the pivotal immune phagocytes that enter the wound after tissue injury to remove the cell debris and invaded microorganisms, which presumably facilitate the regrowth of injured tissues. Taking advantage of the regeneration abilities of zebrafish and the newly generated leukocyte-specific zebrafish lines with labeling of both leukocyte lineages, we assessed the behaviors and functions of neutrophils and macrophages during tail fin regeneration. Live imaging showed that within 6 hours post amputation, the inflammatory stage, neutrophils were the primary cells scavenging apoptotic bodies and small cell debris, although they had limited phagocytic capacity and quickly underwent apoptosis. From 6 hours post amputation on, the resolution and regeneration stage, macrophages became the dominant scavengers, efficiently resolving inflammation and facilitating tissue remodeling and regrowth. Ablation of macrophages but not neutrophils severely impaired the inflammatory resolution and tissue regeneration, resulting in the formation of large vacuoles in the regenerated fins. In contrast, removal of neutrophils slightly accelerates the regrowth of injured fin. Our study documents the differing behaviors and functions of macrophages and neutrophils during tissue regeneration.

Reciprocal regulation between resting microglial dynamics and neuronal activity in vivo.

Microglia are the primary immune cells in the brain. Under physiological conditions, they typically stay in a "resting" state, with ramified processes continuously extending to and retracting from surrounding neural tissues. Whether and how such highly dynamic resting microglia functionally interact with surrounding neurons are still unclear. Using in vivo time-lapse imaging of both microglial morphology and neuronal activity in the optic tectum of larval zebrafish, we found that neuronal activity steers resting microglial processes and facilitates their contact with highly active neurons. This process requires the activation of pannexin-1 hemichannels on neurons. Reciprocally, such resting microglia-neuron contact reduces both spontaneous and visually evoked activities of contacted neurons. Our findings reveal an instructive role for neuronal activity in resting microglial motility and suggest the function for microglia in homeostatic regulation of neuronal activity in the healthy brain.

[Study of a new zebrafish mutant defective in primitive myelopoiesis].

OBJECTIVE: To perform phenotypic identification and characteristic analysis of a new zebrafish mutant 1276 defective in primitive myelopoiesis. METHODS: The AB strain male zebrafish were mutagenized with N-ethyl N-nitrosourea (ENU) to induce mutations in the spermatogonial cells, and the mutations were transmitted to the offsprings. The F3 embryos were screened by neutral red staining for identifying the mutants defective in primitive myelopoiesis. One of the myeloid mutants 1276 was further studied by cytochemistry and whole mount in stiu hybridization (WISH) with different lineage markers. RESULTS: A total of 2140 mutagenized genomes from the 1296 F2 families were analyzed, and 12 mutants were identified to show abnormal signal by neutral red staining. In the primitive hematopoiesis stage, the mutant 1276 showed the absence of neutral red staining-positive cells in the whole body. The expression of microglia marker apoe was totally lost in the head of the mutant, and the expression of the macrophage marker l-plastin was slightly decreased in the head and remained normal in the ventral dorsal aorta region, but the granulocytes and erythrocytes developed normally. in the definitive hematopoiesis stage, the mutant 1276 still showed abnormal macrophages as found in the primitive hematopoiesis stage, but the granulocytes, erythrocytes and lymphocytes appeared normal. CONCLUSION: The zebrafish mutant 1276 shows abnormalities in the function, development and migration of the macrophages in the primitive hematopoiesis stage, which can not be compensated in the definitive hematopoiesis stage.

[Forward genetic screening for zebrafish mutants defective in myelopoiesis].

OBJECTIVE: To identify zebrafish mutants with myelopoiesis defects by ENU mutagenesis and large-scale forward genetic screening. METHODS: Male zebrafish were mutagenized with N-ethyl N-nitrosourea to induce mutations in the spermatogonial cells to generate the founders, which were outcrossed with AB to raise F1 fish. The F1 fish from different founders were mated to generate the F2 families. The F3 embryos from F2 sibling crosses were screened by Sudan black B staining and neutral red staining. RESULTS: A total of 350 F2 families from F1 sibling crosses were screened, and 1424 F2 crosses were analyzed. Six mutations were identified resulting in abnormal Sudan black B staining and neutral red staining, indicating the involvement of neutrophil deficiency or macrophage abnormalities. CONCLUSION: It is simple and cheap to induce and screen myelopoiesis deficiency in zebrafish by ENU chemical mutagenesis and Sudan black B staining and neutral red staining. These mutants shed light on the identification of the genes important to myelopoiesis in zebrafish.

[Forward genetic screening for zebrafish mutants defective in erythropoiesis].

OBJECTIVE: To screen and identify zebrafish mutants with erythropoiesis defects by N-ethyl-N-nitrosourea (ENU) mutagenesis and large-scale forward genetic screening using beta e 1 as the marker. METHODS: The chemical mutagen ENU was used to treat healthy wild-type male fish (AB strain, F0). The surviving ENU-treated fish were mated with wild-type female fish to generate F1, and further F2 family was generated by F1 family intercross. The adult F2 fish were intercrossed within each F2 family and the resulting F3 embryos from each crossing were subjected to whole mount in situ hybridization (WISH) with the beta e 1 probe. Mutagenesis was performed by treating the male zebrafish with ENU to induce mutations in pre-meiotic germ cells to generate the founders, which were outcrossed to obtained the F1 fish. The F1 fish from different founders were mated to generate the F2 families. F3 embryos from the sibling cross in the F2 family were examined by whole mount in situ hybridization using beta e 1-globin probe. The putative mutants were then characterized with different hematopoiesis markers. RESULTS AND CONCLUSION: We identified 4 beta e 1-deficient mutants with erythropoiesis defects, including two with specific erythiod lineage defects and two with concurrent lymphopoiesis defects.

Hematoporphyrin derivative-mediated photodynamic therapy inhibits tumor growth in human cholangiocarcinoma in vitro and in vivo.

AIM: To investigate the effects of hematoporphyrin derivative-mediated photodynamic therapy (HPD-PDT) on cell growth in human cholangiocarcinoma in vitro and in vivo, as well as the underlying mechanisms of these effects. METHODS: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to evaluate growth status of human cholangiocarcinoma cell line (QBC939). Hoechst 33258 staining and flow cytometry assays were applied to determine cell apoptosis. Western blotting analysis was performed to detect the release of cytochrome c in QBC939 cells, and caspases enzymatic assay was used to investigate the activation of caspase-3, -8, and -9. Further, tumor growth after subcutaneous implantation of QBC939 cells in nude mice was monitored. RESULTS: HPD-PDT inhibits QBC939 cell growth via cell apoptosis in vitro, and initiates cell mitochondria apoptosis pathway by the release of cytochrome c and the activation of caspase-9 and -3. Moreover, HPD-PDT also inhibits subcutaneous tumor growth of QBC939 cells and reduces tumor cell mitosis in nude mice. CONCLUSION: HPD-PDT inhibits tumor growth of human cholangiocarcinoma, suggesting that HPD-PDT is useful in cholangiocarcinoma therapy.