Jung et al., 2016 - Impaired Lymphocytes Development and Xenotransplantation of Gastrointestinal Tumor Cells in Prkdc-Null SCID Zebrafish Model. Neoplasia (New York, N.Y.)   18:468-79 Full text @ Neoplasia

Fig. 1 ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

EXPRESSION / LABELING:
Gene:
Antibody:
Fish:
Anatomical Terms:
Stage: Adult
PHENOTYPE:
Fish:
Observed In:
Stage: Adult

Fig. 2

Growth phenotypes and microscopic observations of the Prkdc-null SCID zebrafish. (A and B) Growth retardation. The SCID zebrafish showed a retarded growth in length compared with the age-matched wild-type controls until 3 months. (C) Infectious manifestation. The immune-deficient SCID zebrafish often succumbed to infection. The emaciated zebrafish died within 7 days, whereas the "dropsy" zebrafish died within a couple of days. Red arrow shows a magnified view of the dropsy zebrafish fin. (D) Microscopic findings of the infection and inflammation occurred in the internal organs. The zebrafish with "dropsy" phenotype revealed that the disseminated infection occurred in the internal organs including brain, intestine, and ovary, suggesting septicemia. The emaciated zebrafish showed a predominant muscle necrosis with inflammation.

Fig. 3

Microscopic observation of the hematopoietic organs in the Prkdc-null SCID zebrafish. (A) ISH and H&E images of the thymus. The ISH experiment for Rag1 in the SCID zebrafish embryo at 4 dpf showed that the mutant zebrafish contained a very rudimentary thymus development (dotted red circle). H&E staining images at 3 months of age showed that the number of lymphocytes was severely decreased in the SCID zebrafish (prkdc-/-) compared with the heterozygote (prkdc+/-). (B) Microscopic observation of the zebrafish kidney. The retroperitoneal hematopoietic kidney tissue appears as dark linear structure. The tissue appeared as thinner with less density in the SCID zebrafish compared with that of the heterozygote (red arrowhead). H&E staining of the kidney also showed the consistency in which the kidney of the SCID zebrafish was thinner than that of the heterozygote and contained scanty hematopoietic cells between the renal tubules. (C) Gross and H&E images of spleen. The spleen of the SCID zebrafish was much smaller than that of the heterozygote. Compared with the spleen of the heterozygote, small number of lymphocytes was shown to be scattered between splenocytes in the SCID zebrafish.

EXPRESSION / LABELING:
Gene:
Fish:
Anatomical Term:
Stage: Day 4
PHENOTYPE:
Fish:
Observed In:
Stage Range: Day 4 to Adult

Fig. 4

Hematologic findings of the major blood cell lineages. (A) Wright-Giemsa staining with the smeared cells from the peripheral blood, kidney, and spleen. All images are 1000× view except insets (400×) in middle column. Arrowheads are colored as green for lymphocytes, red for erythroid lineage cells, blue for neutrophils, yellow for thrombocytes, black for monocytes/macrophages, and brown for blasts/precursor cells. Microscopic observation of these organs revealed much decreased number of the lymphocytes in the SCID zebrafish compared with the one in the heterozygote control (detailed counting results are shown in Table 1). The lymphocytes appeared as small cells with a higher nuclear to cytoplasmic ratio, and the monocytes/macrophages appeared as larger cells with a lower nuclear to cytoplasmic ratio and cytoplasmic vacuoles. Direct smear of dissected kidney and spleen also revealed lower lymphocyte fractions in SCID zebrafish. Note the compact cells in the smear of kidney marrow from control and frequent fat globules (*) in the smear from SCID zebrafish. (B) Flow cytometry. Forward scatter (FSC) and side scatter (SSC) are proportional to cell size and cellular granularity, respectively. Peripheral blood and single cell suspensions from the dissected kidney and spleen were processed for flow cytometry. The flow cytometry demonstrated four main cell populations (noted in the box). Note that lymphocyte fractions are markedly decreased in the SCID zebrafish compared with those of the heterozygote control.

PHENOTYPE:
Fish:
Observed In:
Stage: Adult

Fig. 5

IF and RT-PCR analyses for activated lymphocytes. (A) IF detected by CD2AP marker. The CD2AP-reactive lymphocytes are present in the peripheral blood from both SCID and control zebrafish, suggesting that the cells are either activated T lymphocytes or NK cells. DAPI staining was used to detect the blood cells. (B) Double IF detected by CD2AP (Cy2) and NKP46 (Cy3) markers. The NKP46 is a specific marker for NK cells, whereas the CD2AP marker is reactive to both T lymphocytes and NK cells. (C) RT-PCR analysis. Glyceraldehyde 3-phosphate dehydrogenase gene detection was used as control. Nonspecific cytotoxic cell receptor protein-1 and NK specific protein (NK cell triggering receptor) gene detections indicate NK cell. TCR V(D)JC (Vb12-Cb1) and immunoglobulin (IgVH1-Cm) gene transcript detections reveal V(D)J recombination and heavy-chain rearrangement. (D) Counted numbers of cells depending on CD2AP or NKP46 positivity. Green arrowhead, positive for CD2AP; red arrowhead, positive for NKP46; white arrowhead, positive for both CD2AP and NKP46; blue arrowhead, lymphocytes negative for both CD2AP and NKP46; yellow arrowhead; red blood cells.

Fig. 6

Xenograft of tumor cells. Various tumor cells (5 × 104) expressing TurboRFP were injected into intraperitoneal space by using a Hamilton syringe. If not indicated, all H&E and IHC images are 400×. (A) Gross images. The engraftment allows repeated observation of the growing tumors by visualization of transcutaneous RFP. Tumor engraftment was never formed in Prkdc+/-. (B) Dissected images. Bright field (top, asterisk shows a mass formed by the growing tumor) and merged RFP (middle) images showing an engrafted tumor with RFP expression. Dissected fat tissue (bottom left) showed scattered RFP-expressing tumor cells. Metastatic tumor cells (bottom right) at tail fin are also noted. (C) A mass formed in the liver in SK-MEL28 injected zebrafish. Inset is a low-power view showing mass (M) in the liver. HLA-ABC positivity of the tumor cells indicates that the mass originated from injected human melanoma cells. Many of the tumor cells are positive for PCNA and Ki-67, suggesting active proliferation. (D) A discrete mass formed in the Panc-1–injected zebrafish. Inset in the left is a low-power view indicating testis (T), swim bladder (SB), intestine (I), and mass (M). Enlarged view of the boxed area is shown in the middle. Merged TurboRFP and H&E image is shown on the right. Inset in the right is IHC for RFP. Lower column images are findings of IHC stain. Xenografted tumor cells are positive for HLA-ABC, for pan-cytokeratin (Pan-CK), and also for PCNA and Ki-67 frequently. (E) The tumors formed in liver and peritoneal cavity. (F) The xenografted HPAC cells showing the invasive growth, which results in destruction of muscle (M) and skin. The fibroblast with occasional tumor cells (dotted red circle shown in the middle). The proliferating fibroblasts are highly positive to PCNA.

Table 1 ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

PHENOTYPE:
Fish:
Observed In:
Stage: Adult

Table 2 ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

PHENOTYPE:
Fish:
Observed In:
Stage: Adult
Acknowledgments:
ZFIN wishes to thank the journal Neoplasia (New York, N.Y.) for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Neoplasia