FIGURE SUMMARY
Title

Wnt5 signaling in vertebrate pancreas development

Authors
Kim, H.J., Schleiffarth, J.R., Jessurun, J., Sumanas, S., Petryk, A., Lin, S., and Ekker, S.C.
Source
Full text @ BMC Biol.

Time-lapse imaging of insulin:GFP transgenic embryos shows cell migration defects in Fz-2 morphants. (A, C-H) Uninjected insulin:GFP transgenic embryo, (B, I-N) fz-2 MO-injected insulin:GFP transgenic embryo. All panels are dorsal views and anterior is to the left. Scale bar represents 100 μm. (A) Uninjected transgenic embryo, 24 hpf. (B) Fz-2 MO-injected transgenic embryo, 24 hpf. (C) At the 14-somite stage, bilateral patches of GFP-positive cells are visible in uninjected embryo. (D) At the 15–16 somite stage, GFP-positive cells have started proliferating. (E-G) At the 17 somite to 24 hpf stages, GFP-positive cells are aligned in bilateral rows of cells and undergo a medial and posterior migration. (H) At 24 hpf, all GFP-positive cells have merged to form one islet. (I) At the 14-somite stage, bilateral patches of GFP expression are apparent in fz-2 MO-injected embryos similar to uninjected embryos. (J-M) GFP-positive cells migrate in random directions in fz-2 morphant embryos. (N) At 24 hpf, GFP-positive cells have still not merged. (O) Trajectory of GFP-positive cells in uninjected insulin:GFP embryo. Notice that cells are uniformly moving posteriorly. (P) Trajectory of GFP-positive cells in fz-2 MO-injected insulin:GFP embryo. Notice cells are moving in random directions. A: anterior, P: posterior, T: time, L: left, R: right, O: origin.

Migration defects in Fz-2 morphant embryos can be rescued by synthetic fz-2 mRNA. (A) Double in situ hybridization with fz-2 and insulin at 20 somite stage of development. Arrow, insulin; arrowhead, fz-2 expression in the endoderm; dotted line, approximate position of the section in (B). (B) A section of double in situ hybridization with fz-2 and insulin. Fz-2 is expressed more strongly on the surface of mesoderm and entire endoderm. Arrow, insulin; arrowhead, fz-2 expression in the endoderm; a, arteries; asterisk, neural tube; d, pronephric duct. (C) RT-PCR using cDNA made from sorted cells of transgenic insulin:GFP zebrafish embryos. L: ladder; lanes 1-5: GFP-negative cells; lanes 6-10: GFP-positive cells; lanes 1, 6: EF1α lanes 2, 7: insulin; lanes 3, 8: fz-2 primer set #1; lanes 4, 9: fz-2 primer set #2; lanes 5, 10: wnt-5. (D) High-dose injection of either fz-2 MO1 or MO2 resulted in scattered insulin expression, whereas low dose injection of either MO caused such defects in less than 10% of embryos. Co-injection of low dose fz-2 MO1 and MO2 resulted in synergistic increase of percentage of embryos with scattered insulin expression. (E) 80% of fz-2 MO-injected embryos displayed scattered insulin expression. Co-injection of fz-2 MO and fz-2 RNA reduced the percentage of embryos with abnormal insulin expression down to 45%. (F-I) In situ hybridization with insulin at 24 hpf stage, anterior is to the left, (F) fz-2 MO1-injected embryo, (G) fz-2 mismatch MO-injected embryo, (H) fz-2 RNA-injected embryo, (I) fz-2 MO- and fz-2 RNA-co-injected embryo. Notice the compact islet in this embryo that displays an undulated notochord.

EXPRESSION / LABELING:
Genes:
Fish:
Knockdown Reagents:
Anatomical Terms:
Stage Range: 20-25 somites to Prim-5
PHENOTYPE:
Fish:
Knockdown Reagents:
Observed In:
Stage: Prim-5

Wnt-5 has a specific role in islet formation. (A) Double in situ hybridization with pdx-1 and wnt-5, 10 som stage, dorsal view, the anterior is to the left. Arrow, pdx-1 expression, bracket, wnt-5 expression. (B-H) In situ hybridization with insulin at 24 hpf. (B) wild-type, (C) WNT-8 morphant embryos, (D) WNT-11 morphant embryos, (E) WNT-5 morphant embryos, (F)wnt-5 mismatch MO-injected embryos, (G) wnt-5 RNA injected embryo, (H) wnt-5 MO and wnt-5 RNA co-injected embryo. Notice the compact islet in this embryo that displays an undulated notochord. (I) Percentage of embryos with scattered insulin expression resulting from injection of wnt-5 MO reduced significantly from 60% to 10% when wnt-5 RNA was co-injected with wnt-5 MO. (J-L) Morphology at 24 hpf, (J) wild-type, (K) wnt-5 insertional mutant, (L) wnt-5 translation-blocking MO-injected embryos. Notice that wnt-5 MO injected embryos have more severe morphological phenotype than wnt-5 insertional mutant embryos. (M) RT-PCR analysis of wnt-5 transcript in wnt-5 exon-intron MO injected embryos. Injection of wnt-5 exon-intron MO results in severely shortened wnt-5 transcript. L:ladder, 1:EF-1 α control, 2:wnt-5.

Early endoderm markers are not affected in Wnt-5 and Fz-2 morphant embryos. All pictures are dorsal views. (A, D, G, J, M) wild-type, (B, E, H, K, N) Fz-2 morphants, (C, F, I, L, O) Wnt-5 morphants. (A-C) mixer, 50% epiboly, (D-F) sox-17, 90% epiboly, (G-I) fox-A3, 24 hpf, (J-L) anterior endoderm expression of fox-A3, arrow, pancreatic endoderm, 24 hpf, (M-O) gata-6, 24 hpf. Scale bar = 300 µm.

Wnt-5 and Fz-2 morphant embryos exhibit similar pancreatic islet defects at 24 hpf. In all panels, anterior is to the left and 24 hpf. .A-I, dorsal view; J-L, lateral view. (A, D, G, J) Wild-type embryos. (B, E, H, K) Fz-2 morphants. (C, F, I, L) Wnt-5 morphants. In situ hybridization analysis of (A, B, C) somatostatin, (D, E, F) glucagon, notice a hollow spot in the middle of each patch, (G, H, I) islet-1, (J, K, L) fspondin-2b. Note scattered pancreatic cells in Fz-2 and Wnt-5 morphants.

Wnt-5 and Fz-2 morphant embryos have other similar defects. In all panels, view is dorsal, anterior is to the left. (A-I, M-O) 3dpf, (J-L) 24 hpf stage. (A, D, G, J, M) Wild-type embryos. (B, E, H, K, N) Fz-2 morphants. (C, F, I, L, O) Wnt-5 morphants. In situ hybridization analysis of (A-C) insulin, (D-F) carboxypeptidase A, notice the hollow spot indicating the position of the islet, (G-I) ceruloplasmin, (J-O) pdx-1, (M) arrow, pdx-1-staining in islet.

Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ BMC Biol.