FIGURE SUMMARY
Title

Zebrafish cdx1b regulates differentiation of various intestinal cell lineages

Authors
Chen, Y.H., Lu, Y.F., Ko, T.Y., Tsai, M.Y., Lin, C.Y., Lin, C.C., and Hwang, S.P.
Source
Full text @ Dev. Dyn.

cdx1b is expressed in the intestine. A: A 100-hr post-fertilization (hpf) embryo hybridized with a cdx1b antisense RNA probe. B-E: Histological analyses of paraffin sagittal sections of a cdx1b antisense RNA-hybridized embryo. Scale bars = 100 μm.

EXPRESSION / LABELING:
Gene:
Fish:
Anatomical Term:
Stage: Day 4

cdx1b antisense morpholino oligonucleotide (MO) knockdown analyses. A-D: Wild type (A) and cdx1b MO-injected (B-D) 72 hr post-fertilization (hpf) embryos. E-H: Wild-type (E) and cdx1b MO-injected (F-H) 96-hpf embryos. I-L: Histological analyses of paraffin sagittal sections of a 96-hpf cdx1b-4mm-MO-injected embryo. Higher magnifications depicting the intestinal bulb (J), mid-intestine (K), and posterior intestine (L). M-P: Histological analyses of paraffin sagittal sections of a 96-hpf cdx1b morphant. Higher magnifications depicting the intestinal bulb (N), mid-intestine (O), and posterior intestine (P). Arrows indicate locations of goblet cells in the mid-intestine (K,O). Scale bars = 100 μm.

Inhibition of cdx1b function affects the development of goblet cells in the intestine. A-D: Wild type (A), cdx1b-4mm MO-injected (B), and cdx1bMO-injected (C, D) 102-hr post-fertilization (hpf) embryos were hybridized with agr2 antisense RNA probes. E-H: Wild type (E), cdx1b-4mm MO-injected (F), and cdx1b MO-injected (G, H) 102-hpf deyolked embryos that had been hybridized with agr2 antisense RNA probes. I-L: Wild type (I), cdx1b-4mm MO-injected (J), and cdx1b MO-injected (K, L) 102-hpf embryos were stained with alcian blue. M-P: Higher magnifications of alcian blue-stained paraffin sagittal sections corresponding to the mid-intestine (M, O) and posterior intestine (N, P) of 102-hpf cdx1b-4mm MO-injected (M, N) and cdx1b MO-injected (O, P) embryos. Arrows indicate locations of alcian blue-stained goblet cells in the intestine. Q-T: Wild type (Q), cdx1b-4mm MO-injected (R), and cdx1b MO-injected (S, T) 120-hpf embryos were stained with alcian blue. U: Comparison of agr2-expressing goblet cell numbers in the intestine of 102-hpf wild type (n = 28), cdx1b-4mm MO-injected (n = 28), and cdx1b MO-injected (n = 21) embryos. V: Comparison of alcian blue-stained goblet cell numbers in the intestine of wild type (n102 = 20; n120 = 21), cdx1b-4mm MO-injected (n102 = 23; n120 = 21), and cdx1b MO-injected (n102 = 22; n120 = 23) embryos from 102 (red bars) and 120 hpf (green bars). W: Comparison of the percentages of intestinal goblet cell numbers reveals decreased goblet cell numbers in cdx1b MO-injected (n = 7) embryos compared to wild type (n = 5) and cdx1b-4mm MO-injected (n = 5) embryos. Error bars indicate standard errors. Student's t-test was conducted to compare cdx1b MO-injected embryos with either wild type or cdx1b-4mm MO-injected embryos. *P < 0.001 in both comparisons. An, anus; gc, goblet cell; mc, mucus cell; ov, otic vesicle; ph, pharynx. Scale bars = 100 μm.

EXPRESSION / LABELING:
Gene:
Fish:
Knockdown Reagent:
Anatomical Term:
Stage: Day 4
PHENOTYPE:
Fish:
Knockdown Reagent:
Observed In:
Stage: Day 4

Knockdown of cdx1b function affects the development of enteroendocrine cells in the intestine. A, B: cdx1b-4mm MO-injected (A) and cdx1b MO-injected (B) 96-hr post-fertilization (hpf) deyolked embryos hybridized with nkx2.2a antisense RNA probes. C: Comparison of nkx2.2a-expressing enteroendocrine cell numbers in cdx1b-4mm MO-injected (n = 21) and cdx1b MO-injected (n = 21) embryos. D-H: Wild type (D), cdx1b-4mm MO-injected (E, F), and cdx1b MO-injected (G, H) 96-hpf deyolked embryos hybridized with glucagon antisense RNA probes. I: Comparison of glucagon-expressing enteroendocrine cell numbers in wild type (n = 15), cdx1b-4mm MO-injected (n = 31), and cdx1b MO-injected (n = 29) embryos. Error bars indicate standard errors. Student's t-test was conducted to compare cdx1b MO-injected embryos with either wild type or cdx1b-4mm MO-injected embryos. *P < 0.001 in both comparisons. Ac, pancreatic α cells; ee, enteroendocrine cells; p, pancreas. Scale bars = 100 μm.

EXPRESSION / LABELING:
Gene:
Fish:
Knockdown Reagent:
Anatomical Term:
Stage: Day 4
PHENOTYPE:
Fish:
Knockdown Reagent:
Observed In:
Stage: Day 4

Knockdown of cdx1b function affects PepT1 expression in enterocytes. A-C: Wild type (A), cdx1b-4mm MO-injected (B), and cdx1b MO-injected (C) 80-hr post-fertilization (hpf) embryos hybridized with PepT1 antisense RNA probes. D-F: Wild type (D), cdx1b-4mm MO-injected (E), and cdx1b MO-injected (F) 96-hpf embryos hybridized with PepT1 antisense RNA probes. G: Q-PCR indicated decreased PepT1 expression in cdx1b morphants compared to wild type and cdx1b-4mm MO-injected embryos from 80 to 102 hr of development. Error bars indicate standard errors. Results from duplicated experiments are shown. Significant differences between ΔΔCt values based on Student's t-test were used to indicate different gene expressions expressed as fold changes. Student's t-test was conducted to compare cdx1b MO-injected embryos with either wild type or cdx1b-4mm MO-injected embryos.*P < 0.05 in both comparisons. H, I: cdx1b-4mm MO-injected (H) and cdx1b MO-injected (I) 102-hpf embryos hybridized with PepT1 antisense RNA probes. Ib, intestinal bulb. Scale bars = 100 μm.

EXPRESSION / LABELING:
Gene:
Fish:
Knockdown Reagent:
Anatomical Terms:
Stage Range: Protruding-mouth to Day 4
PHENOTYPE:
Fish:
Knockdown Reagent:
Observed In:
Stage Range: Protruding-mouth to Day 4

Transmission electron microscopy (TEM) revealed abnormal ultrastructural morphology in the intestine of cdx1b morphants. A-D: EM sagittal sections of the intestinal bulb (A, C) and mid-intestine (B, D) of 80-hr post-fertilization (hpf) wild type (A, B) and cdx1b MO-injected (C, D) embryos. E-H: EM sagittal sections of the intestinal bulb (E, G) and mid-intestine (F, H) of 96-hpf wild type (E, F) and cdx1b MO-injected embryos (G, H). Arrows indicate the locations of goblet cells in the mid-intestine. Scale bars =10 μm.

Overexpression of cdx1b increased goblet cell numbers in the intestine. A-D: Wild type (A), lacZ mRNA-injected (B), and cdx1b mRNA-injected (C, D) 98-hr post-fertilization (hpf) deyolked embryos hybridized with agr2 antisense RNA probes. E-H: Wild type (E), CMV-GFP-injected (F), and CMV-cdx1b-injected (G, H) 98-hpf deyolked embryos stained with alcian blue. The arrowhead in G indicates secreted mucin in the lumen. I: Comparison of agr2-expressing goblet cell numbers in the intestine of lacZ (n = 20) and cdx1b (n = 20) ectopically expressed embryos. J: Comparison of alcian blue-stained goblet cell numbers in the intestine of GFP (n = 20) and cdx1b (n = 20) ectopically expressed embryos. Error bars indicate standard errors. *P < 0.001. Gc, goblet cell. Scale bars =100 μm.

Effects of ectopic cdx1b expression on the development of enteroendocrine cells in the intestine. A-E: Wild type (A), CMV-GFP-injected (B, C), and CMV-cdx1b-injected (D, E) 96-hr post-fertilization (hpf) deyolked embryos hybridized with glucagon antisense RNA probes. F: Comparison of glucagon-expressing enteroendocrine cell numbers between CMV-GFP-injected (n = 29) and CMV-cdx1b-injected (n = 27) embryos. Error bars indicate standard errors. *P < 0.001. Ac, pancreatic α cells; ee, enteroendocrine cells. Scale bars = 100 μm.

Cell proliferation affected in cdx1b morphants. A-F: BrdU-labeled paraffin transverse sections corresponding to the intestinal bulb (A, D), mid-intestine (B, E), and posterior-intestine (C, F) of respective 72-hr post-fertilization (hpf) cdx1b-4mm-MO-injected (A-C) and cdx1b MO-injected (D-F) embryos. Arrows indicate BrdU-labeled cells. G-L: p-Histone H3-stained cryostat transverse sections corresponding to intestinal bulb (G, J), mid-intestine (H, K), and posterior-intestine (I, L) of respective 72-hpf cdx1b-4mm-MO-injected (G-I) and cdx1b MO-injected (J-L) embryos. Arrows indicate p-Histone H3-stained cells. M: Comparison of the percentages of BrdU-labeled cells in the intestinal bulb, mid-intestine, and posterior-intestine of cdx1b-4mm-MO-injected (n=8) and cdx1b MO-injected (n=9) embryos. N: Comparison of the percentages of p-Histone H3-stained cells in the intestinal bulb, mid-intestine, and posterior-intestine of cdx1b-4mm-MO-injected (n = 5) and cdx1b MO-injected (n = 6) embryos. Total cell number counted for intestinal bulb, mid-intestine, and posterior intestine of both 72-hpf cdx1b -4mm-MO-injected and cdx1b MO-injected embryos are shown. Error bars indicate standard errors. *P < 0.001. Ns, not significant. Scale bars = 100 μm.

TUNEL assay revealed a low level of apoptosis in cdx1b morphants. A-I: Paraffin sagittal sections corresponding to the intestinal bulb (A, D, G), mid-intestine (B, E, H), and posterior-intestine (C, F, I) of respective 72-hr post-fertilization (hpf) wild type (A-C), cdx1b-4mm-MO-injected (D-F), and cdx1b MO-injected (G-I) embryos treated with TUNEL reactions using FITC-dUTP (green). Nuclei were stained with DAPI (blue). Embryos in A-C and G-I are oriented with the anterior to the left, while those in D-F are oriented with the anterior to the right. Scale bars = 100 μm.

PHENOTYPE:
Fish:
Knockdown Reagent:
Observed In:
Stage: Protruding-mouth

Notch signaling was not affected in cdx1b morphants. A-F: Respective 72-hr post-fertilization (hpf) wild type (A, C, E) and cdx1b MO-injected (B, D, F) embryos hybridized with delta D (A, B), notch4/6 (C, D), or her6 (E, F) antisense RNA probes. I, intestine. Scale bars = 100 μm.

Comparison of sox2 expression patterns in wild type and cdx1b MO-injected embryos. A-D: Respective 72-hr post-fertilization (hpf) wild type (A,C) and cdx1b MO-injected (B,D) embryos hybridized with sox2 antisense RNA probes. Higher-magnification images are shown in C and D. Es, esophagus; ph, pharynx; sb, swim bladder. Scale bars= 100 μm.

Acknowledgments
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