1. Volume 135, Issue 5, Pages 1665-1675 (November 2008)
Intestinal Differentiation in Zebrafish Requires Cdx1b, a Functional Equivalent of Mammalian Cdx2 
Maria Vega C. Flores, Chris J. Hall, Alan J. Davidson, Primal P. Singh, Alhad A. Mahagaonkar, Leonard I. Zon, Kathryn E. Crosier, Philip S. Crosier 
Gastroenterology 
Volume 135, Issue 5, Pages (November 2008)
DOI: /j.gastro
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2. Figure 1
Phylogenetic and genomic analysis. (A) Phylogram (branch lengths drawn to scale) generated by bootstrap analysis from 1000 replications. (B) Genetic and radiation hybrid map positions of zebrafish cdx1a and cdx1b showing conservation of syntenic relationships with those of human chromosome (Hs) 5q (C) Alignment of predicted amino acid sequences for the zebrafish Cdx ParaHox family. The conserved homeodomain is underlined.
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
Embryonic expression of cdx1b analyzed by whole mount in situ hybridization. (A–D) Dorsal views of whole embryos at 24, 36, 48, and 72 hpf, respectively; bracket in A marks gut primordium of a 24-hpf embryo (E) 5-dpf larva; upper panel, lateral view; lower panel, dorsal view (F) Lateral view of a 7-dpf larva; inset, magnified lateral view of intestine. All embryos oriented anterior to left.
Gastroenterology  , DOI: ( /j.gastro )
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4. Figure 3
Expression of cdx1a and cdx1b in the adult zebrafish gut. In situ detection of transcripts (purple) on tissue sections of adult gut counterstained with nuclear Fast Red (Vector, Burlingame, CA) shows robust expression of cdx1b (A–C) but no expression of cdx1a (D–F). Scale bars, 50 μm.
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
Specific depletion of Cdx1b by morpholino knockdown. (A) The activity of a cdx1b splice-blocking MO (0.25 pmoles per embryo; ∼2 ng) designed spanning the donor site of the exon 1/intron 1 splice junction was determined by RT-PCR from uninjected, cdx1b sbMO-, and cdx1a MO-treated embryos at 24 hpf and 4 and 6 dpf. The 378-bp product represents intact cdx1b transcript while aberrant intron splicing resulted in a 250 bp (*) or larger molecular size (bracket) PCR product. RT-PCR of 6-dpf MO-injected larva cDNA displayed some recovery of normal cdx1b transcription (arrowhead). (B and C) Dorsal views of uninjected and MO-treated 4-dpf larvae showing expression of fgfrl1a in the pharyngeal region (ph), liver (li), pancreas (pa), and intestinal bulb (ib). Diminished expression of fgfrl1a was detected in the ib of cdx1b sbMO-injected embryos. (D and E) Lateral views of live uninjected and MO-treated embryos at 6 dpf (inset, higher magnification of ib). Folding within the ib of morphant larva (E) appears less distinct, and the rest of the gut tube appears thinner (arrow in E).
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
The intestinal epithelium does not differentiate into absorptive enterocytes in Cdx1b-deficient larvae. All are lateral views, anterior to left. (A–D) Analysis of ifabp expression by in situ hybridization. Normal expression of ifabp in the intestinal bulb of a 3-dpf larva (A) and a cdx1a atgMO-treated larva (0.5 pmol; B). Abrogation of ifabp mRNA in a cdx1b atgMO-injected embryo at 3 dpf (0.5 pmol; C) and in a cdx1b sbMO-injected embryo at 4 dpf (D). (E and F) A 3-dpf embryo hybridized with slc15a1 riboprobe; uninjected larva (E) and a developmentally matched 4-dpf Cdx1b morphant displaying loss of expression (F).
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
Cdx1b morphants do not develop enteroendocrine cells. (A–D) Lateral views of a developmentally matched uninjected (A and C) and a Cdx1b-deficient (B and D) larva showing the intestinal bulb (ib; A and B) and midintestine (mi; C and D). Expression of nkx2.2a in the developing enteroendocrine lineage was only detected in uninjected larva (arrowheads in A and C), although nkx2.2a expression in the pancreas (pa) primordium was normal for both uninjected and morphant embryos. (E and F) Dorsal views of 6-dpf uninjected (E) and cdx1b sbMO-treated (F) larvae displaying expression of nkx2.2a in the ib of uninjected larva (arrowheads).
Gastroenterology  , DOI: ( /j.gastro )
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8. Figure 7
Abnormal intestinal cellular morphology and goblet cell differentiation in Cdx1b morphants. (A–F) Sagittal sections (anterior to left) of 6-dpf larvae stained with Alcian blue-periodic acid Schiff's reagent to highlight mucin in goblet cells. Normal organogenesis in the anterior region of an uninjected larva (A) and a Cdx1b morphant (B). Intestinal sections displayed numerous goblet cells and columnar enterocytes with large supranuclear vacuoles in the midintestine of uninjected controls (C, and higher magnification in E) when compared with those depleted of Cdx1b (D, and higher magnification in F). br, brain; en, enterocyte; es, esophagus; gc, goblet cell; he, heart; ib, intestinal bulb; li, liver; mi, midintestine; pi, posterior intestine; ph, pharyngeal arches; so, somites; sb, swim bladder. Scale bars, 50 μm in A–D; 25 μm in E and F.
Gastroenterology  , DOI: ( /j.gastro )
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9. Figure 8
Proliferation and apoptosis in Cdx1b-deficient larvae. (A–D) Sagittal sections (anterior to left) of midintestinal regions of uninjected (A and C) and cdx1b sbMO-injected larvae (B and D) processed for fluorescent labeling of BrdU incorporation (A and B) and TUNEL reaction (C and D). (E) Graph depicting a comparison of BrdU and TUNEL-positive cells in the midintestine of uninjected (n = 10) and morphant (n = 13) larvae. Numbers represent average counts of high-power fields; 1 field per larva.
Gastroenterology  , DOI: ( /j.gastro )
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