Faas L, Isaacs HV.

Wellcome Trust

	Fig. 5. Effects of Cdx knockdown on Hox gene expression. Cdx function is required for the normal expression of multiple posterior Hox genes. A–E: Whole-mount in situ hybridizations of the indicated Hox genes. A: Late gastrula stage 13 embryos, and B–E show late neurula stage 20 embryos. A–C: Embryos were injected bilaterally with 20 ng of Cdx1, Cdx2, and Cdx4 MOs. A: Panels with HoxA9 shows posterior views with dorsal to the top. Panels with HoxB9 show dorsal views with anterior to the top. Black bars indicate lateral extent of expression domain in neural tube. B: Lateral views with anterior to the left. C: Dorsal views, anterior to the left. D: Representative control embryos and embryos injected unilaterally with 10 ng of Cdx1, Cdx2, Cdx4 MOs, or the triple Cdx MO combination; injected side is indicated with a red asterisk (left panels are dorsal views with anterior to the left, and right panels are posterior views with anterior to the top. E: A control uninjected embryo and embryos injected bilaterally with 20 ng of Cdx1, Cdx2, or the Cdx1 and Cdx2 combination (dorsal views with anterior to the left). Black arrows indicate anterior boundary of expression within the neural tube on injected and contralateral uninjected sides.
	Fig. 6. Effect of Cdx knockdown during gastrulation. A: The major morphogentic movements of gastrulation are not affected in Cdx knockdown embryos. Sagittal sections are shown of a control embryo and an embryo injected with a total dose of 40 ng of the triple Cdx1+2+4 MO combination at late gastrula stage 13 (anterior to the left, dorsal to the top). ar, archaenteron; ntc, prospective notochord; blc, blastocoel; bp, blastopore; end, endoderm. B: Growth factors induce elongation in animal cap explants from Cdx knockdown embryos. Animal hemisphere explants were taken at mid-blastula stage 8 from uninjected embryos and embryos injected with 40 ng of Cdx1, Cdx2, and Cdx4 MOs and cultured to neurula stage 18. Growth factor treatment was with 10 U/ml recombinant FGF4 or 50 U/ml recombinant activin-A (act). C: The expression of early germ layer markers is unaffected in Cdx knockdown embryos. Expression is shown of the endodermal marker Xsox17b and the mesodermal markers Xhox3 and brachyury (Xbra) in triple Cdx knockdown embryos (40-ng total dose) and controls (posterior views, dorsal to the top). Xbra, Xhox3, and Xsox17b embryos are at gastrula stages 11.5, 12.5, and 11, respectively.
	Fig. 7. Effects of Cdx knockdown during post-gastrula stages. All samples are Xenopus tropicalis except (F–H) which are Xenopus laevis explants, which were used because their bigger size allowed easier of processing for in situ hybridization. A: Expression of the trunk/posterior mesodermal markers MyoD and brachyury (Xbra) is unaffected in Cdx knockdown embryos at late neurula stage 20. Embryos were injected unilaterally with 10 ng of combined Cdx1, Cdx2, and Cdx4 morpholino oligos (MOs); red asterisk indicates injected side. Left and middle panels are dorsal views with anterior to the left; right panel is a posterior view with dorsal to the top. B: Expression of the ventral mesodermal markers Scl and Vent2 is down-regulated in Cdx MO knockdown embryos at stage 20. Embryos were injected bilaterally (Scl) or unilaterally (Vent2) with 10 ng per blastomere of the triple Cdx MO combination at the two-cell stage. Left panels are ventral views with anterior to the left; middle panels are dorsal views with anterior to the left and right panels are posterior views with dorsal to the top. Red asterisk indicates the injected side. C: Wnt11 expression is unaffected in triple Cdx knockdown embryos (40-ng total dose) at the early neurula stage 14. Top panels are dorsal views with anterior to the left, and bottom panels are ventral views with anterior to the left. D: Posterior expression of Wnt5A and Wnt3A is down-regulated in triple Cdx knockdown embryos (40-ng total dose) at early neurula stage 14. Dorsal and posterior views are indicated. Dorsal views with anterior to the left and posterior views with dorsal to the top. Black arrows indicate posterior paraxial mesoderm. E: Increasing Cdx activity in embryos up-regulates Wnt3A and Wn5A expression. Embryos were injected with15 pg Xcad-VP16 mRNA. Dorsal views anterior to the left. F: Increasing Cdx function in animal caps up-regulates Wnt3A and Wnt5A expression. Animal cap explants from embryos injected with 50 pg of Xcad-VP16 mRNA were cultured until early neurula stage 14. G: Wnt3A expression is upregulated in animal caps from embryos injected with 250 pg of Cdx1 mRNA. (H) Cdx1 expression is up-regulated in animal caps from embryos injected with 50 pg of Wnt3A mRNA.
	Fig. 9. Effect of Cdx knockdown on endoderm development. A–C: Whole-mount in situ hybridizations to the indicated endodermal markers from neurula to tadpole stages. A,B: Show that expression of the endodermal marker genes Darmin, Vito, and Sox17b is unaffected in (A) early neurula stage 14 embryos (ventral views, anterior to the left and (B) late neurula stage 20 embryos (lateral view, anterior to the left) injected with a total of 20 ng of Cdx1, Cdx2 and Cdx4 MOs into the four vegetal hemisphere cells at the eight cell stage. C: Darmin expression is down-regulated in Cdx knockdown embryos at later stages of development. Lateral views of tail bud stage 32 (left panels) and tadpole stage 42 (right panels) embryos (anterior to the left) are shown.
	Fig. 1. Inhibition of Cdx protein translation by morpholino oligos (MOs). A–C: Western blots showing that the Cdx1, Cdx2, and Cdx2 MOs but not the standard control MO or the corresponding five base mismatch control MOs block the translation of myc-epitope tagged Cdx target proteins in embryos. Early cleavage stage embryos were injected with 10 pg of Cdx-myc mRNA or co-injected with 10 pg of Cdx-myc mRNA+ 10 ng of standard control morpholino (cMO) or 10 ng of the corresponding translation blocking MOs (Cdx1 MO, Cdx2 MO, or Cdx4) or 10 ng of the corresponding five mismatch MOs (Cdx1-mmMO, Cdx2-mmMO, or Cdx4-mmMO). Data are presented for the set-1 morpholinos.
	Fig. 2. The phenotype of Cdx knockdown embryos. A: Single and triple knockdowns of Cdx1, Cdx2, and Cdx4 give rise to a similar range of phenotypes. Single knockdown embryos were injected with 20 ng each of Cdx1-A morpholino oligo (MO), Cdx2-A MO, or Cdx4-A MOs. Triple knockdown embryos were injected with a 20 ng total of the set-1 Cdx1, Cdx2, Cdx4 MOs. B: The phenotypes of larval stage 41 control embryos and embryos exhibiting type 1, 2, and 3 axial defects produced by the knockdown of Cdx function. C: A bar chart showing the proportions of type1, 2, and 3 axial defect embryos in control uninjected embryos, standard control morpholino injected embryos and embryos injected with combined or single set-1 Cdx or set-2 Cdx translation blocking morpholinos. In each case, the total mass injected is made up of equal quantities of each contributing MO. Total injected quantities and n values for each group are indicated on the chart. D: XcadVP16 mRNA rescues the compound Cdx1, Cdx2, and Cdx4 knockdown phenotype. Embryos were injected with a 20 ng of total of the Cdx1, Cdx2, and Cdx4 MO, together with the indicated quantities of XcadVP16 mRNA and cultured until larval stage 41.
	Fig. 3. Histology of Cdx phenotype embryos. Despite shortening of the body axis the main axial and paraxial tissues are present. A–C: Sagittal sections of control, type 2, and type 3 embryos at larval stage 41, respectively. The insets Ai, Bi, and Ci are transverse sections at the indicated axial level. Note the disorganized structure of the anterior central nervous system and enlarged gut cavity in Type 3 embryos. fb, forebrain; gc, gut cavity; hb, hindbrain; mb, midbrain; nt, neural tube; ntc, notochord; sm, somite. Red asterisk indicates enlarged brain ventricle.
	Fig. 4. Overlapping Cdx function. The phenotype of double Cdx knockdown embryos at larval stage 41 can be rescued with a third nontargeted Cdx protein, indicating overlapping function between amphibian Cdx family members. Cdx1+2 knockdown embryos (20 ng of MO total dose) were rescued with the indicated amounts of Cdx4 mRNA. Cdx2+4 double knockdown embryos (20 ng of MO total dose) were rescued with Cdx1 mRNA.
	Fig. 8. Effects of Cdx knockdown on neurula stage explants. A: Cdx knockdown inhibits the elongation of dorsal and ventral explants at control stage 27. Explants were taken at neurula stage 16 from uninjected embryos and embryos injected with a total 40 ng of combined Cdx1, Cdx2, and Cdx4 morpholino oligos (MOs). B: A bar chart showing the average length of dorsal and ventral explants of Cdx knockdown and control embryos at control stage 28 (mean ± SE; n values per group are indicated on the chart).
	Fig. 10. Effects of Cdx knockdown on gut development. A–H: Knockdown of Cdx1 and Cdx2 results in down-regulation of the posterior endodermal marker Intestinal Fatty Acid Binding Protein (IFABP). Exp (E–H). Expression of the anterior gut marker Sox2 is unaffected by Cdx1 and Cdx2 knockdown. Both right lateral and left lateral views are presented of larval stage 41 embryos injected with 20 ng of either Cdx1 morpholino oligo (MO), Cdx2 MO, or their combination. Black arrows indicate anterior gut. I–M: Knockdown of Cdx1 or Cdx2 results in abnormal gut morphogenesis. Embryos shown are at larval stage 45 and were injected with 20 ng of total of either Cdx1 MO, Cdx2 MO, or their combination. Main images are lateral views with anterior to the left. Insets are ventral views of the gut region showing details of gut coiling. M: Shows lateral view of embryos injected into the vegetal cells at the eight-cell stage with 10 ng of total of combined Cdx1 and Cdx2 MOs. Note that targeting the MOs to the presumptive endoderm results in embryos with abnormal gut morphology but normal development of the dorsal axis.
	darmin (darmin) gene expression in Xenopus laevis embryo, NF stage 32, assayed by in situ hybridization,lateral view, dorsal up, anterior left.
	darmin (darmin) gene expression in Xenopus laevis embryo, NF stage 42, assayed by in situ hybridization, lateral view, dorsal up, anterior left.
	darmin (darmin) gene expression in Xenopus laevis embryo, NF stage 14, assayed by in situ hybridization, ventral view, anterior left.
	darmin (darmin) gene expression in Xenopus laevis embryo, NF stage 20, assayed by in situ hybridization, lateral view, dorsal up, anterior left.
	slc5a8 (solute carrier family 5 (sodium/monocarboxylate cotransporter), member 8) gene expression in Xenopus laevis embryo, NF stage 14, assayed by in situ hybridization, ventral view, anterior left.
	slc5a8 (solute carrier family 5 (sodium/monocarboxylate cotransporter), member 8) gene expression in Xenopus laevis embryo, NF stage 20, assayed by in situ hybridization, lateral view, dorsal up, anterior left.

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