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doi: 10.1242/10.1242/dev.00306

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The secreted Frizzled-related protein Sizzled functions as a negative feedback regulator of extreme ventral mesoderm

Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA

View larger version (56K): [in a new window]   Fig. 1. Morphological features of embryos injected with szl RNA or with the antisense morpholino oligo. (A) Sibling embryos derived from a single frog were injected with the morpholino oligo MOSZL in both blastomeres at the two-cell stage, or with Szl RNA in the ventral marginal zone at the four-cell stage. Representative stage 33/34 embryos are sorted along a theoretical gradient of Szl protein levels. (B) Sibling embryos were injected with 20 ng MOSZL in both blastomeres at the two-cell stage, or with 40 pg wSzl RNA in the marginal zone at the four-cell stage. Development of a small group of injected and uninjected embryos was recorded by video time lapse between neurula and tailbud stages. For clarity, a single representative individual is shown at each time point (in h:m:s below each panel).

View larger version (90K): [in a new window]   Fig. 2. Elongation of isolated ventral halves from Szl knockdown and overexpressing embryos. Explants were dissected from stage 15 embryos; video micrographs were taken immediately after dissection (left column), and when sibling embryos reached stage 28 (right column). In all panels, explants are oriented with anterior towards the left.

View larger version (50K): [in a new window]   Fig. 3. Szl negatively regulates development of the ventral blood islands (VBI). (A) Benzidine staining of hemoglobin-containing cells. Embryos were injected with the morpholino oligo MOSZL in both blastomeres at the two-cell stage, or with Szl RNA in the ventral marginal zone at the four-cell stage. (B) In situ hybridization analysis of hematopoietic markers in embryos injected with 20 ng of the morpholino oligos MOSZL or MOC2, or with 40 pg of wSzl RNA in the ventral marginal zone. (a) Expression of xSCL at stage 24. Embryos are shown from the ventral side with anterior upwards. (b) Expression of xGata1 at stage 30. Embryos are shown from the ventral side with anterior downwards. Note that detection of xGata1 in MOC2 injected embryos required a longer reaction time, resulting in darker background staining of head structures. (c) Expression of xSCL at stage 28. (d) Expression of larval globin at stage 33/34.

View larger version (48K): [in a new window]   Fig. 4. Szl affects VBI development in whole embryos, but has no effect on blood differentiation in isolated animal caps. (A) BMP4-induced blood differentiation in animal caps. Embryos were injected in the animal pole at the two-cell stage; caps were explanted at stage 8 to 9 and incubated in saline until sibling embryos reached stage 30 to 35. Erythroid markers xGata1 and T3 globin were analyzed by radioactive, semi-quantitative RT-PCR. EF1 was used as an internal control for RNA levels. Where indicated, 50 ng/ml bFGF were added to the medium. When no bFGF was added (left panel), embryos were injected with 1 ng BMP4 RNA. When explants were treated with bFGF (right panel), embryos were injected with 200 pg of a BMP4 expression plasmid (pCS2-BMP4). Where indicated, embryos were also injected with 20 ng of the morpholino oligo MOSZL or with 1 ng of wSzl RNA. (B) Blood differentiation in whole embryos. 20 ng of MOSZL or increasing amounts of wSzl RNA were injected, respectively, into two- or four-cell stage embryos; expression of erythroid markers was assessed by semi quantitative RT-PCR at stage 30 to 35 as above.

View larger version (84K): [in a new window]   Fig. 5. Patterns of gene expression in szl morphants and Szl-overexpressing embryos. (A) In situ hybridization analysis of marker genes in embryos injected with 20 ng of MOSZL. (a,b) Expression of Xbra at stage 10.5; vegetal view. (c,d) Expression of Chordin (blue), and Sox17ß (magenta) at stage 11. Vegetal view, dorsal is upwards. (e,f) Expression of Vent2/Xom at stage 12. Vegetal/posterior view, dorsal is upwards. (g,h) Expression of Xenopus posterior (Xpo) at stage 10.5. Vegetal view, dorsal is upwards. (i,j) Double in situ hybridization for xMyoD (blue) and szl (magenta) at stage 11. Vegetal view, dorsal is upwards. (k,l) Expression of xMyoD (blue) and szl (magenta) at stage 18. In each panel, the embryo on the left is seen from the top, with anterior towards the left; the embryo on the right is seen form the posterior side, with dorsal upwards. (m,n) Szl expression at stage 24. Embryos are seen from the ventral side, with anterior upwards. (B) RT-PCR analysis of szl expression in embryos injected with 20 ng of the morpholino oligo MOSZL, or increasing amounts of wSzl RNA. Endogenous szl was selectively amplified with primers that do not recognize the injected wSzl RNA. EF1 served as an internal control for RNA levels. (C) In situ hybridization of lateral mesoderm markers in embryos injected with 20 ng of MOSZL. (a) Expression of Xlim-1 at stage 20 in lateral (top) and dorsal view (bottom). Arrowheads indicate the point where expression is shifted dorsally in szl morphants. (b-d) Expression of Flk-1 at stage 26 in lateral view and corresponding transverse section. Arrowheads indicate the presumptive vitelline (yellow) and cardinal (black) veins.

View larger version (84K): [in a new window]   Fig. 6. Rescue of the Szl knockdown phenotype by ventral injection of szl RNA. The experimental procedure is schematically illustrated. (A) Uninjected embryos. (B) Embryos injected with 10 ng of MOSZL. (C) Embryos injected ventrally with 40 pg of wSzl RNA. (D) Embryos injected with 10 ng of MOSZL and subsequently injected ventrally with 40 pg of wSzl RNA. Each panel also shows a representative embryo stained with benzidine to reveal hemoglobin-containing cells.

View larger version (115K): [in a new window]   Fig. 7. Effects of Wnt8 and Wnt11 on development of the VBI. Embryos were injected in the marginal zone at the four-cell stage with the indicated amounts of expression vectors encoding xWnt8 or xWnt11 (pCS2-xWnt8 and pCS2-xWnt11), or the indicated amounts of RNA encoding dominant-negative xWnt8 or xWnt11. Embryonic T3 globin was detected by in situ hybridization at stage 30/35.

View larger version (43K): [in a new window]   Fig. 8. Comparative analysis of szl and other secreted Wnt inhibitors. (A-C) Increasing amounts of RNA encoding Myc-tagged versions of Xenopus Szl, Frzb-1 (Frzb), Crescent (Cres) and Dikkopf1 (Dkk1) were injected in the marginal zone of four-cell stage embryos. (A) Expression of the erythroid markers xGata1 and T3 globin was analyzed by RT-PCR at stage 33/34. EF1 was used as an internal control. (B) Expression of the injected Myc-tagged proteins was analyzed by immunoblotting at stage 11 on embryos from the same round of injections. The same blot was probed for actin as a loading control. (C) At the same time, morphology of the embryos and shape of the VBI were visualized by whole-mount in situ hybridization for embryonic T3 globin. (D) Activity of the injected proteins as inhibitors of the canonical Wnt pathway was assessed in a Luciferase reporter assay. Embryos were injected in the animal pole with 200 pg of pTOP-FLASH, 4 pg of xWnt8 RNA, and increasing amounts of RNA encoding Myc-tagged Wnt inhibitors. Relative LUC activity is normalized to activation of the reporter by xWnt8 alone, and amounts of injected sFRPs are plotted on a logarithmic scale. For each RNA dilution, injected proteins were expressed at comparable levels; i.e. Frzb-MT RNA was injected at a higher concentration to compensate for poor translation.