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First published online 31 October 2007
doi: 10.1242/dev.005942

Development 134, 4255-4263 (2007)
Published by The Company of Biologists 2007
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Kremen is required for neural crest induction in Xenopus and promotes LRP6-mediated Wnt signaling

Christine Hassler1, Cristina-Maria Cruciat1, Ya-Lin Huang1, Sei Kuriyama2, Roberto Mayor2 and Christof Niehrs1,*

1 Department of Molecular Embryology, German Cancer Research Center, Im Neuenheimer Feld 581, 69120 Heidelberg, Germany.
2 Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK.


Figure 1
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  		Fig. 1. In Xenopus, Krm2 is regulated by Wnt signaling and expressed in the NC region. (A-D) Whole-mount in situ hybridizations. (A) Comparison of Xkrm2, XWnt8 and XWnt3a expression patterns in gastrula and early neurula stage embryos. Top: gastrula stage. Vegetal view, dorsal is up. Bottom: neurula stage. Dorsal view, anterior is up. (B) Comparison of Xkrm2 and slug expression patterns at the indicated stages. Dorsal view, anterior is up. Lowermost panel: view of frontally cut stage 16 embryos, dorsal is up. Brackets indicate overlapping expression domains of krm2 and slug. (C-E) Effect of Wnt pathway perturbations on krm2 expression. (C) Embryos at the 32-cell stage were injected equatorially in two opposite blastomeres with 1 ng PPL or dnWnt8 and 250 pg lacZ mRNA and analyzed at gastrula stage. Arrowheads indicate ß-gal lineage tracer staining (blue). Vegetal view, dorsal is up. (D) Embryos at the four-cell stage were injected animally with 200 pg pCS-PPL or pCSKA-Wnt8 DNA or 100 pg pCS-Wnt3a DNA in one blastomere and analyzed at gastrula stage. Lateral view, dorsal to the right. (E) Statistical overview of experiments shown in C and D. (F) Embryos at the four- to eight-cell stage were injected animally with 100 pg XWnt8 or Wnt3a mRNA, or 1 or 2 ng BMP4 mRNA. Animal caps were cut at stage 8-9, cultured until stage 20 equivalent and were analyzed by RT-PCR for expression of the indicated genes. (G) Embryos at the 32-cell stage were treated with 120 mM LiCl for 50 minutes, cultured until stage 11.5 and analyzed by RT-PCR. Histone H4 was used for normalization. -RT, minus reverse transcriptase control.

 

Figure 2
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  		Fig. 2. Krm2 overexpression induces NC-derived structures and NC markers in Xenopus. (A-E) Phenotypic analysis of krm2 overexpression. Anterior to the left. (A-C) Embryos at the 16-cell stage were injected in a single animal (A,B) or ventral equatorial (C) blastomere with 400 pg krm2 mRNA and photographed at tadpole stages. Arrowheads indicate ectopic pigment-containing structures. (D) Uninjected control embryo at tadpole stage. (E) Embryos at the four-cell stage were injected equatorially in both dorsal blastomeres with 400 pg krm2 mRNA each. (F-M) Neurula stage embryos, shown in anterior view. Embryos were injected with 400 pg PPL (F-I) or krm2 (J-M) mRNA into one ventral equatorial blastomere at the 16-cell stage (F,H,J,L) or one dorsal animal blastomere at the 8-16-cell stage (G,I,K,M). In situ hybridizations were performed using slug and sox10 probes as indicated. ß-gal lineage tracer is stained in red. Circles indicate lineage-tracer-positive cells. Black and white arrowheads indicate altered and control marker gene expression, respectively. (N) Scheme and statistical overview of experiment shown in F-M. Red area indicates region of krm2-injected cells. (O,P) Embryos were injected as in C and sox10 expression was analyzed at tailbud stage by whole-mount in situ hybridization. Arrowheads indicate ectopic sox10 expression. Co-injected lacZ was used as lineage tracer (red).

 

Figure 3
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  		Fig. 3. Krm2 and LRP6 are required for NC induction in Xenopus. (A-D) Morpholino-mediated knockdown of krm2 expression. (A) Embryos at the eight-cell stage were injected animally with 10 ng Krm2 MO-1 plus either 0.8 ng PPL (middle panel) or V5-Xkrm2 mRNA (right panel), respectively. Control, PPL mRNA injected. Normal or hyper-pigmentation was observed in 9% (n=35) of (Krm2 MO-1 + PPL) and in 44% (n=27) of (Krm2 MO-1 + V5-Xkrm2) mRNA injected embryos, respectively. (B) Embryos at the eight-cell stage were injected animally with 10 ng Krm2 MO-1 or 20 ng Krm2 MO-2 or control MO (CoMO), respectively. Shown are whole-mount in situ hybridizations for slug expression at neurula stage in anterior view. ß-gal lineage tracer is stained in red. (C) Statistical overview of MO injection experiment in B. (D) Top left: diagram of experiment. Two-cell-stage embryos were injected animally with 7.5 ng CoMO or Krm2 MO-1, and animal caps (ACs) were explanted at stage 8-9 and combined with dorsolateral marginal zones (DLMZs) of uninjected or Krm2 MO-1 (7.5 ng) injected gastrula-stage embryos. Conjugates were assayed at stage 20 for slug expression by in situ hybridization. Top right: conjugates of DLMZs and CoMO-injected caps. Arrowheads indicate slug expression. Inset: ACs injected with CoMO and processed for slug expression. Bottom left: conjugates of ACs and Krm2 MO-1 injected DLMZs. Arrowheads indicate slug expression. Bottom right: conjugates of DLMZs and Krm2 MO-1 injected ACs. (E,F) Phenotypic analysis of LRP6 MO in X. laevis (E) and X. tropicalis (F). All embryos were injected equatorially at the two-cell stage. (E) (a) Injection of 5 ng CoMO. (b,c) Co-injection of 5 ng LRP6 MO and either 1 ng control (PPL) (b) or 400-600 pg human LRP6 mRNA (c). (e) Statistical overview of a-c. (d) Injection of 20 pg dkk1 mRNA. (F) Xenopus tropicalis embryos injected with 1.25 ng CoMO or LRP6 MO show the displayed phenotypes at frequencies of 89%, n=38, upper panel and 98%, n=44, lower panel. (G) Two-cell-stage X. laevis embryos were injected equatorially in one blastomere with 5 ng CoMO or increasing LRP6 MO doses as indicated. Neurula stage embryos were processed for slug expression by in situ hybridization and are shown in anterior view. ß-gal lineage tracer is stained in red.

 

Figure 4
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  		Fig. 4. Krm promotes LRP6-mediated Wnt signaling. (A-D) TOPFLASH luciferase reporter assays in HEK293T cells. Co, empty pCS2 vector. (A) XKrm1 cooperates specifically with LRP6. (B) Co-transfection of mesd and Xkrm1. (C) BMP luciferase reporter assay in HEK293T cells. (D) Effect of XKrm1 on LRP6 signaling is Dkk1-dependent. RLU, relative light units.

 

Figure 5
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  		Fig. 5. Krm binds to LRP6 specifically and directly. (A,A') Co-IP assays of HEK293T cell lysates transfected with krm1-V5 (A) or krm2-V5 (A') and the indicated constructs. Co-IPs were performed with anti-flag antibody and analyzed by SDS-PAGE and western blotting. (B) In vitro binding assay with secreted recombinant proteins as indicated. IPs were performed with anti-V5 antibody and analyzed by SDS-PAGE and western blotting. Upper panel, protein expression. Middle and lower panels, IPs.

 

Figure 6
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  		Fig. 6. Krm increases cell-surface levels of LRP6. (A) Western blot analysis of HEK293T cells transfected with flag-LRP6 and the indicated constructs. Samples were treated with EndoH as indicated. Arrows indicate the EndoH-resistant mature and EndoH-sensitive immature forms of LRP6. (B) Cell surface biotinylation assay. HEK293T cells were co-transfected with flag-LRP6 and the indicated constructs. After cell surface biotinylation, cell lysates were immunoprecipitated with anti-flag antibody and subjected to SDS-PAGE and western blot analysis. Membranes were probed with streptavidin-HRP to detect plasma membrane LRP6 (upper panel) and anti-flag antibody to detect total LRP6 (lower panel). (B') The cytoplasmic protein NME1 is not biotinylated (upper panel). Lower panel: Total NME1 protein. dg, deglycosylated form; im, EndoH-sensitive immature form; ma, EndoH-resistant mature form.

 

Figure 7
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  		Fig. 7. LRP6 protein is reduced in neural crest of Krm2 morphants. (A) Confocal immunofluorescence analysis of Xenopus NC and animal cap (AC) explants. For NC explants, two-cell-stage embryos were injected with 2 ng LRP6 MO, 3 ng Krm2 MO-1 or 7.5 ng Krm2 MO-2 or CoMO per blastomere. Explants were dissected at stage 14-15, immunostained with anti-ß1-Integrin and anti-LRP6 antibodies, and analyzed by confocal laser scanning microscopy. For AC explants, eight-cell-stage embryos were injected animally with 0.025 ng LRP6 MO, 4 ng Krm2 MO-1 or 15 ng Krm2 MO-2 or CoMO. Explants were dissected at stage 9-10 and analyzed like NC explants. (B) Quantification of relative LRP6 protein levels. The graph shows the ratio of ß1-Integrin (R; red in A) to LRP6 (G; green in A) signal intensities. Numbers in bars indicate the number of analyzed explants. Hoechst-stained nuclei are blue. Scale bar: 50 µm.

 

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© The Company of Biologists Ltd 2007