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Figure 2. NumbL gain and loss of function studies in X. laevis embryos. (A-B) Influence of NumbL overexpression on neuronal differentiation in X. laevis embryos at the open neural plate and tailbud stage. Two cell stage embryos were coinjected animally in one blastomere with NumbL mRNA (500 pg) and β-gal mRNA (75 pg). The injected embryos were analyzed by whole mount in situ hybridization for N-tubulin. Shown is a dorsal view of stage 14 embryos, anterior up (A) and the corresponding transversal section (A') as well as a transversal section at the level of the hindbrain of a stage 26 embryo (B). (C-J) Morpholino oligonucleotide mediated knockdown of NumbL. Embryos were injected with 12.5 ng NumbL MO or a mismatched MO (mmMO) together with β-gal mRNA (75 pg) in one blastomere at the two-cell stage and analyzed by for N-tubulin expression at the open neural plate stage. To rescue the NumbL MO phenotype, 100 pg of murine NumbL or Numb mRNA as well as X. laevis NumbL mRNA was coinjected as indicated. Embryos are shown in a dorsal view, anterior up and the injected site is marked by X-Gal staining (light blue) and is always on the right. The number of embryos (n) and percentage of embryos showing the described phenotype are indicated in the lower right corner. The midline is indicated with a red arrowhead. |
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Figure 3. X. laevis NumbL inhibits Notch signaling in reporter assays, but knockdown of NumbL does not influence the expression of Notch target genes in the open neural plate. (A) The influence of NumbL overexpression on a Notch responsive reporter. X. laevis embryos were injected with the Notch-ICD (NICD), Noggin and NumbL mRNA as indicated, together with a Hes1-luciferase reporter. Embryos were cultivated until open neural plate stage and luciferase activity was measured and normalzed to renilla luciferase. Shown is a summary of four independent luciferase experiments. Two batches of embryos containing at least ten embryos per injection mix were collected for each experiment. The error bars represent the standard deviation. (B-E)NumbL MO (12.5 ng) does not lead to an increase in Notch target gene expression at open neural plate stages. (F-I) The NumbL MO induced loss of neuronal differentiation is not rescued by inhibition of Notch signaling. DeltaStu or Su(H)DBM mRNA was injected alone or in combination with 12.5 ng NumbL MO into one blastomere at the two-cell stage and N-tubulin expression analyzed by whole mount in situ hybridization at stage 14. Whole mount in situ probes are indicated in the lower left corner. Embryos are shown in a dorsal view, anterior up. The injected side is marked by X-Gal staining and is always on the right. The midline is indicated with a red arrowhead. |
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Figure 5. NumbL interacts in a tandem affinity purification approach with subunits of the AP-2 complex. (A) Colloidal coomassie blue stained gradient gel of two subsequent immunoprecipitations from X. laevis embryos injected in both blastomeres with 500 pg of NumbL-CTap or CTap mRNA. The bands representing NumbL and major specifically co-immunoprecipitated proteins (i.e. Xenopus proteins that were identified in the NumbL-CTap, but not in the CTap lane) are indicated on the gel as follows: 1, AP-2 beta 1 subunit, AP-2 alpha 2 subunit; 2, NumbL; 3, AP-2 mu1 subunit. (B) Table listing the protein identification details, the numbering of the bands refers to the gel image in Figure 5A. The number of peptides sequenced refers to the set of non-redundant peptides that have been assigned to the protein sequence according to the MASCOT MS/MS ion search algorithm. (C-H) Putative AP-2 binding mutants of NumbL are not able to rescue the MO phenotype. Embryos were injected into one blastomere at the two-cell stage with 500 pg of NumbL mRNA and 12.5 ng NumbL MO as indicated in the upper right corner together with β-gal mRNA (75 pg) and N-tubulin expression was analyzed by whole mount in situ hybridization. Shown are stage 15 embryos in a dorsal view, anterior up. The injected site is marked by X-Gal staining (light blue) and is always on the right. The number of embryos (n) and percentage of embryos showing the described phenotype are indicated in the lower right corner. The midline is indicated with a red arrowhead. |
