Nieber F , Hedderich M , Jahn O , Pieler T , Henningfeld KA .

Xla Wt + {dn}dll1 (Fig 3. F)
Xla Wt + {dn}rbpj (Fig 3. G)
Xla Wt + neurog2 (Fig 1. P, Q)
Xla Wt + numbl (Fig 2. B)
Xla Wt + numbl MO (Fig 2. C, D)
Xla Wt + numbl MO (Fig 4. A)
Xla Wt + numbl MO (Fig 4. B)
Xla Wt + numbl MO (Fig 4. C)
Xla Wt + numbl MO (Fig 4. D)
Xla Wt + numbl MO (Fig 4. E)
Xla Wt + numbl MO (Fig 4. F)
Xla Wt + numbl MO (Fig 4. I)

	Figure 1. Expression and regulation of Xenopus Numb and NumbL. (A) Schematic representation of the mouse Numb proteins. The EH motifs are in orange. (B) RT-PCR analysis using X. tropicalis cDNA. Primer pairs used spanning the putative insert regions of the isoforms are indicated on the right side. Arrows indicate the expected PCR products. (C-N) Whole mount in situ expression analysis of X. laevis embryos using a X. tropicalis Numb or X. laevis NumbL antisense RNA probe. Stage 11 embryos shown in C and I are a blastopore view with the dorsal side up. Stage 15/16 embryos are shown in an anterior view (D and J) a dorsal view anterior up (E and K), as well as in transversal sections (F and L). Stage 30 embryos are shown in a lateral view (G and M) and as a transversal section at the level of the hindbrain (H and N). The plane of the transversal sections of E, G, K and M are indicated with a white dotted line. (O) RT-PCR analysis of X. laevis animal caps injected with 20 pg Neurog1-3 mRNA. Explants were cultured until control siblings reached stage 15. H2O represents a negative control where no RNA was added to the RT reaction. (P-S) Whole-mount in situ hybridization of stage 14 embryos injected with Neurog2 (20 pg) or NICD (50 pg) mRNA together with β-Gal (75 pg) mRNA (light blue staining). The injected side is on the right and embryos are shown as a dorsal view. The red asterisk in R and S marks the intermediate stripe that is inhibited on the NICD-injected side. bp, blastoporous; nc, notochord; m, medial stripe; i, intermediate stripe; l, lateral stripe; ba, branchial arches; ol, olfactory placode; ov, otic vesicle; pn, pronephros; pp, panplacodal primordium; vz, ventricular zone; svz, subventricular zone.
	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.
	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.
	Figure 4. Influence of NumbL knockdown on pan-neural and neuronal differentiation genes as well as proliferation. (A-F)X. laevis embryos were injected into one blastomere at the two-cell stage with of 12.5 ng of NumbL MO together with β-gal mRNA (75 pg), and analyzed at the open neural plate stage for the indicated markers by whole mount in situ hybridization. Embryos are shown in a dorsal view, anterior up; the injected side marked by X-Gal staining is always on the right. Statistics are indicated in the lower right corner. (H) Control embryos and embryos injected with 12.5 ng of NumbL MO, were collected at stage 10 and stage 12 and mitotically active cells visualized by staining for pH3. The graph shows the statistical evaluation of 15 consecutive sections of two embryos per stage, based on the number of pH3 positive cells in a defined area on the injected and uninjected site as indicated in the cross-sections. (I) Black double arrows indicated the size of the neural epithelium on the control side (left) and on the injected side (right) from stage 15 embryos. The midline is indicated with a red arrowhead. s, somite, nc, notochord.
	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.
	Figure S2: Alignment of the mouse Numb 1–4 protein sequences with the predicted amino acid sequences from X. tropicalis (Xt) Numb (NM_001097359). The following mouse reference sequences were used: Numb 1/p66 (NP_001129547.1), Numb2/p72 (NP_035079.1) Numb3/p71 (NP_001258984.1) and Numb4/p65 (NP_001258985.1). Alignment was done using Cluster V method using the DNA Star Lasergene Megalign program. Identical conserved amino acids are highlighted in yellow, the blue bar indicates the putative PTB domain, the green bar marks the insert in the PRR domain and the orange bars mark the alpha-Adaptin binding motif DPF (DQF) and the Eps-15 binding motif NPF, respectively.
	Figure S1: Alignment of the NumbL predicted amino acid sequences from X. laevis (Xl) (KF589315), X. tropicalis (Xt) (XP_002938862) and Mus musculus (m) (NP_035080). Alignment was done using Cluster V method using the DNA Star Lasergene Megalign program. Identical conserved amino acids are highlighted in yellow, the blue bar indicates the putative PTB domain, the green bar marks a Q15 repeat and the orange bars mark the α-Adaptin binding motif DPF (DQF) and the Eps-15 binding motif NPF, respectively.
	Figure S3: Comparative whole mount in situ expression analysis of staged X. laevis embryos using an antisense (A-D) and sense (E-H) X. tropicalis Numb RNA probe. Stage 11 embryos shown in A and E are a blastopore view. Stage 16 embryos are shown in an anterior view (B and F) and a dorsal view anterior down (C and G). Stage 26 embryos are shown in a lateral view (D and H) anterior right.
	Figure S4: Double whole mount in situ hybridization expression analysis of stage 15 X. laevis embryo with an antisense X. tropicalis Numb probe (dark purple) and X. laevis EpiK (red). (A) Shown is a dorsal view, anterior down. The blue arrowheads mark the beginning of the longitudinal stripe of Numb expression. (B) Transversal section of the embryo depicted in (A). Numb expression (blue bracketed) in the superficial layer of the ectoderm is directly flanked laterally by EpiK expression (onset indicated by red arrow), which is excluded from the neural ectoderm.
	Figure S5: Comparative whole mount in situ expression analysis of staged X. tropicalis embryos using an antisense X. tropicalis Numb3 RNA probe (A-I) or a antisense X. laevis NumbL RNA probe (J-R). The embryos in A, H, I, J, Q, and R are shown in a lateral view; in B, D, F, K, M and O in an anterior view; in C, E, G, L, N and P in a dorsal view. The expressions patterns obtained are highly correlative with those shown Figure 1 using X. laevis embryos demonstrating cross-species probe hybridization of Numb3 and NumbL and their conservation in expression.
	Figure S6: Verification of the NumbL MO function in vitro and in vivo. (A) The NumbL MO (MO) but not the NumbL mismatch MO (mmMO) inhibits translation of NumbL in in vitro assays. Per reaction, 500 μg NumbL-pCS2 and 1000 ng, 100 ng or 10 ng of MO were used and analyzed by 12% SDS-PAGE. (B): The NumbL MO inhibits GFP reporter construct activities in X. laevis embryos. Embryos were injected in both blastomeres of the two-cell stage with 100 pg of mRNA encoding for the NumbL-5′UTR-GFP reporter and 5 ng of MO. GFP expression was evaluated at stage 10.5. A schematic representation of the reporter construct is shown below, MO binding site is indicated.
	numb (numb homolog ) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior right, dorsal up.
	numbl (numb homolog-like) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior right, dorsal up.

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