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.

Bani-Yaghoub, A switch in numb isoforms is a critical step in cortical development. 2007, Pubmed

Bani-Yaghoub, A switch in numb isoforms is a critical step in cortical development. 2007, Pubmed
Bellefroid, X-MyT1, a Xenopus C2HC-type zinc finger protein with a regulatory function in neuronal differentiation. 1996, Pubmed , Xenbase
Cayouette, Asymmetric segregation of Numb in retinal development and the influence of the pigmented epithelium. 2001, Pubmed
Chalmers, Intrinsic differences between the superficial and deep layers of the Xenopus ectoderm control primary neuronal differentiation. 2002, Pubmed , Xenbase
Chitnis, Primary neurogenesis in Xenopus embryos regulated by a homologue of the Drosophila neurogenic gene Delta. 1995, Pubmed , Xenbase
Chitnis, Sensitivity of proneural genes to lateral inhibition affects the pattern of primary neurons in Xenopus embryos. 1996, Pubmed , Xenbase
Coffman, Expression of an extracellular deletion of Xotch diverts cell fate in Xenopus embryos. 1993, Pubmed , Xenbase
Colaluca, NUMB controls p53 tumour suppressor activity. 2008, Pubmed
Dent, A whole-mount immunocytochemical analysis of the expression of the intermediate filament protein vimentin in Xenopus. 1989, Pubmed , Xenbase
Dho, Characterization of four mammalian numb protein isoforms. Identification of cytoplasmic and membrane-associated variants of the phosphotyrosine binding domain. 1999, Pubmed
Di Marcotullio, Numb is a suppressor of Hedgehog signalling and targets Gli1 for Itch-dependent ubiquitination. 2006, Pubmed
Fineberg, MiR-34a represses Numbl in murine neural progenitor cells and antagonizes neuronal differentiation. 2012, Pubmed
Gulino, The multiple functions of Numb. 2010, Pubmed
Guo, Control of daughter cell fates during asymmetric division: interaction of Numb and Notch. 1996, Pubmed
Harland, In situ hybridization: an improved whole-mount method for Xenopus embryos. 1991, Pubmed , Xenbase
Hartenstein, Early neurogenesis in Xenopus: the spatio-temporal pattern of proliferation and cell lineages in the embryonic spinal cord. 1989, Pubmed , Xenbase
Jahn, Technical innovations for the automated identification of gel-separated proteins by MALDI-TOF mass spectrometry. 2006, Pubmed
Jarriault, Signalling downstream of activated mammalian Notch. 1995, Pubmed
Kageyama, Dynamic Notch signaling in neural progenitor cells and a revised view of lateral inhibition. 2008, Pubmed
Karaczyn, Two novel human NUMB isoforms provide a potential link between development and cancer. 2010, Pubmed
Kyriakakis, Tandem affinity purification in Drosophila: the advantages of the GS-TAP system. 2008, Pubmed
Kyriazis, Numb endocytic adapter proteins regulate the transport and processing of the amyloid precursor protein in an isoform-dependent manner: implications for Alzheimer disease pathogenesis. 2008, Pubmed
Lamborghini, Rohon-beard cells and other large neurons in Xenopus embryos originate during gastrulation. 1980, Pubmed , Xenbase
Lee, Conversion of Xenopus ectoderm into neurons by NeuroD, a basic helix-loop-helix protein. 1995, Pubmed , Xenbase
Li, Inactivation of Numb and Numblike in embryonic dorsal forebrain impairs neurogenesis and disrupts cortical morphogenesis. 2003, Pubmed
Liu, Epigenetic regulation of miR-184 by MBD1 governs neural stem cell proliferation and differentiation. 2010, Pubmed
Ma, Identification of neurogenin, a vertebrate neuronal determination gene. 1996, Pubmed , Xenbase
McGill, Mammalian numb proteins promote Notch1 receptor ubiquitination and degradation of the Notch1 intracellular domain. 2003, Pubmed
McGill, Numb regulates post-endocytic trafficking and degradation of Notch1. 2009, Pubmed
Nieber, Comparative expression analysis of the neurogenins in Xenopus tropicalis and Xenopus laevis. 2009, Pubmed , Xenbase
Nishimura, Numb controls integrin endocytosis for directional cell migration with aPKC and PAR-3. 2007, Pubmed
Oschwald, Localization of a nervous system-specific class II beta-tubulin gene in Xenopus laevis embryos by whole-mount in situ hybridization. 1991, Pubmed , Xenbase
Patzig, Quantitative and integrative proteome analysis of peripheral nerve myelin identifies novel myelin proteins and candidate neuropathy loci. 2011, Pubmed
Petersen, Continuing role for mouse Numb and Numbl in maintaining progenitor cells during cortical neurogenesis. 2004, Pubmed
Petersen, Progenitor cell maintenance requires numb and numblike during mouse neurogenesis. 2002, Pubmed
Pierfelice, Notch in the vertebrate nervous system: an old dog with new tricks. 2011, Pubmed
Reugels, Asymmetric localization of Numb:EGFP in dividing neuroepithelial cells during neurulation in Danio rerio. 2006, Pubmed
Revinski, Delta-Notch signaling is involved in the segregation of the three germ layers in Xenopus laevis. 2010, Pubmed , Xenbase
Rhyu, Asymmetric distribution of numb protein during division of the sensory organ precursor cell confers distinct fates to daughter cells. 1994, Pubmed
Rupp, Xenopus embryos regulate the nuclear localization of XMyoD. 1994, Pubmed , Xenbase
Santolini, Numb is an endocytic protein. 2000, Pubmed
Sato, Numb controls E-cadherin endocytosis through p120 catenin with aPKC. 2011, Pubmed
Schlosser, Induction and specification of cranial placodes. 2006, Pubmed , Xenbase
Shen, Asymmetric Numb distribution is critical for asymmetric cell division of mouse cerebral cortical stem cells and neuroblasts. 2002, Pubmed
Smith, Secreted noggin protein mimics the Spemann organizer in dorsalizing Xenopus mesoderm. 1993, Pubmed , Xenbase
Souopgui, XPak3 promotes cell cycle withdrawal during primary neurogenesis in Xenopus laevis. 2002, Pubmed , Xenbase
Spana, Asymmetric localization of numb autonomously determines sibling neuron identity in the Drosophila CNS. 1995, Pubmed
Tokumitsu, Phosphorylation of Numb regulates its interaction with the clathrin-associated adaptor AP-2. 2006, Pubmed
Tokumitsu, Phosphorylation of Numb family proteins. Possible involvement of Ca2+/calmodulin-dependent protein kinases. 2005, Pubmed
Tonissen, Two neural-cell adhesion molecule (NCAM)-encoding genes in Xenopus laevis are expressed during development and in adult tissues. 1993, Pubmed , Xenbase
Toriya, Distinct functions of human numb isoforms revealed by misexpression in the neural stem cell lineage in the Drosophila larval brain. 2006, Pubmed
Uemura, numb, a gene required in determination of cell fate during sensory organ formation in Drosophila embryos. 1989, Pubmed
Vaira, Regulation of lung cancer metastasis by Klf4-Numb-like signaling. 2013, Pubmed
Verdi, Distinct human NUMB isoforms regulate differentiation vs. proliferation in the neuronal lineage. 1999, Pubmed
Verdi, Mammalian NUMB is an evolutionarily conserved signaling adapter protein that specifies cell fate. 1996, Pubmed
Vernon, The cdk inhibitor p27Xic1 is required for differentiation of primary neurones in Xenopus. 2003, Pubmed , Xenbase
Wakamatsu, NUMB localizes in the basal cortex of mitotic avian neuroepithelial cells and modulates neuronal differentiation by binding to NOTCH-1. 1999, Pubmed
Wang, Only a subset of the binary cell fate decisions mediated by Numb/Notch signaling in Drosophila sensory organ lineage requires Suppressor of Hairless. 1997, Pubmed
Wettstein, The Xenopus homolog of Drosophila Suppressor of Hairless mediates Notch signaling during primary neurogenesis. 1997, Pubmed , Xenbase
Zhong, Mouse numb is an essential gene involved in cortical neurogenesis. 2000, Pubmed
Zhong, Differential expression of mammalian Numb, Numblike and Notch1 suggests distinct roles during mouse cortical neurogenesis. 1997, Pubmed
Zhong, Asymmetric localization of a mammalian numb homolog during mouse cortical neurogenesis. 1996, Pubmed