EMBO/SourceData huggingface dataset:
Figure 2A-D Wild type (WT), single (Ltk-/- and Alk-/-) and double (DKO) knockout embryos were injected with BrdU on day E14.5 and fixed cortices analyzed at E17.5, P2 and P7. A-C Coronal sections of BrdU labeled WT and mutant cortices at the indicated ages. Dotted lines mark the upper and lower cortical limits determined for each section. The location of the cortical plate (CP), intermediate zone (IZ), subventricular/ventricular zone (SVZ/VZ) in E17.5 and P2 mice and Layers I-VI in P7 WT mice are indicated. Scale bars are 50 µm. B, D Quantitation in E17.5 (B) or P2 and P7 (D) cortices of BrdU-positive cells in the indicated bins (marked on left of images) is plotted. Data are presented as the mean ± SEM from 3 (DKO, Ltk-/-) or 2 (WT, Alk-/-) independent biological experiments (B) or 3 (DKO and WT) independent biological experiments (D). Statistical significance: ****p<0.0001, ***p<0.001, ** p<0.005, *p<0.05 by Student's t-test.
Figure 3B Dissociated E16 cortical neurons were cultured for 38 h and then fixed. Representative images of cortical neurons from WT, single or DKO mice, stained for Tau-1, an axonal marker (red) and MAP2, a dendritic marker (green). Arrowheads mark axons. Data are presented as percent of neurons with multiple, single and no axons and are plotted as the mean ± SEM of 150 of neurons per condition from 3 independent biological experiments. Statistical significance: ****p<0.0001, **p<0.01, *p<0.05 by one-way ANOVA, Dunnett's test.C Representative images of dissociated WT and DKO cortical neurons, electroporated with plasmids encoding EGFP alone or together with HA-tagged LTK-WT. Cells were fixed 38 h after plating and then stained for Tau-1 (axons, red) and MAP2 (dendrites, green). Arrowheads mark axons. Scale bars are 20 µm. Quantitation of the percent of neurons with multiple, single and no axons in EGFP positive neurons is plotted. Data are presented as percent of neurons with multiple, single and no axons and are plotted as the mean ± SEM of 150 of neurons per condition from 3 independent biological experiments. Statistical significance: ****p<0.0001, **p<0.01, *p<0.05 by one-way ANOVA, Dunnett's test.D Representative images of WT cortical neurons, treated with the ALK inhibitors, TAE684 and crizotinib or DMSO as a control at 4 h after plating. Neurons were stained for Tau-1 (red) and MAP2 (green). Arrowheads mark axons. : Data are presented as percent of neurons with multiple, single and no axons and are plotted as the mean ± SEM of 150 of neurons per condition from 3 independent biological experiments. Statistical significance: ****p<0.0001, **p<0.01, *p<0.05 by one-way ANOVA, Dunnett's test.E Golgi stains of adult cortices in wild-type, single (Ltk-/- and Alk-/-) and double (DKO) knockout mice show severe defects in neuronal projections and the callosal tract. Representative coronal sections of cortical layers I-V in WT and DKO mice (left), Layers IV-V in WT, single and double knockout cortices (center) and higher magnification images of WT and DKO neurites (right). Examples of disrupted directional growth and extension of projections in pyramidal neurons in knockout mice are marked (red arrowheads).F Representative coronal sections of the medial region of the corpus callosum in wild-type and mutant cortices. Red line delineates the upper and lower regions of the corpus callosum (CC) with higher axon density within the upper corpus evident in WT mice. Prominent defects in the callosal fibers including substantial deviations and incoherent projections from the major tract in knockout mice compared to WT mice are marked (red arrowheads). Schematics of the regions represented in images are shown on the right. CC: Corpus callosum, Py: pyramidal neurons.
Figure 4C Schematic of the puzzle box arena consisting of a white, light compartment (start box) and a black compartment (goal box) divided by black barrier. Latency to reach the goal box during the 9 trials of the test is plotted for each individual 2-3 m old mouse of the indicated genotypes.
Figure 5Representative images of cortical neurons fixed at 38 h and stained with Tau-1 (axons, red) and MAP2 (dendrites, green) are shown. Arrowheads mark axons. C, D Treatment of cortical neurons from WT or KO mice with PI3 kinase inhibitors reverses polarity defects caused by loss of Ltk/Alk. Dissociated E16 cortical neurons were treated with LY294002 (10 µM; C), BKM120 (1 µM; D) or DMSO control, 4 h after plating. : In quantitation of the percent of neurons with multiple, single or no axons is plotted as the mean ± SEM of n=150 neurons per condition from , C-WT) or 4 (C-DKO independent biological experiments. Statistical significance: ****E A representative immunoblotting showing the levels of pAKT (pAktS473), Akt and GAPDH (loading control) in lysates of cortical neurons from WT, single and DKO mice. Quantitation of pAktS473 to Akt levels relative to WT is plotted.
Figure 6A, B IGF-1R inhibitors rescue the multiple axon phenotype in DKO cortical neurons. Cortical neurons, isolated from WT and DKO mice were treated with 1 µM AG1024 (A), varying concentrations of Picropodophylin (PPP; B) or DMSO as control 4 h after plating, fixed at 38 h, and stained for Tau-1 (axons, red) and MAP2 (dendrites, green). Arrowheads mark axons. Scale bars are 20 µm. Quantitation of the percent of neurons with multiple, single or no axons is plotted. Data are presented as the mean ± SEM, n ≥ 150 neurons per condition from 4 independent biological experiments.C Blocking Igf-1r activation with a neutralizing anti-IGF-1Rα antibody rescued the multiple axon phenotype in DKO cortical neurons. Neurons isolated from WT or DKO mice were treated anti-IGF-1Rα antibody (1.5 µg/ml), BSA as control (1.5 µg/ml), or PPP (200 nM) 4 h after plating. Representative images of Tau-1 and MAP2 stained cells with arrowheads marking axons are shown. Scale bars are 20 µm. Quantitation of the percent of neurons with multiple, single or no axons is plotted.F Inhibition of Igf-1r blocks neuronal migration in vivo. Pregnant E14 WT or DKO mice were injected (i.p) with BrdU and 24 h later were injected with PPP or saline as control. Brains from P2 pups were subjected to staining with anti-BrdU antibody. Quantitation in P2 cortices of BrdU-positive cells in the indicated bins (marked on left) is plotted. The location of the cortical plate (CP), intermediate zone (IZ), subventricular/ventricular zone (SVZ/VZ) in P2 mice and are indicated. Scale bars are 50 µm. Data in (F) are presented as the mean ± SEM from 3 (DKO) and 2 (WT) independent biological experiments. Statistical significance: ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05 by one-way ANOVA, Dunnett's test by Student's t-test (F), where WT vs WT+PPP or DKO vs DKO+PPP were compared.
Figure 7A Representative immunoblot showing the levels of phosphorylated (pIgf-1rY1135/1136), total Igf-1r and Gapdh (as loading control) in lysates of isolated cortical neurons from WT and DKO mice. Band intensities for Igf-1r were normalized to Gapdh (left plot) and for pIgf-1r were normalized either to Gapdh (middle plot) or to Igf-1r (right plot) and the fold increase relative to WT is plotted. Data are presented as the mean ± SEM from 3 (A independent experiments. Representative blots from 3 independent biological experiments are shown in (AC Representative image showing phosphorylation of recombinant His-tagged IGF-1R (kinase domain and C-terminal region) by recombinant GST-LTK (kinase domain) in an in vitro radioactive kinase assay. Phosphorylation of IGF-1R was quantitated as fold change relative to auto-phosphorylation of IGF-1R alone. Data in (C are plotted as the mean ± SEM from 6 (C) independent biological experiments.E Endogenous ALK was immunoprecipitated from lysates of IMR-32 cells, treated with control (CCM) or ALKAL2 (ACM) conditioned media and co-precipitated endogenous IGF-1R was detected by immunoblotting.G Cell-surface expression of Igf-1r is increased in DKO cortical neurons. Cell-surface Igf-1r (NA ppt) is normalized to totals and the fold change relative to WT samples is plotted.I WT cortical neurons were labeled with biotin and incubated at 37ºC for 15 or 30 min in the presence of CCM or ACM conditioned media. Cell-surface Igf-1r (NA ppt) normalized to totals as the fold change relative to controls at time 0 is plotted.J WT and DKO cortical neurons were treated with control (CCM) or ALKAL2 (ACM) conditioned medium 4 h after plating and IGF-1 was added 1 hour later. Representative images of cells fixed at 36 h and stained for Tau-1 (axons; red) and MAP2 (dendrites: green) are shown. Arrowheads mark axons. Scale bars are 20 µm. In (J) quantitation of the percent of neurons with multiple, single or no axons is plotted as the mean ± SEM of n=150 neurons per condition from 3 independent experiments.
Figure 1To monitor the progression of the CLL-like population, blood was collected from the animals every four weeks up to an age of 60 weeks and analyzed by flow cytometry. Representative FACSplots show examples of stainingsof B220lowCD5+B cells (CLL-like cells) and the selected gate for B).To monitor the progression of the CLL-like population, blood was collected from the animals every four weeks up to an age of 60 weeks and analyzed by flow cytometry. The diagram displays the percentage of CLL-like cells in relation to all lymphocytesover a period of 60 weeks. Shown are mean values with ±SD. Significant differences between groups were determined by one-way ANOVA with Kruskal-Wallis test (no normal distribution) and corrected for multiple comparison with Dunn's test, **p<0.01, ***p<0.001. Summarized data from more than 40 independent experiments for individual time points, comprising n=4-75 animals per time pointand genotype. X = no surviving TCL1 x Siglecg-/-animals at the ageof 60 weeks. + indicates that in these groups only 4 animals were used for the analysis and therefore no calculation of significance was possible.
Figure 2The absolute cell numbers of B220low CD5+ lymphocytes at different time points in the spleen are shown as mean values with ±SD. Significant differences between groups were tested by one-way ANOVA with Kruskal-Wallis test and corrected for multiple comparison with Dunn's test. The different time points were tested separately. Alongside representative dot plots show selection and percentages of B220low CD5+ cells in the spleen pre-gated on single cells, living cells and lymphocytes.The percentage of B220low CD5+ leukocytes in the liver at different time points is illustrated. Besides representative dot plots show the selection of B220low CD5+ cells in the liver. Cells were pre-gated on single cells, living cells and CD45+ leukocytes. Shown are mean values with ±SD. Significant differences between groups were tested by ordinary one-way ANOVA with Šídák's post-hoc test. Data are from at least 10 independent experiments for individual time points, comprising n=5-12 animals per time point and genotype. X = no surviving TCL1 x Siglecg-/- animals at the age of 60 weeks. *p<0.05, **p<0.01, ****p<0.0001.The survival of TCL1 x Siglecg-/- mice compared to TCL1 mice is illustrated with the Kaplan-Meier survival curve. (n=81 for TCL1, n=101 for TCL1 x Siglecg-/-). The groups were compared for significant differences via log-rank test.
Figure 4To monitor the progression of the CLL-like population, blood was collected from the animals every four weeks up to an age of 48 weeks and analysed by flow cytometry. Genotypes of the mice are: controls (Siglecg-R26ki/ki), Siglec-G overexpressing mice (Siglecg-R26ki/ki mb1cre), transgenic TCL1 controls (TCL1 x Siglecg-R26ki/ki) and transgenic TCL1 Siglec-G overexpressing mice (TCL1 x Siglecg-R26ki/ki mb1cre). Representative FACSplots show examples of stainingsof B220lowCD5+B cells (CLL-like cells) and the selected gate for B)To monitor the progression of the CLL-like population, blood was collected from the animals every four weeks up to an age of 48 weeks and analysed by flow cytometry. Genotypes of the mice are: controls (Siglecg-R26ki/ki), Siglec-G overexpressing mice (Siglecg-R26ki/ki mb1cre), transgenic TCL1 controls (TCL1 x Siglecg-R26ki/ki) and transgenic TCL1 Siglec-G overexpressing mice (TCL1 x Siglecg-R26ki/ki mb1cre). The diagram displays the percentage of B220lowCD5+cells in respect to lymphocytesover a time periodof 48 weeks. Shown are mean values with ±SD. Significant differences between groups were determined by one-way ANOVA with Kruskal-Wallis test (no normal distribution) and corrected for multiple comparison with Dunn's test. All time pointswere tested separately. *p<0.05, **p<0.01, ***p<0.001. Summarized data from more than 20 independent experiments with n=5-20 animals per time pointand genotype are shown.
Figure 5(C) Absolute cell numbers of B220low CD5+ lymphocytes at 36 and 48 weeks of age in the spleen are shown as mean values with ±SD. Significant differences between groups were tested by one-way ANOVA with Kruskal-Wallis test and corrected for multiple comparison with Dunn's test. The different time points were tested separately. n=8-13 animals per time point and genotype. (D) Representative dot plots show selection and percentages of B220low CD5+ B cells in the spleen pre-gated on single cells, living cells and lymphocytes. Data are from at least 10 independent experiments for individual time points. *p<0.05, **p<0.01, ***p<0.001. n=48 for TCL1 x Siglecg-R26ki/ki, n= 52 for TCL1 x Siglecg-R26ki/ki mb1cre(E)The percentage of B220low CD5+ lymphocytes in the liver at 36 and 48 weeks of age is illustrated. Shown are mean values with ±SD. Significant differences between groups were tested by ordinary one-way ANOVA with Šídák's post-hoc test. n=7-14 animals per time point and genotype. (F) Representative dot plots for the selection of B220low CD5+ cells in the liver. Cells were pre-gated on single cells, living cells and CD45+ leukocytes. Data are from at least 10 independent experiments for individual time points. *p<0.05, **p<0.01, ***p<0.001. n=48 for TCL1 x Siglecg-R26ki/ki, n= 52 for TCL1 x Siglecg-R26ki/ki mb1cre(G) The differences in survival are shown as Kaplan-Meier plots and log-rank test n=59 for TCL1 x Siglecg-R26ki/ki , n=57 for TCL1 x Siglecg-R26ki/ki mb1cre).
Figure 7To measure calcium influx cells were loaded with Indo-1. Shown is the mean calcium concentration as ratio of bound to unbound Indo-1 versus time in s. First the basal level was monitored for 50 s in Krebs-Ringer solution. Subsequently, the BCR was stimulated with 13 µg/ml anti-IgM F(ab)2 (shown by the black arrow). Shown is one representative result of four independent experiments. Mice were between 36 and 48 weeks of age. To measure intracellular signaling splenic cells were pre-gated on single, living cells and lymphocytes, then B220+ cells, including B220low CD5+ lymphocytes were selected. TCL1 and TCL1 x Siglecg-/- B cells were analyzed by intracellular staining with phopho-specific antibodies (or total protein detecting antibody Bcl2).TCL1 x Siglecg-R26ki/ki or TCL1 x Siglecg-R26ki/ki mb1-cre B cells were analyzed by intracellular staining with phopho-specific antibodies (or total protein detecting antibody Bcl2).
Figure 8Peripheral blood B cells of CLL patients are pre-gated on single, living B lymphocytes (CD19+). The mean fluorescence intensity (MFI) of surface Siglec-10 is given from 35 IgV-mutated (left) and 18 IgV-unmutated CLL cases (right), always including tumor cells (CD20lowCD5high) and normal residual B cells (CD20highCD5-) as paired samples.The transcript expression (normalized counts from bulk mRNA sequencing) of SIGLEC10 is given from five healthy donors, discriminating naïve CD5- (five donors), mature CD5+ 86 donors) and CD27+ memory B cell (eight donors) subsets, from CLL tumor cells (n=9) and from five paired normal residual B (NRB) cells (n=5).
Figure 1ivMCs induce differentiation of co-cultured intestinal organoids as determined by qRT-PCR for stem cell (Lgr5, Olfm4) and proliferation (Ki67, Pcna) and differentiation markers (Muc2-goblet cells, Krt20-differentiated epithelial cells, Sim and Alpi-enterocytes). Enhanced terminal enterocytic differentiation was checked by villus center markers (Fabp1, Fabp2, G6pc, Leap2) and villus tip markers (Ada, Lama3, Lgals3, Cdh1). (Expression levels normalized to Ubiquitin B (Ubb), ENR parallel set as 1, error bars show sd, each dot represents one technical replicate of one representative experiment). If n = 2 technical replicates, error bars, and mean are not shown.The impact of individual Bmp ligands on the growth of freshly established intestinal organoids. (After 96 hours in 500 ng/ml of indicated Bmp. Brightfield images. Scale bar, 200 μm) Proliferation of freshly established intestinal organoids cultivated with indicated Bmp for 96 hours determined by quantification of EdU incorporation (% of EdU positive cells). Each dot represents one cultured organoid. Average value for each treatment indicated by line, error bars show sd.Relative expression of differentiation (Muc2-goblet cells, Krt20-differentiated epithelial cells, Sim and Alpi-enterocytes) and stem (Lgr5, Olfm4) markers in intestinal crypts upon 96 hours cultivation with indicated Bmp (500 ng/ml). (qRT-PCR, expression levels normalized to Ubiquitin B (Ubb), error bars show sd, Untreated parallel set as 1, each dot represents one technical replicate of one representative experiment.).
Figure 2Heatmap showing changes in expression of indicated genes after 24 hours of indicated Bmp (500 ng/ml) treatment in the intestinal organoids. Hierarchical clustering of Bmp treatments shows differences in gene regulation by Bmp2 vs. Bmp4. (n= at least 3 per each treatment and/or control, top 10 differentially expressed genes per each treatment were selected and are shown. Z-score was calculated using standard formula z = (x - μ) / σ).Whereas Bmp4 triggers expression of villus center genes connected to lipid metabolism, Bmp2 preferentially activates the expression of villus tip genes. (Graphs show normalized counts determined by RNAseq, n= at least 3 for each treatment, timepoint: 24 hours, error bars denote sd).
Figure 3Bmp2 treatment induces the expression of the villus tip markers Nt5e (Cd73 protein). (Graph shows normalized counts determined by RNAseq, n= at least 3 for each treatment, timepoint: 24 hours, error bars denote sd).Expression of Cd73 (Nt5e gene) (green) and Fabp2 (red) upon the Bmp2 and Bmp4 treatment in the intestinal organoids. Immunocytochemistry, DAPI (blue) counterstains nuclei, timepoint: 48 hours. (Scale bar,100 μm).Quantification of Cd73 (Nt5e gene) and Fabp2 immunostainings determined as the ratio of specific signal to DAPI ratio. (Each dot represents one organoid, timepoint: 48 hours, n= at least 20 organoids, graphs show: min.,1st quartile, median, 3th quartile, max., p values counted using t-test).Relative Ada enzymatic activity in intestinal organoids after Bmp2 and Bmp4 treatment (Each dot represents individual measurement, timepoint: 48 hours, sd.).
Figure 4Tgfβ induces the expression of villus tip genes and represses villus center genes. (Graphs show normalized counts determined by RNAseq, n=3 for each treatment, timepoint: 24 hours, error bars denote sd.).
Figure 5Four days treatment with LDN-193189-4HCl (50 mg/kg) reduces the expression of Ada (green) protein at the villus tips. (Immunohistochemistry, DAPI (blue) counterstains nuclei. E-cadherin (white) marks epithelial cells, scale bar, 100 μm).Quantification of Ada signal range at the villus tip as visualised in B. (Each dot represents one villus, n= at least 20 villi, graphs show: min.,1st quartile, median, 3th quartile, max., p values counted using t-test).Changes in the expression of villus tip and villus center genes after LDN-193189-4HCl (150 mg/kg) treatment determined by qRT-PCR normalized to β-actin (β-act). (Each dot represents one technical replicate of one representative experiment, error bars show sd.).
Figure 6Induction of shRNA against Smad4 upon the doxycycline (+Dox) treatment (3 days) followed by Bmp2 treatment for additional 24 hours. shRNA activation is visualised by the expression of coupled TdTomato (red), scale bar, 200 μm.Smad4 knock-down (shRNA-ON) alleviates Bmp2-induced differentiation marked by relative expression of villus tip genes. (qRT-PCR, expression levels normalized to Ubiquitin B (Ubb), each dot represents one technical replicate of one representative experiment, error bars show sd, untreated parallel set as 1, treatment as in 6A).Long term (4 weeks) organoid cultivation in the presence of Bmp2 (10 ng/ml) enriches the expression of villus tip genes. (qRT-PCR, expression levels normalized to Ubiquitin B (Ubb), each dot represents one technical replicate of one representative experiment, error bars show sd, untreated parallel set as 1, P2 - collected 4 days after 2nd passage, P4 -collected 4 days after 4th passage).
Figure 7qRT-PCR for villus tip genes and villus center genes in freshly established organoids cultivated 24 or 96 hours with Bmp2 (500 ng/ml), Wnt5a (500 ng/ml) or both Bmp2 + Wnt5a (each 500 ng/ml). Whereas Bmp2 induces villus tip genes by its own, it can act synergistically with Wnt5a. For the short-term cultivation, the effect is specific for villus tip genes (notice the different scale in left graphs). (Expression levels normalized to Hmbs, untreated parallel set as 1, each dot represents one technical replicate of one representative experiment, error bars show sd). If n = 2 technical replicates, error bars and mean are not shown.Expression of E-cadherin (red) and Bmp2, Bmp4, Wnt5a, Bmp2 + Wnt5a treatment in the intestinal organoids. Immunocytochemistry, DAPI (blue) counterstains nuclei. (Immunocytochemistry, timepoint: 24 hours, 500 ng/ml each protein, scale bar, 200 μm).
Figure 1SDS-PAGE analysis of the binding of Strep-tagged REC114 variants (using a single Strep-tag (WSHPQFEK)) to co-expressed His-MEI41-127 after purification on Strep-Tactin resin. SDS-PAGE analysis of the binding of His-MBP-REC114226-254 to co-expressed His-MEI4 variants after purification on Amylose resin.Native MS spectrum of co-purified Strep-REC114 and MEI41-127. The main complexes are Strep-REC114:MEI41-127 in ratios 2:1 and 4:2. There are also free Strep-REC114 and 1:1 Strep-REC114-MEI4 complex.Sedimentation velocity profiles of the REC114226-254-MEI41-43 complex obtained at 280 nm at three concentrations (0.2 mg/ml, 1 mg/ml and 4.5 mg/ml) in a solution containing 20 mM Tris pH 8.0 and 200 mM NaCl. Expected sedimentation coefficient for the 2:1 complex is 1.13S and for 4:2 is 1.58S. The sedimentation coefficient of the main peak increases with increasing protein concentration: s=1.2S at 0.2 mg/ml, s=1.44S at 1 mg/ml and s=1.64S at 4 mg/ml. These results suggest a fast exchange between the two forms.
Figure 2Native MS spectrum of His-MBP-REC114226-254. This protein is a dimer.
Figure 3E. Pull-down experiments of full-length Strep-tagged REC114 mutants indicated above the lanes with His-MBP-MEI41-43. All proteins were first purified by affinity chromatography and gel filtration. A total of 0.9% of the input (lanes 1-6) and 1% of the eluates (lanes 7-12) were analyzed on 15% SDS-PAGE gels stained with coomassie brilliant blue. Control lanes 1-2 and 7-8 show inputs­ and elutions of REC114 and MEI4 alone, respectively. The MEI4 sample contains also free MBP tag. The REC114 sample also contains a degradation product, likely corresponding to its PH domain.F. Pull-down experiments of Strep-tagged REC114 with His-MBP-MEI41-43 mutants indicated above the lanes. All proteins were first purified by affinity chromatography and gel filtration. A total of 0.9% of the input (lanes 1-4) and 1% of the eluates (lanes 5-8) were analyzed on 15% SDS-PAGE gels stained with coomassie brilliant blue. The MEI4 sample contains also free MBP tag. The REC114 sample also contains a degradation product, likely corresponding to its PH domain.
Figure 4Molecular mass determination of IHO11-281, IHO1125-281 and IHO1196-245 by MALLS using Superose 6 column. The measured molecular mass of the main peak of each construct is 120 kDa, 67 kDa and 24 kDa, respectively. These values correspond to calculated molecular weights of IHO1 tetramers (125 kDa, 72 kDa and 24 kDa, respectively). The samples were injected at 5-10mg/ml.