EMBO/SourceData huggingface dataset:
Figure 6.(A) Ovaries were stained with TUNEL (green) to detect DNA fragmentation. Clusters of cysts with TUNEL staining were observed in region 2 in nutrient-deprived w1118 files. In Dcp-1Prev flies, fewer TUNEL-positive cysts in region 2 were observed. Under well-fed conditions, numerous TUNEL-positive cysts were observed in BruceE81 flies. DAPI staining of nuclei is shown in white. (B) Numerous degenerating stage 8 egg chambers (arrows) with Bruce-positive staining (green) were observed in well-fed BruceE81 flies. DAPI staining of nuclei (white) is shown on the right.
Figure 7.(A) TUNEL-positive staining was observed in dying stage 8 egg chambers (arrows) of starved control flies (CG5335d30/Atg7d14). DAPI staining of nuclei (white) is shown on the right. (B) In nutrient-deprived Atg7 mutants (Atg7d77/Atg7d14), degenerating stage 8 egg chambers (arrows) showed no or low levels of TUNEL staining. Nuclear DNA condensation, detected by DAPI, was still observed. (C) Dying stage 8 egg chambers (arrows) from nutrient-deprived control siblings (Atg1Δ3D/TM3) generated from the same cross in D had abundant TUNEL-positive staining. (D) In nutrient-deprived Atg1 GLCs, degenerating stage 8 egg chambers (arrows) showed no or low levels of TUNEL staining. Nuclear DNA condensation (DAPI, right) in degenerating egg chambers appeared to occur as in the controls.
Figure 1CSTB gene expression analysis in hCOs, starting from day (d) 16 until d140. For every time point, at least 3 different samples were analyzed; each sample was made by a pool of 3 to 4 hCOs. Data are represented as mean ± SEM. Unpaired t-test (*P < 0.05, **P < 0.01).Micrographs of sections of control hCOs immunostained with CSTB antibody at different time points in culture. The dashed lines represent the apical surface of the cavities/ventricles (V-like). The cortical plate like side is indicated (CP-like). Nuclei (blue) are stained with DAPI.Real time qPCR results of PAX6, NEUN, NESTIN, CSTB gene expression levels in PAX6- and NEUN- FACS sorted nuclei from f-CTRL d135 hCOs. Data are represented as mean ± SEM. Statistical significance was based on T-student test (*P < 0.05, **P < 0.01). For PAX6, 10 samples were analyzed as biological replicates: 5 for Pax6+sorted Nuclei and 5 for Neun+sorted Nuclei; for NEUN, NESTIN and CSTB, 9 samples were analyzed as biological replicates: 5 for Pax6+sorted Nuclei and 4 for Neun+sorted Nuclei.Western blot analysis and relative quantifications (arbitrary units, a.u.) on protein extracts from cortical (Cx) and ganglionic eminence (GE) tissues for Cstb using Actb as a loading control. Cx and GE tissues from mouse embryos at different time of development as indicated in figure.Micrograph of coronal sections of E16 mouse cerebral cortices immunostained for Cstb and acetylated tubulin (acTub) as marker for stabilized microtubules. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Nuclei (blue) are stained with DAPI.
Figure 2Micrograph sections of d40 hCOs electroporated with GFP-empty vector control or GFP-CSTB and analyzed 5 dpe. Sections were then immunostained for GFP and KI67. The dashed lines represent the apical surface of the cavities/ventricles (V-like). The cortical plate like side is indicated (CP-like). Quantification of the total number of proliferating KI67+ cells/area (µm2) of V-like structures transfected with GFP-empty vector control or GFP-CSTB in (A).Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP-empty vector or GFP-Cstb and analyzed 2 dpe and immunostained with GFP,Ki67 and BrdU 30 minutes after BrdU injection. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Quantification of the total number of proliferating Ki67+ cells/area(µm2)(D) and Ki67+Brdu+ cells/area(µm2)(E) of ventricles transfected with GFP-empty vector or GFP-Cstb in (C).Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP-empty vector or GFP-Cstb, analyzed 2 dpe and immunostained with GFP and Phospho-Histone H3 (PH3)(F) and with GFP, Pax6 and Tbr2 (G). Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles.Distribution of proliferating Ki67+/BrdU+cells (H) and mitotic progenitor PH3+ cells (I) in the mouse cortex. The cortex was subdivided in 5 equal Bins -Bin1 corresponded to apical side and Bin5 to pial side of the cortex and PH3+ cells or Ki67+/BrdU+ cells calculated in each Bin.Distribution of Tbr2+ intermediate progenitors (J) and Pax6+Tbr2+ cells (K) in the mouse cortex. The cortex was subdivided in 5 equal Bins -Bin1 corresponded to apical side and Bin5 to pial side of the cortex and Tbr2+ cells or Pax6+Tbr2+ cells calculated in each Bin.
Figure 3Western blot analysis for CSTB in the CSF from 2 different E14 mouse embryos (A), in the CM from E14 cortical cells in culture for 4 daysWestern blot analysis for CSTB in the CM from hCOs after 4 days in culture (C). The CM was collected from hCOs at different time points in culture (d19, d38, d60, d80). Protein extract (PE) from 60 days hCOs was used as a positive control. GAPDH immunostaining was used as a negative control on hCOs CM. Ponceau red staining is shown to indicate the presence of proteins at comparable levels in the CM samplesMicrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co-electroporated with mCherry expressing vector and HA-empty vector or -Cstb, and analyzed 3 dpe. Immunostaining with RFP to identify electroporated cells and GFP to identify migrating interneurons in the GAD65-GFP transgenic mouse line. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Distribution of GFP+ interneurons in the 5 equal Bins of the mouse developing cortex-Bin1 corresponded to apical side and Bin5 to pial side. Ventricle (V) is indicated. The dashed lines represent the apical surface of the ventricles.Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co-electroporated with mCherry and HA-Cstb expressing vector, analyzed 3 dpe. Immunostaining with RFP to identify electroporated cells and GFP to identify migrating interneurons in the GAD67-GFP transgenic mouse line. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Distribution of GFP+ interneurons in the 5 equal Bins of the mouse developing cortex comparing electroporated sides with the contralateral ones in the same section to avoid different distribution of interneurons rostro-caudally.
Figure 5Left panel shows representative images of hCOs from the different iPSC lines at the analyzed time points. Right panel shows the difference of the areas of f-CTRL, b-CTRL, UL1 and UL4 hCOs at different time in culture. Data shown as Z-scores relative to the mean of b-CTRL.Micrograph sections of d30 b-CTRL, UL1 and UL4 hCOs immunostained for proliferation markers KI67 and PH3. The dashed lines represent the apical surface of the cavities/ventricles. Quantification of the total number of proliferating Ki67+ cells/area (µm2) of ventricles like structures and of the number of PH3+ cells facing the ventricle/apical surface length (µm). Data shown as Z-scores relative to the mean of b-CTRL. Nuclei (blue) are stained with DAPI.Micrograph sections of d60 b-CTRL, UL1 and UL4 hCOs immunostained for proliferation markers KI67 and PH3. The dashed lines represent the apical surface of the cavities/ventricles. Quantification of the total number of proliferating KI67+ cells/ area of ventricles like structures and of the number of PH3+ cells facing the ventricle/the apical surface length (µm). Data shown as Z-scores relative to the mean of CTRL. Nuclei (blue) are stained with DAPI.Scheme of the experiment with conditioned medium exchange between b-CTRL and UL4 hCOs and quantification of the Ki67+ cells by FACS analysis on 36 days old dissociated hCOs. Four samples were analyzed as biological replicates.
Figure 6Micrograph sections of d30 b-CTRL, UL1 and UL4 hCOs immunostained for the neuronal marker DCX. The dashed lines represent the apical surface of the cavities/ventricles. Quantification of the DCX+ area. The area of DCX+ cells was calculated and related to the total area (µm2) of the ventricular structure. Data shown as Z-scores relative to the mean of b-CTRL. Nuclei (blue) are stained with DAPI.Micrograph sections of d60 b-CTRL, UL1 and UL4 hCOs immunostained for neuronal marker DCX. The dashed lines represent the apical surface of the cavities/ventricles. Quantification of the DCX+ area. The area (µm2) of DCX+ cells was calculated and related to the total area of the ventricular structure. Data shown as Z-scores relative to the mean of b-CTRL. Quantification of the number of neuronal DCX+ cells by FACS sorting on 60 days old dissociated hCOs. Data shown as Z-scores relative to the mean of b-CTRL. Nuclei (blue) are stained with DAPI.Micrograph sections of d30 fused-organoid; ventral GFP-f-CTRL and dorsal UL1.Micrographs of sections from d30 fused organoids, showing ventral GFP+ cells in the dorsal area. The migrating GFP+ cells were counted starting from 100 µm far from the ventral/dorsal border. Quantification of ventrally-derived GFP+ cells in dorsal/thickness of the counted area (µm3). The counted area was always 100 µm far from the border between ventral and dorsal area as indicated in Fig.EV5D. Every dot represents a different hCOs and different areas were counted for each hCO. Nuclei (blue) are stained with DAPI.Volcano plot representing proteins identified by high-resolution nanoLC MS/MS analysis. Up- and down-regulated proteins (-0.3≥log2FC≥0.3) in human cerebral organoids (UL1 vs b-CTRL) are shown in red and green, respectively.
Figure 3A) TUBB1 is expressed in the developing human thyroid at 8 gestational weeks (GW), 10 GW, and 12 GW and in the adult human thyroid: quantitative PCR results normalized to one of three thyroid tissues at 8 GW value (in black) and peptidylprolyl isomerase A. Experiments with at least three tissues per stage. Statistical comparisons versus 8 GW (in black) using ANOVA test showed no significant differencesB) Tubb1 is expressed in the developing mouse thyroid at E13.5, E15.5, and E17.5 and in the adult mouse thyroid: quantitative PCR results normalized to one of three thyroid tissues at E13.5 value and peptidylprolyl isomerase A. Experiments with at least three tissues per stage. Statistical comparisons versus E13.5 (black), **p<0.01 using ANOVA testC) Sorted mouse thyroid cells, Tubb1 expression in endothelial cells (Pecam-positive cells), epithelial cells (Epcam), fibroblasts (Pdgfra), and leucocytes (CD45) at E17.5 and adulthood: quantitative PCR results (normalized to Epcam-positive cells at adult stage and peptidylprolyl isomerase A). Mean±SEM of three independent cell-sorting experiments. Statistical comparisons versus epithelial cells (in black) at each stage, *p<0.05 using ANOVA testD) Immunohistofluorescence of β1-tubulin (in red), thyroglobulin (TG, in green), and both merged (from left to right) at 12 GW in human thyroid tissue. The boxes delineate the enlarged areas shown belowF) Localization of transfected Myc-tagged wild-type and mutant (P160L) β1-tubulin in Nthy cells. Immunostaining with anti-Myc antibody (in green) and microtubule cytoskeleton stained with α-tubulin antibody (in red). Note that β1-tubulin -P160L is found throughout the cytoplasm showing puncta appearance, whereas wild-type β1-tubulin colocalizes with the microtubules
Figure 4A) Thyroid morphology was investigated using Nkx2-1 staining at E9.5 and E11.5 (sagittal sections) and at E13.5, E15.5, and E17.5 (transverse sections) in wild-type (wt) and Tubb1-/- littermates. Nkx2-1was used as a marker of early thyrocyte progenitors and differentiated thyrocytes. Delays in thyroid migration and in fusion of the median anlage to the ultimobranchial bodies were observed at E11.5 and E13.5 (arrow), respectively, in Tubb1-/- mice. At E11.5, stained migrating thyroid cells were visible along the tract only in Tubb1-/- littermates (arrow). At E17.5, a pyramidal lobe was visible in Tubb1-/- littermates (arrow). tr: trachea; ub: ultimobranchial body. Scale bar: 50 μB) Total thyroid surface area (μm2) was quantified using Nkx2-1 staining at each embryonic stage. Top: thyroid surface area normalised for weight of each embryo. Bottom: thyroid surface area in μm2. Right: proliferation ratio calculated as the proportion of Nkx2-1-positive cells labelled with Ki67 at E9.5 in wt and Tubb1-/- thyroid anlages. ma: median anlage. Note that the thyroid ma is larger and the proliferation ratio higher at E.9.5 in Tubb1-/- mice, whereas from E13.5 onwards the Tubb1-/- thyroids were hypoplastic. Wt in black, KO, Tubb1-/- in greC) Thyroid marker expression by quantitative PCR in thyroid tissue at E15.5 and E17.5 in Tubb1-/- versus wt mice normalised for peptidylprolyl isomerase A. Note the decreases in thyroid differentiation markers at E15.5 and E17.5 and also in thyroid transcription factors at E17.5D) Staining of Nkx2-1 and T4 at E17.5 and surface area quantification demonstrating T4 retention in Tubb1-/- versus wt thyroids. The data are the percentage of T4 or calcitonin (CT) surface area versus total thyroid area (estimated from the Nkx2-1-stained surface area). Scale bar: 50 μm. Student"s test, *p<0.05, **p<0.01 and ***p<0.001. Experiments were done on at least three tissues per stage for each gen
Figure 5A) Serum TSH and T4 levels in 3-month-old Tubb1-/- and wild-type mice. Numbers of animals tested were 10 wild-type males and 14 Tubb1-/- males for TSH and for 14 wild-type and 22 Tubb1-/-males for T4. Tubb1-/- mice had hypothyroidism with elevated TSH and decreased T4 versus wild-type mice. Student"s test, ** p<B) Nkx2-1 (in brown) immunostaining of adult thyroid tissue. Note the disorganisation of the thyroid tissue in Tubb1-/- versus wild-type miceC) Ultrastructural alterations shown by electron microscopy in wild-type and Tubb1-/- thyroid tissues. In Tubb1-/- tissues, note the disorganisation of secretion vesicles (white asterisks) and rods of identical density to secretion vesicles (white arrow). The ER is considerably dilated in Tubb1-/- thyrocytes. Representative views. Scale bars at the bottom left for each view. Co: colloiD) ER stress in Tubb1-/- thyroid tissue. Top: Chop and XBPs expression by quantitative PCR in adults, normalized to peptidylprolyl isomerase A. Experiments with at least three tissues per stage for each genotype; wt in black and Tubb1-/- in grey. Bottom: Chop and Actin protein expression by western blotting in three representative wild-type and Tubb1-/- mice. The Chop band quantification normalised for Actin confirms the increased Chop expression demonstrated by quantitative PCR in Tubb1-/- versus wild-type thyroids. All lanes are from the same blot, which was cut where indicated. Student"s test, *p<0.05 and ***p<E) Co-immunostaining of endoplasmic reticulum (ER) marker (KDEL, in red), thyroglobulin (Tg in green), and both merged (from left to right) in adult thyroid tissue. Note the thyrocyte disorganisation in Tubb1-/- mice
Figure 6A) Representative blood smears of patients with TUBB1 mutations (P1 and P3 homozygous for p.P160L, P4 heterozygous for p.Y106X, and P6 and P7 heterozygous for c.35delG) after May-Grünwald Giemsa staining. Variable platelet size in patients was observed with all three mutations with presence of macroplatelets (arrows)B) Transmission electron microscopy (TEM) analysis of platelet ultrastructure in the patients. The results confirm the abnormal platelet morphologyC) Representative images of proplatelet formation in P1 and P4. Cultured control and patient megakaryocytes (MKs) after thrombopoietin-induced differentiation were spread over a BSA-coated coverslip on day 10. On day 13 or 14, MKs were fixed (4% paraformaldehyde) and proplatelet structure was observed after β-tubulin staining. Scale bars: 20 μm. Only P1 and P4 could be studied. The graphs show the mean ± SEM of the shaft thickness of proplatelet extensions (left) and the diameter of coiled elements (right) of the future proplatelets, both of which were significantly increased in the patients, indicating that the TUBB1 mutations (homozygous p.P160L and heterozygous p.Y106X) affected proplatelet morphology. Statistical significance was determined by one-way ANOVA, followed by Dunnett"s multiple comparisons test (*p<0.05, ***p<0.001; 20-60 MKs were analysed/pat
Figure 7A) Relative expression of α-tubulin and β1-tubulin in platelets from P1 and P3 (homozygous p.P160L), P4 (heterozygous p.Y106X), and P6 and P7 (heterozygous c.35delG) was quantified by Western blotting using specific antibodies against α-tubulin or β1-tubulin. With both mutations, α-tubulin expression was normal but β1-tubulin expression was significantly decreased. The β1-tubulin antibody recognizes C-terminal domainB) αIIbβ3 integrin activation was evaluated by flow cytometry (% of positive platelets and mean fluorescence intensity) in whole blood from P1 and P3 (homozygous p.P160L) and from P6 and P7 (heterozygous c.35delG); P4 could not be studied. While no αIIbβ3 integrin activations were detected in P6 and P7 (family 3), P1 and P3 (family 1) exhibited a significant increase of αIIbβ3 activation, indicating basal platelet activationC) Aggregation of washed platelets induced by ADP (5 or 10 μM) or collagen (0.6, 0.8 or 2 μg/mL) was evaluated in at least one member of each family. Patients from families 1 and 2 had increased platelet aggregation in response to low doses of agonist versus controls, suggesting that TUBB1 affected by homozygous p.P160L and heterozygous p.Y106X mutations affected platelet function. Platelet aggregation to both agonists was normal in both patients from family 3 (heterozygous c.35delG). Family F1, n=3 independent experiments; Family F2, n=1 experiment; Family F3, n=2 independent experiments. Statistical significance was determined by one-way ANOVA, followed by Dunnett"s multiple comparisons test (*p<0.05, **p<0.01, ***p<0.001, nd: statistical significance not determined because
Figure 1(C-C''') Terminal cell expressing reporters for membrane (C), ER (C') and Golgi apparatus (C''). Red arrowheads: CD4::mIFP vesicles and associated markers at the tip of the cell.(D) Mean and 95% confidence interval of tube and branch length during the first hour of growth. Numbers represent the rate of growth estimated from the slope of the curve +/-SD, p=0.39, Mann-Whitney test, n=6.(E) Distance of the tip of the tube, and the leading ER and Golgi accumulations from the cell junction that defines the base of the cell. Line and shading represent mean and 95% confidence interval, n=4.(F-I) Terminal cells expressing the general membrane marker CD4::mIFP in combination with different membrane or polarity markers: PH::mCherry, a PIP2 sensor commonly used as apical plasma membrane marker (F'), an endogenously GFP-tagged Crb (G'), polarity proteins Par3::YFP (H') and Par6::mCherry (H''). The boxed region in (H''') is shown at higher magnification in (I) over 4 time points. Red arrowheads: CD4::mIFP vesicles and associated markers at the tip of the cell.
Figure 2TEM tomograms from high-pressure frozen embryos. (A-A') Low magnification (2.5 nm voxels) tomogram and 3D reconstruction from 10 serial 300 nm sections that cover one terminal cell. (A''-A''') 3D reconstructions derived from higher resolution tomograms showing different organelles in the cell.  (B) Example of an individual high-resolution (0.78nm voxels) tomograms used to generate 3D reconstructions.(C) Low resolution tomogram of another cell; the dotted box is magnified in (D). (D) High-resolution tomogram. (D') Model derived from 4 serial 3D reconstructions including the one shown in (D).
Figure 3(B) Distribution of three commonly seen classes of vesicles, expressed as their density per area of cytoplasm screened, in the two regions of the cell. Box plots represent median, interquartile range (IQR), and IQR*1.5 below and above the IQR. n=62 tomograms from two different cells. (C) Representative examples for each vesicle class are shown next to the quantification plots. (D) Distribution of endocytic events at the apical or basal membrane compartment, expressed as density per membrane surface screened. Left shows tomograms corresponding to the cell body and branch, and right shows tomograms corresponding to the tip of the cell. Box plots represent median, interquartile range (IQR), and IQR*1.5 below and above the IQR. n=48 tomograms from two different cells.
Figure 5 (A-F) TEM tomograms and 3D reconstructions of terminal cells in shibirets embryos. (A) Control. (B-F) Kept at 34ºC for 1 (B, E) or for 2 hours (C, D, F). Boxed regions in (A) and (B) are magnified in (A'') and (B''). (A', B') Reconstructed 3D models. The tube membrane (cyan) is characterized by the electron-dense apical extracellular matrix (aECM). Invaginations with connections that can be traced to the tube membrane are red, invaginations from the basal plasma membrane are green. The basal membrane is light pink. (C-D') shibirets terminal cell after 2 hours at 34ºC. (C') 3D reconstruction, color coding for basal and tube membrane is the same as above. Membrane that is continuous with both the apical and the basal membrane is shown in green, purple and dark blue. (D-D') higher magnification of a region in the same cell at a level where membrane sheets (purple, dark blue) bridge the apical and basal plasma membrane domains. (E-E'') Two focal planes and model of a tomogram from the cell shown in (B) where a basal (pink arrowhead) and an apical invagination (blue) are seen in close proximity (closest distance is marked by cyan and blue arrowheads). (F-F') apical membrane overgrowth regions of the cell shown in (C). The genotype of the embryos was shibirets; btl>KDEL::RFP, Par3::YFP. The cell shown in (C-D) was found and acquired without the CLEM approach.
Figure 6(A-B) Terminal cells expressing the membrane marker CD4::mIFP under btl-gal4 and FGFR::GFP under its own promoter (from the fTRG library). (A) Time lapse imaging of a control cell. Blue arrowheads point to filopodia and basal plasma membrane and red ones point to puncta containing CD4::mIFP and FGFR::GFP. (B) shibirets cell imaged before dynamin inactivation, after 23 and 45 minutes of inactivation, and after 30 minutes of recovery. White arrowheads point to FGFR::GFP accumulation at the apical compartment.(C-E) Single confocal planes of terminal cells expressing PH::mCherry and stained for βPS-Integrin. The outline of the cells was traced using the PH::mCherry signal and is shown as a blue dashed line. (C) Control. Blue arrowheads: βPS-Integrin signal in filopodia. (D) 1 hour at 34ºC, (E) 2 hours at 34ºC. (C''-E'') Orthogonal views of the lines shown in white in (C-E).(F-G) Quantification of fluorescence intensity from stained embryos of FGFR::GFP (F) and of βPS-Integrin (G). Data are plotted as mean +/- SD; significance was assessed using one-way ANOVA with Dunnett's correction for multiple comparisons. **p=0.0022, ***p=0.0003, ****p<0.0001. Number of cells analyzed, for (F): Control n=8, 1hr at 34ºC n=6, Recovery n=8. (G). For (G): Control= 7, 1hr at 34ºC= 8, 2hrs at 34ºC= 8.