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. 2023 Jun 5;12:e81716. doi: 10.7554/eLife.81716

Figure 2. WDR62 is associated with the Golgi apparatus in neural progenitors during interphase.

(A) Schematic illustration of Neuroepithelial Stem Cell derivation protocol from induced Pluripotent Stem Cells (iPSCs) (iPS-NES). (B) Immunofluorescence analysis of WDR62 and GOLGA1 in interphase and metaphase iPS-NES cells. During interphase, the WDR62 subcellular localization is comparable in Iso 2 and Mut iPS-NES cells. In metaphase, the WDR62 signal is localized to the spindle poles in Iso 2 iPS-NES cells, but remains diffuse and co-localized with GOLGA1 in Mut iPS-NES cells. Orthogonal projections indicate WDR62, GOLGA1, and DAPI signals. (C) Quantification of WDR62 and GOLGA1 signal co-localization during interphase (top left histogram) and mitosis (top right histogram); WDR62 mean fluorescence in interphase (bottom left histogram) shows no differences in Iso 2 versus Mut iPS-NES cells. Fluorescence distribution in mitotic cells (bottom right histogram) (skewness, arbitrary units) show differences in Iso 2 versus Mut cells (top left histogram: replicates n=3, total cells N=60, p-value >0.05, unpaired Student’s t-test; top right histogram: replicates n=3, total cells N=33, p-value <0.0001, Kolmogorov-Smirnov test; bottom left histogram: replicates n=3, total cells N=164, p-value >0.05, unpaired Student’s t-test; bottom right histogram: replicates n=3, total cells N=21, p-value <0.0001, unpaired Student’s t-test). (D) Schematic illustration of cerebral organoid (CO) derivation protocol from iPSCs. (E) Immunofluorescence analysis of WDR62, GOLGA1, and TUBA1A in Iso 2, Het, and Mut COs at 30 days in vitro (DIV30). WDR62 and GOLGA1A signals co-localize to the apical domain of neural progenitors in COs during interphase. 3D surface plots show WDR62 and GOLGA1 signal distribution within the apical process. (F) Quantification of WDR62 and GOLGA1 signal co-localization in neural progenitors of Iso 2, Het, and Mut COs at DIV30 (COs n=6, total cell N=193, p-value >0.05, Kruskal-Wallis test, post hoc Dunn’s test). (G) Quantification of WDR62 fluorescence within the apical process versus the basal process and the soma of neural rosettes of Iso 2, Het, and Mut COs at DIV30 (COs n=6, total cell N=54, p-value <0.05, p-value <0.001, and p-value <0.0001, two-way ANOVA, post hoc Tukey’s test top histogram; p-value >0.05, one-way ANOVA, post hoc Tukey’s test bottom histogram). Data are shown as mean ± SD. Scale bar = 5 μm in (B) and 20 μm (E).

Figure 2.

Figure 2—figure supplement 1. iPS-NES cells are polarized progenitors.

Figure 2—figure supplement 1.

Morphology of Neuroepithelial Stem cells derived from induced Pluripotent Stem cells (iPS-NES cells), live imaging and representative immunofluorescence assay for SOX2, nestin (progenitor markers), and CTNNB1 (beta catenin, apical domain marker) showing iPS-NES cells at early passages self-organizing into neural rosettes. Scale bars = 50 μm.
Figure 2—figure supplement 2. iPS-NES cells express mutant WDR62.

Figure 2—figure supplement 2.

(A) WDR62 mRNA expression in Neuroepithelial Stem cells derived from induced Pluripotent Stem cells (iPS-NES cells). Quantitative analysis of mRNA expression levels detected by RT-qPCR reveals no differences in Iso 2 and Mut iPS-NES cells. Data are shown as fold change in mRNA expression of WDR62 relative to GAPDH, a housekeeping gene, according to the 2−ΔΔCT method. (B) Western blot showing full-length (165 KDa) and mutant (118 KDa) WDR62 protein expression. CTRL iPS-NES cells were transfected with WDR62 or WDR62 D955AfsX112 expression plasmids. Transfected cell lysates were immunoblotted with antibodies against WDR62 and TUBA1A.
Figure 2—figure supplement 2—source data 1. Uncropped images of western blots.
Figure 2—figure supplement 3. WDR62 knockdown supports localization to the Golgi apparatus.

Figure 2—figure supplement 3.

(A) siRNA-mediated knockdown of WDR62 leads to loss of endogenous signal. Immunofluorescence assay for WDR62, GOLGA1, and TUBA1A shows no WDR62 signal in CTRL Neuroepithelial Stem cells derived from induced Pluripotent Stem cells (iPS-NES cells) transfected with WDR62-siRNA. In non-transfected cells (arrowhead), the WDR62 signal is present at the Golgi apparatus. Magnification of WDR62-siRNA transfected cells in mitosis (A’) and interphase (A’’). (B) Immunofluorescence assay for WDR62 and TUBA1A in CTRL iPS-NES cells co-transfected with WDR62- and LMNA-Cy3 siGLO (positive control for transfection) siRNAs. No WDR62 signal is detected in co-transfected cells (Cy3 fluorescence). (C) Immunofluorescence assay for WDR62, LMNA-Cy3, and TUBA1A in non-transfected CTRL iPS-NES cells. Representative confocal images of WDR62 signal in both mitotic and interphase cells. (D) Representative confocal images of LMNA and LMNA-Cy3 in CTRL iPS-NES cells transfected with LMNA-Cy3 siGLO siRNA. Contrary to non-transfected cells, transfected cells show no LMNA signal (arrowhead). Scale bars = 10 μm.
Figure 2—figure supplement 4. Supporting evidence for WDR62 localization to the Golgi apparatus via Golgi fragmentation.

Figure 2—figure supplement 4.

(A) Schematic illustration of the protocol for Golgi apparatus fragmentation in CTRL Neuroepithelial Stem cells derived from induced Pluripotent Stem cells (iPS-NES cells) through nocodazole treatment and formation of Golgi ministacks. (B) Representative confocal images of GOLGA2 and GOLGA1 in untreated (top), and of WDR62 and GOLGA1 in nocodazole-treated cells showing Golgi apparatus fragmentation. In nocodazole-treated cells, WDR62 follows the same pattern as GOLGA1, indicating WDR62 association with the Golgi ministacks. Arrowheads indicate the areas magnified in the insets. Replicates n=4, total cell N=8. Scale bars = 20 μm.
Figure 2—figure supplement 5. Localization pattern of full-length and mutant WDR62 in iPS-NES cells.

Figure 2—figure supplement 5.

(A) CTRL Neuroepithelial Stem cells derived from induced Pluripotent Stem cells (iPS-NES cells) were co-transfected with WDR62-FLAG and GALT1-mWasabi expression plasmids. Representative confocal images for FLAG, mWasabi (a green fluorescent protein), and GOLGA2. Representative magnified views of a metaphase cell (top row) and of an interphase cell (bottom row) showing WDR62 localization to the mitotic spindle poles and to the Golgi apparatus during interphase. (B – D) Cells were co-transfected with microcephaly (MCPH)-associated WDR62 D955AfsX112-FLAG (B), WDR62 V1402GfsX12-FLAG (C), or WDR62 W224S-FLAG (D) and GALT1-mWasabi expression plasmids. Representative confocal images for FLAG, mWasabi, and GOLGA2. Magnified views of mitotic (top rows) and interphase cells (bottom rows) showing that mutant WDR62 do not localize to the mitotic spindle poles and that the FLAG and mWasabi signals overlap in the Golgi apparatus in interphase cells in a manner similar to the endogenous WDR62/GOLGA1 signals. Scale bars = 10 μm.
Figure 2—figure supplement 6. Characterization of cerebral organoids (COs).

Figure 2—figure supplement 6.

(A) Schematic representation of CO generation (LSBX) via inhibitors of SMAD (LDN193189, SB431542) and WNT (XAV939) from Iso 2, Het, and Mut induced Pluripotent Stem Cell (iPSC) lines. (B) Immunofluorescence assay for nestin and SOX2 in sections of Iso 2, Het, and Mut COs at 30 days in vitro (DIV30) and representative confocal images. (C) Immunofluorescence assay for WDR62 and TUBA1A in Iso 2, Het, and Mut COs at DIV30 and representative confocal images showing the formation of neural rosettes. Boxed areas indicate the magnification in the insets, and white arrowheads indicate mitotic cells. Scale bars = 50 μm in (B) and 20 μm in (C).
Figure 2—figure supplement 7. WDR62 and GOLGA1 localize to the apical domain of radial glia (RG)-like progenitor cells in cerebral organoids (COs).

Figure 2—figure supplement 7.

(A) Immunofluorescence analysis for ZO-1 and GOLGA1 in Iso 2, Het, and Mut COs at 30 days in vitro (DIV30) identifies the position of the Golgi apparatus within the apical process of RG-like cells in neural rosettes. (B) Immunofluorescence analysis for WDR62 and CTNNB1 showing apical localization of WDR62 in rosettes of Iso 2, Het, and Mut COs at DIV30. White dotted lines outline individual cells lining the rosette lumen; yellow lines outline the apical process of RG-like cells. Pink arrowheads indicate the basal process of RG-like cells. 3D surface plots show fluorescence distribution of WDR62 in the apical process of Iso 2, Het, and Mut COs. Scale bars = 20 μm.
Figure 2—figure supplement 8. Full-length and mutant WDR62 overexpression in mouse neural progenitors supports localization to the Golgi apparatus.

Figure 2—figure supplement 8.

(A–D). Immunofluorescence analysis of E14.5 mouse brains for FLAG and mWasabi following in utero electroporation (IUE) at E13.5 with GALT-mWasabi and WDR62-FLAG (A), WDR62 D955AfsX112-FLAG (B), WDR62 V1402GfsX12-FLAG (C), or WDR62 W224S-FLAG (D) expressing plasmids. (A’–D’) Representative confocal images and magnified views of the boxed areas (left) showing overlapping mWasabi (green) and FLAG (magenta) signals in radial processes. Scale bars = 10 μm in (A–D) and 2 μm in (A’–D’).