Journal: bioRxiv

Article Title: Assembly of neuron- and radial glial cell-derived extracellular matrix molecules promotes radial migration of developing cortical neurons

doi: 10.1101/2022.09.03.506497

Figure Lengend Snippet: Histochemical analysis of radial glial cells, intermediate progenitor cells, and the morphology of radial fibers. ( A ) Immunostaining of Pax6-positive radial glial cells and Tbr2-positive intermediate progenitor cells in the VZ of WT and DKO cerebral cortices at E16. Bar graphs show the numbers of Pax6- and Tbr2-positive cells per 150 × 150 μm 2 . N = 5 mice per group. Mean ± SD; ns > 0.05; the Student’s t -test. ( B ) Immunostaining of nestin-positive radial fibers in WT and DKO cerebral cortices at E16. ( C ) High magnification images of mCherry-positive bipolar neurons and radial fibers in the WT and DKO at E16, 2 days after in utero labeling. Bipolar neurons in the SP/IZ-attached radial fiber, irrespective of genotypes. Scale bars represent 20 μm (A), 50 μm (B), and 5 μm (C).

Article Snippet: Sections were incubated at 4°C overnight with biotinylated HA-binding protein (b-HABP) (1:400, Hokudo), anti-NCAN (sheep, 1:400, R&D), anti-TNC (rat, 1:400, R&D), anti-Pax6 (rabbit, 1:400, Invitrogen), anti-Tbr2 (mouse, 1:400, Wako), anti-Nestin (mouse, 1:400, Millipore), and Alexa Fluor 488-conjugated anti GFP (rabbit, 1:1000, Invitrogen).

Techniques: Immunostaining, In Utero, Labeling

Journal: PLoS ONE

Article Title: Radmis, a Novel Mitotic Spindle Protein that Functions in Cell Division of Neural Progenitors

doi: 10.1371/journal.pone.0079895

Figure Lengend Snippet: ( A – C ) E10.5 neural tube of the metencephalon immunostained for radmis ( A , green ) and counterstained with propidium iodide (PI) ( B , red ), showing uniformly distributed radmis in neuroepithelial cells throughout the neural tube. Note the significantly lower immunoreactivity of radmis in the connective tissue outside the neural tube. ( D – F ) Higher magnification view of the ventricular region of an E10.5 neural tube double-stained for radmis ( D , green ) and nestin ( E , red ). Radmis is expressed in the mitotic spindles ( arrowheads ) and nestin-positive radial fibers ( arrows ) of neuroepithelial cells. ( G – J ) E12.5 forebrain immunostained for radmis ( green ) and PI stained ( red ). ( H ) Cerebral neocortex at E12.5. ( I ) Magnified view of the ventricular surface of (H) showing accumulation of radmis in the mitotic spindles ( arrowhead , prometaphase; arrows , metaphase; double arrowhead , anaphase) of dividing NSPCs located at the ventricular surface, and their radial fibers ( double arrows ). ( J ) A dividing NSC at metaphase showing robust distribution of radmis in the mitotic spindle and its extending radial fiber ( double arrows ). ( K – L ) E15.5 neocortex immunostained for radmis ( green ) and Tbr2 ( red ). ( L , L ’) Higher magnification of the boxed area in K. Arrows depicted the radmis immunoreactivity ( L ) in mitotic spindles of Tbr2 ( L ’)-positive dividing cells within the SVZ. Chromosome staining ( blue ) indicated that these two dividing cells were in anaphase. Scale bars: A – C , 250 μm; D – E , 20 μm; G , 200 μm; H , 31 μm; I , 12 μm; J , 3 μm; K , 250 μm; L , 5 μm. lv , lateral ventricle; mge , medial ganglionic eminence; lge , lateral ganglionic eminence.

Article Snippet: EGFP (rabbit polyclonal, Life Technologies, 1:1000), EGFP (chick IgY, AvesLab, Oregon, 1:1000), Tbr2 (chick IgY, Merck Millipore, 1:1000), Pax6 (rabbit polyclonal, MBL, Nagoya, Japan, 1:1000), anti-bromodeoxyuridine (BrdU) (sheep polyclonal, Abcam, 1:1500), Ki-67 (rabbit monoclonal clone SP6, Lab Vision, CA, 1:1000).

Techniques: Staining

Journal: PLoS ONE

Article Title: Radmis, a Novel Mitotic Spindle Protein that Functions in Cell Division of Neural Progenitors

doi: 10.1371/journal.pone.0079895

Figure Lengend Snippet: In utero electroporation of EGFP-radmis, EGFP-radmis KEN mut (radmis-mut), or control EGFP was performed at E14.5, followed by analysis at 24 h post-electroporation (E15.5). ( A – C ) Distribution of cells electroporated with EGFP-radmis ( B ), or control EGFP ( A ) in E15.5 neocortex. Sections were stained for EGFP ( green ) and phosphoH3 ( red ). Arrows indicate EGFP and phosphoH3 double-positive cells in the VZ/SVZ. The ventricular surface is at the bottom, and the pial surface is at the top. ( C ) Magnified view of the ventricular surface of an EGFP-radmis electroporated brain. ( D – G ) Distribution of cells electroporated with control EGFP ( D , F ) or EGFP-radmis-mut ( E , G ). ( D , E ) Double immunostaining for EGFP ( green ) and phosphoH3 ( red ). Hatched areas in panels E and E’ denote the distribution of cells electroporated with EGFP-radmis-mut. NSPCs overexpressing radmis-mut remain mostly in the SVZ/VZ, and many of them express phosphoH3. ( F , G ) Confocal images of electroporated sections stained for EGFP and Tbr2 ( red ). Many cells expressing control EGFP are translocated to the upper IZ toward the pial surface, whereas cells expressing EGFP-radmis-mut remain in the SVZ/VZ and most of them show immunoreactivity for Tbr2. ( H , I ) Representative magnified images of radial glia cells that overexpressed EGFP-radmis-mut. EGFP-radmis-mut expression is frequently found in radial fibers ( arrow in H) and mitotic spindles ( arrowhead ) of radial glial cells, and induces the formation of a monopolar mitotic spindle during mitosis (hatched circle in I) in the VZ. EGFP-radmis-mut ( green ), phosphoH3 ( red ), and DNA ( blue ). ( J ) Quantification of electroporated NSPCs that are positive for phosphoH3 or Tbr2. The ratio of phosphoH3-positive M phase cells, or Tbr2-positive cells, to total EGFP-positive cells in the neocortex was calculated for each electroporation construct, and is presented as the mean ± SEM (%) (group, number of embryo analyzed; EGFP, n = 15; EGFP-Rad, n = 4; EGFP-Rad mut, n = 12). Student’s t -tests; * < 0.01, and **p < 0.001 vs. control EGFP. Scale bars: A – B , 50 μm; C , 20 μm; D – E , 50 μm; F – G , 20 μm; H, I, 10 μm. IZ , intermediate zone; VZ , ventricular zone ; SVZ , subventricular zone.

Article Snippet: EGFP (rabbit polyclonal, Life Technologies, 1:1000), EGFP (chick IgY, AvesLab, Oregon, 1:1000), Tbr2 (chick IgY, Merck Millipore, 1:1000), Pax6 (rabbit polyclonal, MBL, Nagoya, Japan, 1:1000), anti-bromodeoxyuridine (BrdU) (sheep polyclonal, Abcam, 1:1500), Ki-67 (rabbit monoclonal clone SP6, Lab Vision, CA, 1:1000).

Techniques: In Utero, Electroporation, Staining, Double Immunostaining, Expressing, Construct

Journal: PLoS ONE

Article Title: Radmis, a Novel Mitotic Spindle Protein that Functions in Cell Division of Neural Progenitors

doi: 10.1371/journal.pone.0079895

Figure Lengend Snippet: Control EGFP or EGFP-radmis-mut constructs were electroporated into E14.5 embryonic brains that were analyzed after 72 h (E17.5). ( A , B ) Confocal images of E17.5 brain sections electroporated with control EGFP ( A ) or EGFP-radmis-mut ( B ). Sections through the neocortex were stained for EGFP ( green ), Tbr2 ( red ), phosphoH3 ( light blue ), and DNA ( blue ). Hatched areas in panels B and B’ show the cell distributions of cells electroporated with EGFP-radmis-mut. By E17.5, the Tbr2-positive SVZ layer became much thinner upon electroporation of EGFP-radmis-mut (compare with the thickness of the neighboring Tbr2-positive SVZ outside the hatched areas in panel B”). ( C , D ) Magnified views of VZ/SVZ regions in A and B, respectively. Virtually none of the cells overexpressing EGFP-radmis-mut are Tbr2-positive. ( E ) Confocal image of electroporated brain immunostained for EGFP-radmis-mut ( green ), Pax6 ( red ), and phosphoH3 ( blue ). Compared with the control, the majority of cells overexpressing EGFP-radmis-mut no longer express Tbr2, and most of them translocated to the IZL. Arrow indicates persistent expression of radmis-mut in Pax6-positive radial glial cells in the VZ. ( F – I ) Prolonged expression of radmis-mut results in reduced cell proliferation, and increased cell-cycle exit and cell death. E14.5 brains were electroporated with control EGFP ( F ) or EGFP-radmis-mut ( G ) constructs. BrdU was consecutively administered into dams after 24 h post-electroporation (E15.5–E17.5), followed by analysis of brains at 72 h (E17.5). Left column shows merged confocal images of sections stained for EGFP ( green ), BrdU ( red ), and Ki67 ( blue ). Middle and right columns show BrdU-, and Ki67-labeled cells, respectively. Arrowheads indicate EGFP, BrdU, and Ki67 triple-positive cells, and arrows indicate EGFP- and BrdU-positive, but Ki67-negative, cells. ( H , I ) Apoptotic cells ( arrows ) were detected by TUNEL staining in control ( H ) or radmis-mut electroporated brains ( I , I ’). EGFP ( green ) and TUNEL-positive cells ( red ). ( J ) Quantification of 72 h BrdU labeling index. Data are presented as mean ± SEM (%). Student’s t -tests (n = 5 embryos); *p < 0.001. ( K ) Quantification of the cellcycle exit index. The cell cycle exit index was calculated as follows; [(number of EGFP + , and BrdU + cells) – (number of EGFP + , BrdU + , and Ki67 + cells)] /(total number of EGFP + , and BrdU + cells), and presented as the mean ± SEM (%). Student’s t -tests (n = 5 embryos); *p < 0.001. ( L ) Quantification of apoptotic cells. The ratio of TUNEL-positive cells to total EGFP-positive cells was calculated, and is presented as the mean ± SEM (%). Student’s t -tests (n = 6 embryos); *p < 0.01. Scale bars: A – B , 100 μm; C – G , 50 μm; H–I ; 50 μm. IZL , lower intermediate zone; VZ , ventricular zone ; SVZ , subventricular zone.

Article Snippet: EGFP (rabbit polyclonal, Life Technologies, 1:1000), EGFP (chick IgY, AvesLab, Oregon, 1:1000), Tbr2 (chick IgY, Merck Millipore, 1:1000), Pax6 (rabbit polyclonal, MBL, Nagoya, Japan, 1:1000), anti-bromodeoxyuridine (BrdU) (sheep polyclonal, Abcam, 1:1500), Ki-67 (rabbit monoclonal clone SP6, Lab Vision, CA, 1:1000).

Techniques: Construct, Staining, Electroporation, Expressing, Labeling, TUNEL Assay