The Journal of Neuroscience, January 18, 2006, ():
Molecular and Morphological Heterogeneity of Neural Precursors in the Mouse Neocortical Proliferative Zones
J. Neurosci. Gal et al.
26: 1045
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplemental Figure 1: In utero electroporation of mammalian expression plasmids. Plasmid DNA mixed with fast green dye was injected into the lateral ventricle using a pulled glass capillary tube. Electrodes were placed outside the uterine muscle with the positive electrode above the head. Uterine horns were replaced within the abdomen and the dam sutured to allow for an additional 24-48 hrs of in vivo development. Electroporation resulted in VZ-specific transfection in tissue analyzed 14 hours post-electroporation. See Supplementary Movie#1 for a 3D animation of this Z-stack.
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Supplementary Figure 2: A metaphase cell with long process from mouse E13.5 dorsal telencephalic VZ. Image A represents a 3D reconstruction of a fragment of the cell body with a long radial process. The reconstruction was made from 33 serial sections. Electron micrographs in B1-B6 represent some of these sections (not adjacent; 1 to 5 sections in between). The dotted lines delineate the outline of the cell membrane in B and C. The mitotic cell demonstrates a fusiform cell body situated at the ventricular surface. At its apical pole, it is coupled to neighboring cells with adherens junctions (arrowheads). The process emanates from the opposite side of the cell body and traverses several cell layers. Cell nuclei indicated in serial images (n1-n8) can be used as landmarks. The total length of the process extends 50µm from the ventricular surface. The process terminates with a shallow growth cone (diameter about 1.5µm) possessing three filopodia. One filopodium (f) can be seen in A and B6. Another filopodium (arrow) contains several vesicles characteristic for sites of active growth and elongation of cell membrane as seen in high power micrograph in C. Approximately 10µm below the growth cone, the process contains a varicosity (v) with several mitochondria inside (seen in B6). No mitochondria were found in the growth cone or in the fragment of the process between the varicosity. Scale bars: B, 1µm ; C, 0.5µm.
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Movie #1: 3D projection of an E12.5 neocortex electroporated with pEGFP-C2. Fourteen hours after surgery, VZ-specific transfection is evident by EYFP fluorescence. Transfection efficiency in this preparation was greater than 95%.
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Movie #2: Transfection with the pActin:YFP fusion construct labeled interphase and mitotic VZ cells 24 hrs later. In this projected stack, a long RGC is clearly evident. In addition, a rounded mitotic VZ cell lacking an ascending fiber is found at the ventricular surface.
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Movie #3: In this time series experiment, an organotypic slice transfected 24 hrs previously with pActin:YFP was imaged for 8.5 hrs using multiphoton excitation. In the series of collapsed stacks, the cell body of a VZ cell descends to the ventricular surface before dividing (arrowhead). A 3D projection at metaphase identifies an ascending process behind the cell that is maintained throughout mitosis.
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Movie #4: In this series of collapsed z-stacks, acquired over 4.2 hrs, a dividing VZ cell (arrowhead) descends and completes mitosis at the ventricular surface. As the cell body descends, the short ascending process is retracted prior to cell cleavage.
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Movie #5: 3D projection of pTα1:hGFP-transfected cells (red) in E14.5 VZ counterstained with propidium iodide (blue). Two mitotic divisions of transfected cells (arrowheads) are present at the surface of the lateral ventricle 24 hrs after electroporation. The division on the left is in telophase, with two identifiable daughter cells, neither of which possesses ascending processes. Similarly, the cell dividing on the right of the panel is in metaphase and also lacks processes. Interestingly, a long VZ cell whose ventricular foot process abuts the metaphase cell (on the right side) has a clearly defined ascending process that exits the VZ. The mitotic figure in the SVZ (seen in blue) clearly demarcates the superficial extent of the VZ.
