The Journal of Neuroscience, July 26, 2006, ():
Neural-Specific Inactivation of ShcA Results in Increased Embryonic Neural Progenitor Apoptosis and Microencephaly
J. Neurosci. McFarland et al.
26: 7885
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplemental Figure 1 Neural-specific recombination and removal of the STOP cassette in the neural tissue of nestin-Cre; STOPfloxShcFFF embryos.
Illustration of STOP cassette PCR primers in relation to the ShcFFF transgene. (A) The STOP cassette is flanked by loxP sites and is located downstream of the EF-1α promoter and upstream of the ShcFFF transgene. PCR primers (STOP-F and STOP-R) are located within the STOP cassette. (B) STOP cassette excision by Cre recombinase. Note the lack of priming sites for the STOP-F and STOP-R primers. (C) PCR results showing excision of the STOP cassette (a lack of STOP-F/STOP-R PCR product) in the DNA isolated from neural tissue of nestin-Cre; STOPfloxShcFFF embryos at E14.5 and E12 (asterisks) while the STOP cassette is still present in DNA isolated from non-neural tissues of the same animals in the adjacent lanes. ShcFFF indicates animals genotyped by tail biopsy as doubly positive for nestin-Cre and STOPfloxShcFFF transgenes. Positive (+) control indicates animals genotyped by tail biopsy as STOPfloxShcFFF positive, nestin-Cre negative. Negative (-) control indicates animals genotyped by tail biopsy as nestin-Cre positive, STOPfloxShcFFF negative.
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Supplemental Figure 2 The position of the somatosensory cortex is not altered in ShcFFF brains and the reduction in cortical thickness brains is not confined to the S1 cortex
GABAA receptor alpha 2 staining in control (A) and ShcFFF brains (B) in adult brains. GABAA receptor alpha 5 staining in control (C) and ShcFFF (D) adult brains. Lines delineate areas of dark versus light staining in the somatosensensory cortex. Scalebar is 100 μm Cytochrome oxidase staining in control (E) and ShcFFF (F) coronal sections in adult brains. Arrows indicate barrel staining. Total number of adult brains observed: control, n=5; ShcFFF, n=4. Note the thinner cortex in the barrel region of the somatosensory cortex. (G) Coronal sections of the brains of ShcFFF and control littermate brains are shown at the indicated levels in reference to Bregma. Note the generalized cortical thinning of the ShcFFF brains.
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Supplemental Figure 3 Cleaved Caspase-3 staining marks cells with small, pyknotic nuclei
DAPI staining of the pons in a control animal (A) and a higher magnification (B) at E12. DAPI staining of the pons in a ShcFFF animal (C) and a higher magnification (D) at E12. In C-D, arrowheads indicate small, pyknotic nuclei. (E) DAPI staining of the pons of a ShcFFF animal at E12. (F) Cleaved caspase-3 staining of the same location. (G) Merged image of E and F (DAPI, blue; CC3, red). (H) Higher magnification of DAPI nuclei of the pons in a ShcFFF animal. (I) Cleaved Caspase-3 staining of the same location. (J) Merged image of H and I (DAPI, blue; CC3, red). In E-I, arrows indicate pyknotic nuclei that are positive for Cleaved Caspase-3 and arrowheads indicate pyknotic nuclei that are negative for Cleaved Caspase-3. Scale bar is 100 μm in B and D and 200 μm in A and C.
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Supplemental Movie 1. Confocal microscopy reveals colocalization of Cleaved Caspase-3 and Nestin in E12 ShcFFF cortex. A z-series stack of the cell indicated in Figure 9E clearly shows bipolar Nestin-positive (green) cell containing a small intracellular focus of Cleaved Caspase-3 immunoreactivity (red). The ventricular surface is indicated to the right of the figure. Refer to Figure 9 for scalebar.
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Supplemental Movie 2. Confocal microscopy reveals colocalization of Cleaved Caspase-3 and Nestin in E12 ShcFFF cortex. A z-series stack of the cell indicated in Figure 9F clearly shows bipolar Nestin-positive (green) cell containing a small intracellular focus of Cleaved Caspase-3 immunoreactivity (red). The ventricular surface is indicated to the right of the figure. Refer to Figure 9 for scalebar.
