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Figure 5 | Neural Development

Figure 5

From: Dicer is required for neural stem cell multipotency and lineage progression during cerebral cortex development

Figure 5

Birthdating of Tbr1+ neuron genesis in the Dicer null cortex. (A) Experimental design of neuronal birthdating experiment. BrdU was injected into timed-pregnant dams at E14.5, when the generation of Tbr1+ neurons is drawing to a close in wild-type embryos. BrdU incorporates into cycling progenitor cells and is subsequently maintained in newly born, postmitotic neurons. Tissue was collected at birth and coronal sections were analysed by immunofluorescence to determine the cell types generated over this period. (B) Immunofluorescence for BrdU and Tbr1 showed an increase in the number of double labelled cells in the Dicer null. (C) The cortex was divided into six regions of equal size and the BrdU + cells in each fraction were counted. In the Dicer null cortex a higher percentage of BrdU + cells were found in the bins located proximal to the VZ. (D) In the Dicer null cortex 63% of the cells labelled with BrdU were Tbr1+, compared with 19% in the wild-type (P = 0.03). There was no difference in neuronal output in the Dicer null cortex, as indicated by the total number of BrdU + cells per 100 μm. Counts were performed on three sections from two wild-type and two Dicer null brains. (E) Immunofluorescence at E17.5 showed an increase in apoptotic, aCasp3+ cells in both the VZ and the cortical plate. (F) Quantification of immunofluorescence images confirmed that, the Dicer null cortex contained a greater number of aCasp3+ cells per 100 μM of VZ (P = 0.04) and a higher proportion of these were located in the VZ (P = 0.01). (G) Gross anatomy of the brains of 2-month-old (adult) mice reveals a striking reduction in the size of the Dicer null cortex, which is almost completely absent with the ventral forebrain visible through the transparent cortical remnant. Scale bars 50 μm.

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