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PLoS One
2013 Jun 11;86:e66487. doi: 10.1371/journal.pone.0066487.
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The neurogenic factor NeuroD1 is expressed in post-mitotic cells during juvenile and adult Xenopus neurogenesis and not in progenitor or radial glial cells.
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In contrast to mammals that have limited proliferation and neurogenesis capacities, the Xenopus frog exhibit a great potential regarding proliferation and production of new cells in the adult brain. This ability makes Xenopus a useful model for understanding the molecular programs required for adult neurogenesis. Transcriptional factors that control adult neurogenesis in vertebrate species undergoing widespread neurogenesis are unknown. NeuroD1 is a member of the family of proneural genes, which function during embryonic neurogenesis as a potent neuronal differentiation factor. Here, we study in detail the expression of NeuroD1 gene in the juvenile and adult Xenopus brains by in situ hybridization combined with immunodetections for proliferation markers (PCNA, BrdU) or in situ hybridizations for cell type markers (Vimentin, Sox2). We found NeuroD1 gene activity in many brain regions, including olfactory bulbs, pallial regions of cerebral hemispheres, preoptic area, habenula, hypothalamus, cerebellum and medulla oblongata. We also demonstrated by double staining NeuroD1/BrdU experiments, after long post-BrdU administration survival times, that NeuroD1 gene activity was turned on in new born neurons during post-metamorphic neurogenesis. Importantly, we provided evidence that NeuroD1-expressing cells at this brain developmental stage were post-mitotic (PCNA-) cells and not radial glial (Vimentin+) or progenitors (Sox2+) cells.
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Figure 2. Expression pattern of NeuroD1 in the juvenile (F2âJ2) and adult (F3âJ3) X. laevis brains.(F1âJ1) Schematic coronal illustrations of the corresponding transverse sections of a juvenile X. laevis brain (NF stage 66). The letters correspond to the rostro-caudal location of sections as depicted in the whole brain drawing (see Figure 1). Arrows and arrowheads in G2, G3, J2 and J3 highlight less conspicuous areas of labeling. Abbreviations are defined in Table 1. The anatomical drawings, with the exceptions of G1 and J1, are from [55], with modifications of basal ganglia subdivisions according to [56]. For all images, dorsal is to the top. Scale barâ=â400 µm in F2âJ2, and 100 µm in F3âJ3.
Figure 3. Expression patterns of NeuroD1, Sox2 and Vimentin in the juvenile X. laevis brain.In situ hybridizations on coronal sections of cerebral hemispheres (AâG) and cerebellum (HâN). In cerebral illustrations, D, E and F are high magnifications of A, B and C, respectively. In cerebellum illustrations, K, L and M are high magnifications of H, I and J, respectively. To allow merge with the DAPI staining, colors of high magnification illustrations D and K were negatively inverted in photos G and N, respectively. For all images, dorsal is to the top. Scale barâ=â220 µm in AâC and HâJ; 95 µm in DâG and KâN.
Figure 4. NeuroD1/PCNA and Vimentin/PCNA double stainings in the cerebellar and pallial regions of juvenile X. laevis brain.Coronal sections at the level of cerebellum (AâH) or pallium (IâK). In situ hybridization using a NeuroD1 (AâD and IâK) or a Vimentin (EâH) probe combined with PCNA immunohistochemistry. For all images, dorsal is to the top. Scale barâ=â30 µm.
Figure 5. NeuroD1/BrdU double stainings on telencephalic and cerebellar sections of a juvenile X. laevis brain.(AâJ) Telencephalon high magnifications of NeuroD1 in situ hybridizations (A, C, F and H) combined with BrdU immunodetections (B, C, F and H) after 14-days BrdU post-administration time. Arrows indicate double stained cells. DAPI stainings are indicated to certify the presence of the nucleus. (KâT) Low magnifications of the above NeuroD1/BrdU/DAPI triple labelling experiments showing larger view of telencephalon (KâO) and cerebellum (PâT). For all images, dorsal is to the top. Scale barâ=â15 µm in AâJ; 45 µm in KâT.
Figure 1. Expression pattern of NeuroD1 in the juvenile (A2–E2) and adult (A3–E3) X. laevis brains.A1–E1) Schematic coronal illustrations of the corresponding transverse sections of a juvenile X. laevis brain (NF stage 66). The drawing at the top of the figure shows a dorsal view of the X. laevis brain. The letters correspond to the rostro-caudal location of sections as depicted in the whole brain drawing. Arrows and arrowheads in C2, C3, D2, and D3 highlight less conspicuous areas of labeling. Abbreviations are defined in Table 1. The anatomical drawings are from [55], with modifications of basal ganglia subdivisions according to [56]. For all images, dorsal is to the top. Scale bar = 400 µm in A2–E2, and 100 µm in A3–E3.
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