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Experiment details for neurog2

Schlosser G et al. (2008) Assay

Eya1 and Six1 promote neurogenesis in the cranial placodes in a SoxB1-dependent fashion.

Gene Clone Species Stages Anatomy
neurog2.L laevis NF stage 14 chordal neural plate , trigeminal placode , neuron , otic region
neurog2.L laevis NF stage 26 midbrain , hindbrain , spinal cord , trigeminal ganglion , lateral line placode

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  Fig. 11. Role of SoxB1 genes in placodal neurogenesis. (A) Immunoblot showing that Sox2MO and Sox3MO specifically and efficiently block protein synthesis from co-injected utr-Sox2 and utr-Sox3 mRNA, respectively. (B–E, F–I) Effects of unilateral injection (lower half) of Sox2+Sox3MO (B–E), or Sox3 mRNA (F–I) on various neurogenic markers at neural plate stage. Embryos are shown superimposed with green fluorescent channel to reveal distribution of myc-GFP co-injected as lineage tracer. Asterisks indicate reductions, while arrows mark increased or ectopic marker gene expression in the placodal or non-neural ectoderm. (J–L) Effects of unilateral injection of GR–Sox3 and DEX activation at stage 16–18 on neurogenic markers at tail bud stages (J1–L1: injected side; J2–L2: control side). Asterisks indicate reductions in the placodal or non-neural ectoderm. (M, N) Ectopic neuronal differentiation (arrows) after injection of GR–Eya1+GR–Six1 mRNAs (M1: inj. side; M2: control side) and DEX activation at stage 16–18 is significantly reduced after co-injection of Sox2/Sox3 MO (M3: inj. side; M4: control side; N: quantitation, χ2 test; : p < 0.001). (O–R) Ectopic expression (arrows) of Sox3 (O1, O3: inj. side; O2, O4: control side) and Sox2 (P1, P3: inj. side; P2, P4: control side) but not of NeuroD (Q1, Q3: inj. side; Q2, Q4: control side) and p27Xic1 after injection of GR–Eya1+GR–Six1 mRNAs and DEX activation at stage 16–18 persists and is more extensive with CHX treatment (R: quantitation; χ2 test; : p < 0.05, : p < 0.001).

Gene Clone Species Stages Anatomy
neurog2.L laevis NF stage 17 chordal neural plate , trigeminal placode , neuron , otic region

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  Fig. 2. Effects of Eya1 and Six1 knockdown on markers of neurogenesis and placodal ectoderm. (A) Immunoblot showing that Eya1MO1 but not control MO blocks synthesis of Eya1 protein. (B) Co-injecting Eya1MO2 or Eya1MO1+2 with myc-tagged Eya1 mRNA significantly restores NeuroD and prevents ectopic p27Xic1 expression (χ2 test; *: p < 0.05, **: p < 0.001) in three independent rescue experiments. (C–Q) Neural plate stage embryos after unilateral injection (lower half) of Eya1MO1+2 (C–L) or Six1MO1+2 (M–Q). In all cases, where myc-GFP was co-injected as lineage tracer (C–K), embryos are shown superimposed with green fluorescent channel. Asterisks indicate reductions, arrows and arrowheads indicate increased marker gene expression. For Delta1 after Eya1MO1+2 injections two different phenotypes are depicted (J, K). R: Tail bud stage embryo after unilateral injection (R1, R3, R5: injected side; R2, R4, R6: control side) of Eya1MO1+2 reveals reduction of NeuroD expression in all neurogenic placodes or derivative ganglia. R3 and R4 depict a section at the level indicated (white line) with boxed areas shown magnified in R5 and R6, respectively, superimposed with green (myc-GFP co-injected with Eya1MO1+2) and blue (DAPI) fluorescence. Residual NeuroD expression is confined to cells receiving little or no MO. Bar in R4: 100 μm (for R3, R4). Abbreviations: e: eye; epVII+AV: facial epibranchial and anteroventral lateral line placode; epIX: glossopharyngeal epibranchial placode; gVII/AV/AD: ganglion of the facial, anteroventral and anterodorsal lateral line nerve; gVIII: vestibulocochlear ganglion; nt: neural tube; pAD: anterodorsal lateral line placode; pM: middle lateral line placode; pOl: olfactory placode; pP: posterior lateral line placode; pPr: profundal placode; pV: trigeminal placode; vOt: otic vesicle.

Gene Clone Species Stages Anatomy
neurog2.L laevis NF stage 17 chordal neural plate , anterior neural fold , otic region

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  Fig. 3. Effects of Eya1 and Six1 overexpression on markers of neurogenesis and placodal ectoderm at neural plate stages. Embryos after unilateral injection (lower half) of Eya1 (A–J), Six1 (K–N) or Eya1+Six1 (O) mRNA. In all cases, where myc-GFP was co-injected as lineage tracer (A–D, F–I, K, L, N, O), embryos are shown superimposed with green fluorescent channel. Black asterisks indicate reductions in the placodal or non-neural ectoderm. Arrows mark increased or ectopic marker gene expression in the placodal or non-neural ectoderm; black arrowhead identifies deformed blastopore due to gastrulation defects (O). For Sox3 (C, D) and p27Xic1 (I, J) after Eya1 injections two different phenotypes are depicted.

Gene Clone Species Stages Anatomy
neurog2.L laevis NF stage 26 olfactory placode , hindbrain , epibranchial placode

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  Fig. 4. Effects of Eya1, Six1 and Eya1+Six1 overexpression on various neurogenesis markers at tail bud stages (stage 26). Injected (A1–N1) and control (A2–N2) sides of tail bud stage embryos after unilateral injection of Eya1 (A–H), Six1 (I–L) or Eya1+Six1 (M, N) mRNAs. In all cases, where myc-GFP was co-injected as lineage tracer (A–G, M–N), embryos are shown superimposed with green fluorescent channel. Black asterisks indicate reductions, while black arrows mark increased or ectopic marker gene expression in the placodal or non-neural ectoderm. Yellow asterisks identify expression in pharyngeal pouches. For Sox3 (A, B) and NeuroD (C, D) after Eya1 injections two different phenotypes are depicted. Abbreviations as in Fig. 2. gPrV: profundal-trigeminal ganglionic complex; L: lens placode; PN: pronephros.

Gene Clone Species Stages Anatomy
neurog2.L laevis NF stage 26 retina , spinal cord , dorsal , facial nerve

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  Fig. 5. Effects of overexpression of GR–Eya1 or GR–Eya1+GR–Six1 on various neurogenesis markers at tail bud stages (stage 26). Injected (A1–N1) and control (A2–N2) sides of tail bud stage embryos after unilateral injection of GR–Eya1 (A–H) or GR–Eya1+GR–Six1 (I–N) and DEX activation at stage 16–18. In all cases, where myc-GFP was co-injected as lineage tracer (A–F, I–M), embryos are shown superimposed with green fluorescent channel. Black asterisks indicate reductions, while black or white arrows mark increased or ectopic marker gene expression in the placodal or non-neural ectoderm. Yellow asterisks identify expression in pharyngeal pouches. For NeuroD (C, D) and p27Xic1 (G, H) after GR–Eya1 injection two different phenotypes are depicted. Abbreviations as in Fig. 2 and Fig. 4.

Gene Clone Species Stages Anatomy
neurog2.L laevis NF stage 26 hindbrain , facial nerve

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  Fig. 7. Dose-dependent effects of Eya1 on neurogenesis markers as revealed in tissue sections. Distribution of N-Tubulin (A), NeuroD (B), Ngnr1 (C) and Delta1 (D) in stage 26 embryos after injection of myc-Eya1 or co-injection of Eya1 with myc-GFP mRNA. Embryos are shown as wholemounts (injected side: A1–D1; control side: A2–D2) and in transverse sections (at level indicated by black line) shown in bright field (A3–D3), superimposition of green (myc-immunostaining) and blue (DAPI) fluorescence (A4–D4, no DAPI image available in panel D), and as a merged image (A5–D5). Boxed areas are magnified in inserts. The DAPI channel is not shown in the inserts of panel C to make Ngnr1 staining more clearly visible. Hatched boxed area in panels C3–C5 shows Ngnr1 positive epibranchial placode on control side in an adjacent section. Asterisks indicate reductions, while arrows mark ectopic marker gene expression in the placodal or non-neural ectoderm. Double arrows identify areas of residual expression. Arrowheads mark displaced otic vesicle on injected side. Bar in panel D5: 100 μm (for A3–5 – D3–5).