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First published online 30 April 2008
doi: 10.1242/dev.015115

Development 135, 2031-2041 (2008)
Published by The Company of Biologists 2008
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Neurogenin 2 has an essential role in development of the dentate gyrus

Christophe Galichet, François Guillemot* and Carlos M. Parras{dagger}

Division of Molecular Neurobiology, National Institute of Medical Research, The Ridgeway, Mill Hill, NW7 1AA London, UK.


Figure 1
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  		Fig. 1. Ngn2 is expressed in the different progenitor populations of the developing dentate gyrus. (A,B) Sections through the hippocampus of E18.5 wild-type embryos showing the expression of Ngn2 transcripts by in situ hybridization (A) and of Ngn2 protein by immunohistochemistry (B). The different progenitor populations of the developing DG (1ry, primary matrix; 2ry, secondary matrix; 3ry, tertiary matrix) are outlined. (C) Histograms of the number of Ngn2-expressing cells in the different progenitor populations of the DG. (D-G) Sections through the hippocampus of E18.5 brains double-labelled for Ngn2 and BrdU following a 30 minute pulse (D-D''), Prox1 (E-E''), Neurod1 (F-F'') and Ki67 (G). Pictures were taken at the level of the primary matrix (D,E,F), secondary matrix (D',E',F') or tertiary matrix (D'',E'',F'',G). Arrows indicate double-labelled cells, while arrowheads indicate Ngn2-expressing cells only. (D''') Histogram representing the percentage of Ngn2-expressing cells having incorporated BrdU (blue bars) and the percentage of BrdU-labelled cells expressing Ngn2 (red bars). (E''') Histogram representing the percentage of Ngn2+ cells expressing Prox1 at a low level (blue bars) and the percentage of Prox1low cells expressing Ngn2 (red bars). Ngn2 is expressed only in progenitors expressing Prox1 at a low level and not in post-mitotic neurons expressing Neurod1 and Prox1 at a high level. Scale bars: 20 µm.

 

Figure 2
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  		Fig. 2. Ngn2-expressing progenitors give rise to post-mitotic dentate granule neurons. (A-C) Sections through the hippocampus of Ngn2KIGFP/+ embryos at E18.5 (A), P1 (B) and P5 (C) immunolabelled for GFP. (D-G') Hippocampal sections of P1 Ngn2KIGFP/+ brains showing the secondary matrix immunostained for GFP and BrdU after a 30 minutes pulse (D,D'), Prox1 (E,E'), HuC/D (F,F') and Neurod1 (G,G'). Arrows indicate double-labelled cells. (D'',E'',F'',G'') Histograms representing (D'') the percentage of GFP-expressing cells that have incorporated BrdU (blue bars) and the percentage of BrdU-labelled cells expressing GFP (red bars), (E'') the percentage of GFP+ cells expressing Prox1 at a low level (light blue bars) or at a high level (blue bars) and (F'',G'') the percentage of GFP+ cells expressing HuC/D (F'') and Neurod1 (G''). The high level of GFP expression in Ngn2KIGFP/+ mice (A) compared with Ngn2 expression (Fig. 1B) reflects the greater stability of GFP. Almost all post-mitotic dentate granule neurons are GFP+ and therefore originate from Ngn2-expressing progenitors. Differences in GFP labelling in E-E' and other panels are due to the lower signal obtained with chicken anti-GFP antibody. Scale bars: 100 µm in A-C; 20 µm in D-G. 1ry, primary matrix; 2ry, secondary matrix; 3ry, tertiary matrix; DG, dentate gyrus.

 

Figure 3
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  		Fig. 3. Ngn2-expressing progenitors generate dentate granule neurons at the onset of dentate gyrus formation. (A-C) Sections through the hippocampus of wild-type (A,B) and Ngn2KIGFP/+ (C) embryos at E16.5. (A1-C1) High magnification of the areas outlined in A-C. (A1'-C1'') Single-channel images of the pictures in A1-C1. Ngn2-expressing cells in primary and secondary matrices incorporate BrdU after a 30-minute pulse (A,A1), and co-express Ki67 (B,B1). (C,C1) GFP+ cells in Ngn2KIGFP/+ dentate gyrus co-express Prox1 at low or high level. Almost all dentate granule neurons (Prox1high) are GFP+ and therefore originate from Ngn2-expressing progenitors. Arrows indicate double-labelled cells. The different progenitor populations of the developing DG (1ry, primary matrix; 2ry, secondary matrix; 3ry, tertiary matrix) are outlined. Scale bars: 50 µm.

 

Figure 4
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  		Fig. 4. Abnormal development of the dentate gyrus in Ngn2 mutant mice. (A,A') Haematoxylin/Eosin staining of hippocampal sections of E18.5 Ngn2 mutant (A') and wild-type (A) embryos show the lack of a morphologically distinct dentate gyrus (asterisk) in mutant brains at this stage. (B,B') Sections through the hippocampus of 1-month-old (P28) Ngn2 mutant (B') and wild-type mice (B) show the absence of the lower blade of the DG (asterisk) in mutant brains. (C,C') High magnification of the upper blade of the wild-type (C) and Ngn2 mutant (C') dentate gyrus. Note the disorganized appearance of the mutant DG. Scale bars: 100 µm.

 

Figure 5
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  		Fig. 5. Reduction in the number of progenitors in the dentate gyrus of Ngn2 mutant mice. (A-B') Hippocampal sections of E16.5 (A,A') and E18.5 (B,B') Ngn2KIGFP/+ (A,B) or Ngn2KIGFP/GFP (A',B') brains immunolabelled for GFP, Ki67 and BrdU following a 30-minute pulse. Arrows indicate double labelled cells. (A1,A'1,B1-B3,B'1-B'3) High magnification of the areas outlined in A-B'.(C,E) Histograms representing the total number of BrdU-containing cells (C) or Ki67-expressing cells (E) within the different matrices of Ngn2KIGFP/+ (blue bars) and Ngn2KIGFP/GFP (red bars) DG. The number of BrdU+ progenitors is reduced in the three matrices of the mutant DG. The fimbria, which is not affected by the loss of Ngn2, is used as a control. (D) Histogram representing the percentage of GFP-expressing cells that have incorporated BrdU. (F) Histogram representing the ratio of BrdU- and Ki67-expressing cells. *P≤0.05, **P≤0.01. Scale bars: 50 µm.

 

Figure 6
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  		Fig. 6. Reduction in the number of granule neurons in the dentate gyrus of Ngn2 mutant mice. (A-B') Sections through the hippocampus of E16.5 Ngn2KIGFP/+ (A,B) or Ngn2KIGFP/GFP (A',B'). (A,A') Sections showing Prox1high- and Prox1low-expressing cells. Both Prox1low progenitors and Prox1high granule neurons are reduced in Ngn2KIGFP/GFP. The areas outlined in A,A' indicate the regions shown in B,B'. (B,B') Brains immunolabelled for GFP and Prox1. Insets B1,B'1 show Prox1high granule neurons expressing GFP at a higher magnification. Arrows indicate double-labelled cells. (C,D) Histograms representing (C) the number of cells expressing Prox1low (light colour) or Prox1high (dark colour) and (D) the number of Casp3-positive cells at E15.5 and E16.5 in Ngn2KIGFP/+ (blue bars) and Ngn2KIGFP/GFP (red bars) dentate gyrus. Fewer (Prox1low progenitors and Prox1high granule neurons are present in Ngn2KIGFP/GFP embryos. *P≤0.05, **P≤0.01.

 

Figure 7
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  		Fig. 7. Neuronal differentiation defects in Ngn2 mutant dentate gyrus. (A,A',B,B',C,C') Sections through the hippocampus of P1 Ngn2KIGFP/+ (A,B,C) or Ngn2KIGFP/GFP (A',B',C') brains immunolabelled for GFP and HuC/D (A,A'), Prox1 (B,B') or Neurod1 (C,C'). The insets show a high magnification of the secondary matrix. (A'',B'',C''). Histograms representing (A'') the percentage of GFP+ cells expressing HuC/D, (B'') the percentage of GFP+ cells expressing Prox1low (light colour) or Prox1high (dark colour), and (C'') the percentage of GFP+ cells expressing Neurod1 in Ngn2KIGFP/+ (blue bars) and Ngn2KIGFP/GFP (red bars) dentate gyri. Fewer post-mitotic HuC/D, Prox1high-expressing granule neurons are present in the Ngn2KIGFP/GFP DG, while Ngn2 loss of function does not affect Neurod1 expression. *P≤0.05, **P≤0.01. Scale bars: 50 µm.

 

Figure 8
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  		Fig. 8. Mash1 is expressed but not required in dentate granule progenitor generation. (A-C) Sections through the dentate gyrus of P1 wild-type mice immunolabelled for (A,A1) Ngn2 and Mash1, showing that many Mash1+ progenitors co-express Ngn2 (arrows), and for (B,B1) Mash1 and Prox1, showing that most Mash1+ cells co-express Prox1low. (C,C1) Section through the dentate gyrus of P1 Mash1::GFP transgenic mice immunolabelled for Ngn2 and GFP shows that many Ngn2+ progenitors co-express GFP. (D,D1) Section through the dentate gyrus of P1 Ngn2KIGFP/+ mice immunolabelled for Mash1 and GFP, show that many Mash1+ progenitors co-express GFP. (A1-D1) High magnifications of the areas outlined in A-D. Arrows indicate co-expression of markers and arrowheads indicate cells expressing only one marker. (E-F''') Sections through the dentate gyrus of E18.5 wild-type (E,F), Mash1 mutant (E',F'), Ngn2 mutant (F'') and Mash1; Ngn2 double mutant (F''') embryos showing the expression of Neurod1 transcripts by in situ hybridisation (E,E') and Prox1 protein (F-F'''). Mash1 mutant and wild-type embryos have similar patterns of Neurod1 (E,E') and Prox1 (F,F') expression. (G,H) Histograms representing the number of cells expressing Prox1high (G) and Prox1low (H) in the different genotypes shown in F-F'''. The defect in Prox1 expression is not more severe in the dentate gyrus of Mash1{Delta}/{Delta}; Ngn2KIGFP/GFP double mutants than in Ngn2KIGFP/GFP single mutants. Scale bars: 20 µm in A-D; 50 µm in E-F'''.

 

Figure 9
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  		Fig. 9. Abnormal distribution of neuronal progenitors in Ngn2 mutant dentate gyrus. (A-D') Sections through the hippocampus of Ngn2KIGFP/+ (A-D) or Ngn2KIGFP/GFP (A'-D') mice at E18.5 (A,A'), P3 (B,B') and P5 (C,C',C1,C'1,D,D'), immunolabelled for GFP together with Neurod1 (A,A',D,D') or BrdU and GFAP (C,C',C1,C'1). (C1,C1') High magnification of the areas outlined in C,C' showing green/red and white channels separately. (C1,C'1) The radial glial scaffold labelled by GFAP is absent from the Ngn2KIGFP/GFP DG. Note the lack of GFP-expressing cells at the position of the tertiary matrix in Ngn2KIGFP/GFP mice. Square brackets indicate cells in an outer position. (E) Histogram representing the number of GFP+ cells in secondary, tertiary and outer positions at E18.5. (F) Histogram representing the percentage of Ngn2-expressing cells in outer positions of the wild-type forming DG, labelled with BrdU (30-minute pulse) or Prox1low at E18.5. (G) Histogram representing the percentage of GFP-expressing cells labelled with BrdU (30 minutes), Neurod1, Prox1low (light colour) or Prox1high (dark colour) in Ngn2KIGFP/+ (blue bars) and Ngn2KIGFP/GFP (red bars) in outer positions of the dentate gyrus at E18.5. DG, dentate gyrus; 3ry, tertiary matrix; Out, outer position. Scale bars: 100 µm in A-C'; 20 µm in C1-D'.

 

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© The Company of Biologists Ltd 2008