Yeo W and Gautier J (2005), XNGNR1-dependent neurogenesis mediates early ne...

XB-ART-2053

Mech Dev 2005 May 01;1225:635-44. doi: 10.1016/j.mod.2004.12.010.

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XNGNR1-dependent neurogenesis mediates early neural cell death.

Yeo W , Gautier J .

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Early neural cell death is programmed cell death occurring within proliferating and undifferentiated neural progenitors. Little is known about the regulation and role of early neural cell death. In Xenopus embryos, primary neurogenesis is disrupted following the inhibition of early neural cell death, indicating that it is required for normal primary neurogenesis. Here we show that early neural cell death is dependent on primary neurogenesis. Overexpression of XSoxD concomitantly reduced N-Tubulin expression and early neural cell death, as seen by reduced TUNEL staining in stage 15 embryos. Conversely, overexpression of XNgnr1 led to ectopic N-Tubulin expression and TUNEL staining. However, XNeuroD overexpression, which induces ectopic N-Tubulin expression downstream of XNgnr1, had no effect on early neural cell death. E1A12S differentially inhibits the differentiation pathway induced by XNGNR1 protein. E1A12S-mediated inhibition of XNGNR1 neurogenic activity resulted in the reduction of N-Tubulin expression and TUNEL staining. Taken together, our data establish that primary neurogenesis induced by XNGNR1 promotes early neural cell death. This indicates that XNgnr1 positively regulates early neural cell death. We propose that early neural cell death might eliminate cells with abnormally high levels of XNGNR1, which can result in pre-mature neuronal differentiation.

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Species referenced: Xenopus laevis
Genes referenced: dll1 elavl1 myt1 neurod1 neurog2 notch1 sox15 sox2 tubb2b

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	Fig. 1. Consequences of XSoxD overexpression on primary neurogenesis and early neural cell death. Dorsal views of stage 15 (A) and stage 23 (K, L) embryos with anterior to the left. The dorsal midline is indicated in red (A, D, E, I, J). Embryos here were microinjected (see Section 3) with: (A) GFP (50 pg/nl), (E) XSoxD (100 pg/nl) and GFP (50 pg/nl) mRNAs as indicated in the top left corner. The injected half is shown as the bottom half (red arrow) visualized with immunocytochemistry with anti-GFP antibodies which resulted in a blue-coloration (A, I, J). Immunocytochemistry staining using FASTRED (Roche) was washed off (see Section 3) to allow better visualization of in situ hybridization and TUNEL (E, K, L). Embryos were also subjected to whole-mount TUNEL (D, I, J, L) and in situ hybridization with probes specific for XSox2 (A, E), XNgnr1 (B, F), XMyT1 (G) and N-Tubulin (C, H, K) as indicated in bottom right corner. The numbers of TUNEL-positive nuclei in the injected and uninjected are compared to assess the effect on early neural cell death (see text and Table 1).
	Fig. 2. Consequences of overexpression of neurogenic bHLH proteins on primary neurogenesis and early neural cell death. All embryos are shown in the dorsal views with anterior to the left, except for (C) and (D) which are shown in the dorso-lateral view. The dorsal midline is indicated in red (B). (A, B) Control embryos. (C) Embryos were microinjected with: (C, E) XNgnr1 (10 pg/nl) and GFP (50 pg/nl), (D) XNgnr1 (0.5 pg/nl) and GFP (50 pg/nl) (F, G) XNeuroD (100 pg/nl) and GFP (50 pg/nl) and (H, I) XNgnr1 (10 pg/nl) and E1A12S (0.2 pg/nl) and GFP (50 pg/nl) mRNAs as indicated in the top left corner (as in Fig. 1). Ngnr1 lowand Ngnr highdistinguishes the different doses of XNgnr1 microinjected. The injected half is shown as the bottom half (red arrow) in all injected embryos and was visualized by immunocytochemistry with anti-GFP antibodies, which resulted in the blue-green coloration. Immunocytochemistry staining using FAST-RED (Roche) was washed off to allow better visualization of in situ hybridization and TUNEL (D, G, H). Embryos were also subjected to whole-mount TUNEL (B, D, G, H, K, M) and whole-mount in situ hybridization with probes specific for (A, C, E, I, J) N-Tubulin, (F) XNgnr1 as indicated in the bottom right corner.
	Fig. 3. Consequences of HAT inhibition on primary neurogenesis and early neural cell death. Panel 3-I. Embryos were not treated with HUA. All embryos are shown with anterior to the left in their dorso-lateral views (A, C, E and G) and dorsal views for (B, D, F and H). The dorsal midline is indicated in red (A, E and G). (A, E) Embryos were microinjected with: (A, E) XNgnr1 (10 pg/nl) and GFP (50 pg/nl), (B, F) E1A12S (0.2 pg/nl) and GFP (50 pg/nl) and (C, G) XNgnr1 (10 pg/nl) and E1A12S (0.2 pg/nl) and GFP (50 pg/nl) mRNAs as indicated in the top left corner (as in Fig. 1). The injected is shown as the bottom half (red arrow). Embryos were subjected to whole-mount in situ hybridization with probes specific for (A) N-Tubulin and (E) XMyT1 as indicated in the bottom right corner. Panel 3-II. Embryos were treated with HUA. Dorsal views of stage 15 embryos with anterior to the left. The dorsal midline is indicated in red (A, D, E, F, H, I). Embryos were microinjected with: (A) E1A12S (0.2 pg/nl) and GFP (50 pg/nl), (F) E1A12S (0.2 pg/nl) and XSoxD (100 pg/nl) and GFP (50 pg/nl) mRNAs as indicated in the top left corner. The injected half is shown as the bottom half (red arrow) visualized by immunocytochemistry with anti-GFP antibodies, which resulted in the blue-green coloration. The embryos were also subjected to whole-mount TUNEL (D, E, H, I) and whole-mount in situ hybridization with probes specific for XSox2 (A, F), XDelta1 (B) and N-Tubulin (C, G) as indicated in the bottom right corner. (A, F) The widths of XSox2 expression domains are represented by a yellow line (uninjected) and a green line (injected).
	Fig. 4. Early neural cell death is positioned along the HAT-dependent pathway of neuronal differentiation, induced by XNGNR1. The bold dotted line represents the position at which cells are susceptible to programmed cell death. XNOTCH1 inhibits neurogenesis by repressing XNgnr1 expression, resulting in reduction of neurogenesis-dependent early neural cell death. Early neural cell death is insensitive to interference to neurogenesis occurring downstream of lateral inhibition. High levels of XNGNR1 confer insensitivity to lateral inhibition, disrupting the temporal progression of neurogenesis and resulting in pre-mature formation of neurons. Early neural cell can be used as a corrective mechanism against such errors.