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First published online 11 January 2006
doi: 10.1242/dev.02236

Development 133, 581-590 (2006)
Published by The Company of Biologists 2006
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Motoneurons and oligodendrocytes are sequentially generated from neural stem cells but do not appear to share common lineage-restricted progenitors in vivo

Sen Wu1,2, Yuanyuan Wu2 and Mario R. Capecchi1,2,*,{dagger}

1 Interdepartmental Program in Neuroscience, University of Utah, Salt Lake City, Utah 84112, USA.
2 Department of Human Genetics, University of Utah, Salt Lake City, Utah 84112, USA.


Figure 1
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  		Fig. 1. Different models explaining how NSCs in the pMN domain might generate differentiated cell types. (A) NRP/GRP model. (B) MNOP model. (C) Sequential model. Ast, astrocyte; MN, motoneuron; MNOP, motoneuron and oligodendrocyte common progenitor; N, neuron; NA, neuron and astrocyte common progenitor; OPC, oligodendroctye precursor; O, oligodendrocyte; pA, astrocyte precursor; pM, motoneuron precursor. Olig+ cells are orange; NSCs at different stages in the sequential model are coded in different colors to reflect restrictions in competence.

 

Figure 2
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  		Fig. 2. Dynamic expression of Olig1 and Olig2 in normal embryos. Cross sections from the presumptive brachial region of E8.5, and the brachial region of E10.0 to E13.25, heterozygous Olig1-Cre knockin mice were used for immunohistochemical analysis of Olig1 (Cre) (A-H) and Olig2 (I-Q) expression. Olig1 (Cre) and Olig2 are first detectable at E8.5 (A,I). They reach their expression peak in the ventricular zone at E10.0, and double staining indicates a complete overlap of the two expression patterns (B,J,K). From E10.5 to E12.0, more Olig2+ than Olig1+ (Cre+) cells can be detected (C-F,L-O). Both Olig1+ and Olig2+ cells start to migrate out of the ventricular zone starting from E12.5 (G,P). By E13.25, most Olig1+ (Cre+) and Olig2+ cells are also Pdgfr{alpha}+ OPCs (H,Q). Scale bar: 50 µm for A,B,H,I-K,Q; 100 µm for C-G,L-P.

 

Figure 3
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  		Fig. 3. Generation of a conditional cell-ablation mouse by gene targeting. (A) The DTA176 coding sequence is targeted into the Rosa26 locus, and is not expressed until the transcription stop is removed by Cre-mediated recombination. (B) Southern blot screen for ES cells and PCR genotyping for animals. EcoRV digestion and a 5' external probe were used to identify targeted ES cells, which have a 4.1 kb band in addition to the wild-type 11.5 kb band.

 

Figure 4
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  		Fig. 4. Absence of motoneurons in Olig1-DTA embryos. Immunohistochemistry with antibodies against Isl1 and Hb9 was used to analyze cross sections of the brachial region of control and Olig1-DTA spinal cords from E10.0 to E13.0. Isl1+/Hb9+ motoneurons (A,B,E,F,I,J,M,N) are largely missing in the ventral horns of Olig1-DTA mice (C,D,G,H,K,L,O,P) from E10.0 to E13.0. Although a few Isl1+/Hb9+ motoneurons are still detectable around the ventricular zone of Olig1-DTA embryos from E11.0 to E12.0 (G,H,K,L, arrowheads), they are essentially undetectable by E13.0 (O,P). Scale bar: 100 µm.

 

Figure 5
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  		Fig. 5. Olig1-DTA-mediated cell ablation is specific to the Olig1-Cre expression domain. (A-E,G-K) Expression of Irx3 and Shh (A,E) is largely unchanged in Olig1-DTA embryos (G,K). Pax6 expression in the p2 and more dorsal domains of Olig1-DTA embryos is unchanged, but, normally, weak expression in the pMN domain is not detected in Olig1-DTA embryos (B,H). Nkx6.1 and Nkx2.2-expressing p3 progenitor cells (A,C,D) are missing in Olig1-DTA animals (G,I,J). (F,L) The ventral spinal cord patterning defect at E10.5 is summarized in the schematics. (M-O) Lineage analysis using Olig1-Cre and Rosa26-eYFP indicates that the Nkx2.2+ p3 domain has expressed Olig1 (Cre) prior to E10.5. (P-R) The numbers (mean±s.d., left hemisection) of p2 domain-derived Chx10+ V2 interneurons are not significantly different in control (P) and Olig1-DTA (Q) embryos. Scale bars: in O, 50 µm for M-O; in K, 100 µm for A-E,G-K; in Q, 100 µm for P,Q.

 

Figure 6
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  		Fig. 6. Oligodendrocytes are absent from Olig1-DTA embryos but astrocytes are generated normally. Antibodies against Pdgfr{alpha} (A-F), Olig2 (G-L), MBP (M-P) and Gfap (Q-T) were used for immunostaining sections from the thoracic region of the spinal cord. OPCs expressing Pdgfr{alpha} (A-C), and mature oligodendrocytes expressing MBP (M,N, arrowheads), are missing in Olig1-DTA embryos (D-F,O,P). Olig2-expressing cells, normally scattered in gray and white matter (G-I), are greatly reduced at E14.0 to E18.0 (J; K,L, insets, arrowheads) in Olig1-DTA embryos. Astrocytes expressing Gfap are visible both in control (arrowheads in Q,R) and in Olig1-DTA (arrowheads in S,T) animals. Scale bars: in J, 100 µm for A,D,G,J; in T, 200 µm for B,C,E,F,H,I,K-T.

 

Figure 7
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  		Fig. 7. Time course of TUNEL and Olig1/2 expression in the spinal cord of normal and Olig1-DTA embryos. (A-G) Few apoptotic cells detected by TUNEL staining are found in E9.0 to E12.5 control spinal cords. (H,O) Olig1-DTA-mediated cell death is first detected at E9.0. (I-L,P-S) Massive cell death continues until E11.5. (M,T) E12.0 Olig1-DTA embryos have very few TUNEL-positive cells. (N,U) From E12.5 on, dying cells are seen on the edges of the Olig gene expression domain. Olig2+ cells seen in controls (C-E) are always present from E9.0 to E12.5 in Olig1-DTA embryos (H-N). In particular, similar numbers of Olig2+ cells are found in E12.0 control and Olig1-DTA animals (F,M). Scale bars: in O, 50 µm for A,B,H,I,O,P; in U, 100 µm for C-G,J-N,Q-U.

 

Figure 8
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  		Fig. 8. Normal Olig2 expression in the pMN domain and absence of Nkx2.2 expression in the p3 domain of the Olig1-DTA embryo suggests that NSCs persist in the pMN domain but are absent from the p3 domain. (A-C) The numbers of pMN-derived Olig2+ cells (mean±s.d., left hemisection) are not significantly different in control (A) and Olig1-DTA (B) embryos. (D-I) Early expression of Olig1-Cre in the p3 domain is responsible for the absence of the Nkx2.2+ p3 domain throughout embryogenesis in Olig1-DTA mice. Nkx2.2 expression in the p3 domain seen in control embryos (D-F) is largely absent from Olig1-DTA embryos (G-I), but some residual expression in the pMN domain can be detected at E12.5 and E13.25 (H,I). (J,K) Normal expression of RC2 in the p3 domain (J, yellow brackets) is missing in Olig1-DTA animals (K, yellow brackets). RC2 expression in the pMN domain (J, white brace) is relatively normal in Olig1-DTA animals (K, white brace). Scale bars: in B, 50 µm for A,B; in I, 100 µm for D-I; in K, 50 µm for J,K.

 




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