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First published online 16 April 2008
doi: 10.1242/dev.020180

Development 135, 1843-1851 (2008)
Published by The Company of Biologists 2008
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SoxB1 transcription factors and Notch signaling use distinct mechanisms to regulate proneural gene function and neural progenitor differentiation

Johan Holmberg1, Emil Hansson2, Michal Malewicz1, Magnus Sandberg1, Thomas Perlmann1, Urban Lendahl2 and Jonas Muhr1,*

1 Ludwig Institute for Cancer Research, Karolinska Institute, Box 240, SE-171 77 Stockholm, Sweden.
2 Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, SE-171 77 Stockholm, Sweden.


Figure 1
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  		Fig. 1. Both Notch and Sox3 block neurogenesis. (A-E) Notch1 (A), Hes5 (B), Sox3 (C), Ngn2 (D) and E47 (E) exhibited complementary expression patterns in the embryonic day 4.0 chick spinal cord. (F-H) Expression of the intracellular domain of Notch1 (NICD) (F) or Sox3 (G) for 42 hours significantly reduced the generation of Tuj1+ neurons (H). Data are represented as percentage of electroporated cells expressing Tuj1 (mean±s.e.m.). **P<0.01 relative to EGFP control transfected cells, Student's t-test. Scale bars: 50 µm.

 

Figure 2
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  		Fig. 2. Notch-mediated block of neurogenesis depends on intact SoxB1 function. (A-C,Q) Misexpression of NICD for 42 hours prevented the generation of neurons expressing NeuN (C,Q) but retained expression of the progenitor protein Sox3 (A) and the incorporation of BrdU (B). (D-F,Q) Misexpression of HMGSox3-EnR for 24 hours caused cells to downregulate Sox3 (D), exit the cell cycle (E) and upregulate the expression of NeuN (F,Q), even in the presence of NICD misexpression. (G-I,Q) Twenty-four hours after transfection, expression of a dominant-negative version of CSL (dnCSL), which is unable to bind DNA, had induced cells to downregulate Sox3 (G), exit the cell cycle (H) and upregulate the expression of NeuN (I,Q). (J-L,Q) Combined expression of Sox3 and dnCSL for 42 hours efficiently blocked the generation of NeuN+ cells (L,Q) and maintained cells in a self-renewing (K) and Sox1 expressing (J) state. Black and white representations of A-L are shown in Fig. S3 (see supplementary material). (M-P) The gamma secretase inhibitor DAPT acted in a concentration-dependent manner and caused neural cells to downregulate Sox3 expression (M), exit the cell cycle (P) and upregulate the expression of Tuj1 and NeuN (M,O). Neural explants transfected with Sox3 did not upregulate neuronal marker expression (N,O) or exit the cell cycle (P), regardless of the DAPT concentration. (Q) Statistical representation of NeuN-expressing cells in neural tubes transfected with EGFP, NICD, dnCSL, dnCSL/Sox3 or NICD/HMGSox3-EnR. Data are represented as mean±s.e.m. **P<0.01, ***P<0.001, Student's t-test relative to 0 µm DAPT control in O and P and relative to EGFP-transfected control cells in Q. Student's t-test. Scale bars: 40 µm in L; 10 µm in N.

 

Figure 3
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  		Fig. 3. Notch, but not Sox3, attenuates expression of Ngn2 and E47. (A,B) Expression of NICD for 42 hours attenuated Ngn2 (A) and E47 expression (B). (C,D) Transfection of dnCSL for 10-20 hours increased Ngn2 (C) and E47 expression (D). (E,F) Misexpression of Sox3 for 42 hours did not alter the levels of Ngn2 (E) or E47 expression. Black and white representations of A-F are shown in Fig. S4 (see supplementary material). Scale bar: 40 µm.

 

Figure 4
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  		Fig. 4. Ngn2 and E47 can rescue NICD induced block of neurogenesis. (A-C,T) Cells transfected with NICD (0.7 µg/µl) for 42 hours were Sox3+ (A) and incorporated BrdU (B), but had failed to upregulate the expression of the neuronal markers NeuN and Tuj1 (C,T). (D-I,T) Co-transfection with either Ngn2 or E47 (0.7 µg/µl) did not block the capacity of NICD to maintain cells in an undifferentiated and self-renewing state. (J-L,T) Forty-two hours after electroporation, neural cells co-transfected with Ngn2, E47 and NICD (0.7 µg/µl of each expression vector) had downregulated Sox3 (J), exited the cell cycle (K) and upregulated the expression of neuronal markers (L,T). (M) The Notch responsive reporter construct, 12xCSL-DsRed, was highly activated in NICD-transfected cells. (N,O) These cells were maintained in a Sox3-expressing state (N) and failed to upregulate the expression of Tuj1 (O). (P-R) In cells co-transfected with NICD/NGN2/E47 the 12xCSL-DsRed reporter construct was expressed in cells located in the marginal zone (P) that had downregulated Sox3 (Q) and instead upregulated the expression of Tuj1 (R). (S) Misexpression of Sox3 efficiently blocked the generation of neurons, even when co-transfected with high levels of Ngn2 and E47. (T) Quantification of the number of electroporated cells expressing the neuronal marker NeuN. The white square in A indicates regions analyzed. The figures in T are represented as percentage of electroporated cells expressing NeuN, mean±s.e.m. **P<0.01, ***P<0.001, relative to EGFP-transfected control cells, Student's t-test. Scale bar: 40 µm in P; 10 µm in S.

 

Figure 5
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  		Fig. 5. Hes repress proneural proteins, but not E-protein expression. (A-D) Electroporation of NICD induced Hes1 (A) and Hes5 (B) expression. Misexpression of dnCSL downregulated Hes1 and Hes5 (C,D). (E,F) Misexpression of Hes5 efficiently attenuated Ngn2 expression (E) but had no effect on E47 transcription (F). (G-I) Misexpression of Hes5 suppressed Ngn2 (G) and Tuj1 expression (H,I). (J-O) Co-transfection of Ngn2 and Hes5 promoted electroporated cells to differentiate into post-mitotic neurons (J-L), whereas misexpression of Hes5 in combination with E47 blocked neuronal differentiation (M-O). Embryos were analyzed 24 hours after electroporation. Black and white representation of G,H,J,K,M,N are shown in Fig. S7 (see supplementary material). Data in I,L,O are represented as percentage of electroporated cells expressing Tuj1, mean±s.e.m. *P<0.05, **P<0.01, relative to EGFP-transfected control cells, Student's t-test. Scale bar: 50 µm.

 

Figure 6
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  		Fig. 6. A dominant-active version of Hes5 upregulates Ngn2 but not E47 expression. (A) NICD and Hes5 repressed the activity of the Ngn2 reporter construct in 293 HEK cells, whereas this reporter was activated by Hes5{Delta}ct-VP16. Data are represented as a logarithmic scale where mock is set to one. (B-E) Within 5 hours, Hes5{Delta}ct-VP16 transfected cells had upregulated the expression of Ngn2 (B,C) but not that of E47 (D); ectopic expression of neuronal markers could be detected 12 hours after transfection (E). (F) Quantification of electroporated cells (GFP+) in G-J expressing Tuj1. Data are represented as mean±s.e.m. ***P<0.001, relative to NICD electroporated cells, Student's t-test. (G) Forty-two hours after electroporation, a majority of cells expressing Hes5{Delta}ct-VP16 was terminally differentiated and expressed Tuj1. (H) Cells co-transfected with NICD together with Hes5{Delta}ct-VP16 cells remained undifferentiated. (I) Similarly, cells co-transfected with Ngn2, NICD and Hes5{Delta}ct-VP16 remained undifferentiated and failed to upregulate the expression of Tuj1. (J) Misexpression of E47, Hes5{Delta}ct-VP16 and NICD promoted cells to commit to neurogenesis. (K) Proposed molecular pathway regulating neurogenesis in the vertebrate CNS. The proneural bHLH protein Ngn2 acts together with the E-protein E47 to drive the differentiation of neural progenitor cells by promoting cell cycle exit and the upregulation of neuronal protein expression. Notch signaling maintains neural cells in an undifferentiated state via the activation of CSL. Activated CSL is, in turn, inducing the expression of Hes1/5 and an alternative repressor (designated X), which subsequently represses the expression of Ngn2 and E47, respectively. SoxB1 transcription factors are according to this model, maintaining progenitor cells in an undifferentiated state by activating the expression of progenitor features and, in addition, blocking the activity of Ngn2 and E47. Scale bars: 20 µm in D; 50 µm in G.

 

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