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First published online 16 October 2008
doi: 10.1242/dev.021899

Development 135, 3687-3696 (2008)
Published by The Company of Biologists 2008
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Wnt signaling determines ventral spinal cord cell fates in a time-dependent manner

Weiying Yu1, Kristen McDonnell1, Makoto M. Taketo2 and C. Brian Bai1,*

1 Department of Genetics, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106, USA.
2 Department of Pharmacology, Kyoto University, Yoshida-Konoé-cho, Sakyo, Kyoto 606-8501, Japan.


Figure 1
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  		Fig. 1. Wnt signaling is active in the mouse ventral spinal cord at E8.5 but is downregulated by E9.5. (A-D) X-Gal staining of coronal sections of E8.5 TCF/LEF-lacZ embryos. Sections in A-D are shown from anterior to posterior, with the plane of section shown on an E8.5 embryo above the panels. (E-K) RNA in situ hybridization of Axin2 in coronal sections of E8.5 and E9.5 embryos. At E8.5, Axin2 is expressed in a similar pattern to lacZ in TCF/LEF-lacZ embryos (E-H). By E9.5, Axin2 expression is more restricted in anterior (I) than intermediate (J) or posterior (K) sections (compare brackets). Red dashed line, margin of neural epithelium. Scale bars: 50 µm.

 

Figure 2
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  		Fig. 2. Expression of stabilized β-catenin in the mouse ventral spinal cord inhibits ventral progenitor cell fates and promotes dorsal progenitor cell fates. Tamoxifen (TM) was given at 150 mg/kg body weight at 09:00 h on E8 and the embryos were analyzed at E11. (A,D) X-Gal staining of the Rosa26 reporter. (B,E) The dorsal marker Pax7 was induced ventrally. (C,F) Msx1/2 proteins were also ectopically induced in the ventral spinal cord. (G) Schematics of the molecular markers used in this figure. White box indicates normal expression domains. Red ovals and circles indicate ectopic expression of dorsal markers in the ventral spinal cord. Stippled box indicates downregulation of ventral markers. (H-U) Expression of progenitor or neuronal markers. (N-U) Pax7 and Msx1/2 were used as markers for cells expressing stabilized β-catenin. Mash1 was ectopically expressed in the ventral spinal cord (yellow bracket in L). By contrast, Math1 was not ectopically induced (compare H with K). (N,O,R,S) Markers for dorsal neurons were ectopically induced. A small number of Foxd3+ (yellow asterisk in R) or Lbx1+ (arrowhead and white box in S) cells were also ectopically induced. White box indicates the area shown in the inset. Markers for ventral progenitors, such as Nkx2.2 (M), Olig2 (T) and Nkx6.1 (U), were inhibited. Note that the assignment of progenitor domains is based on residual wild-type (WT) pattern. Scale bar: 100 µm.

 

Figure 3
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  		Fig. 3. Expression of stabilized β-catenin can induce different sets of targets in a time-dependent manner. (A-H) TM was used at 50 mg/kg body weight and embryos were analyzed at E10.5. Early induction (TM E7 at 12:00 h) induces Pax7 and Msx1/2 (B,F). Subsequent induction (TM E8 at 18:00 h) induces Msx1/2 but not Pax7 in the ventralmost cells, although dorsal cells still express both Pax7 and Msx1/2 (C,G). Late induction (TM E9 at 09:00 h) does not induce Pax7 or Msx1/2 (D,H). White lines indicate dorsoventral boundary. White box indicates the region shown in the insets. (I) Schematic of spinal cord divided into five ventral domains. (J,K) Distribution of ectopic cells within five ventral domains. Each dot in J and K represents the percentage of ectopic cells falling in one domain from one embryo (n represents the total number of ectopic cells counted). Thick lines represent the average from three different embryos. Scale bar: 100 µm.

 

Figure 4
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  		Fig. 4. Activation of Wnt signaling using Olig1-Cre promotes V2 neurons at the expense of MNs and V3 neurons at E10.5. (A-L) The ventral expression limits of Pax6 (A,E, arrowheads) and Irx3 (B,F, arrows) were extended ventrally, and ectopic Mash1+ cells were also found (C,G red arrowhead). At the same time, there were reductions in the number of Olig2+ pMN and Nkx2.2+ pV3 (D,H). The number of Chx10+ V2 interneurons was increased (I,K) and the number of Isl+ MNs or Isl+ Lim3+ differentiating neurons reduced (J,L). (M) Quantification of different cell types in WT and Olig1-Cre;Ctnnb1gof mouse embryos. Bar represents average ± s.e.m. **, P<0.01; *, P<0.05. Scar bar: 50 µm.

 

Figure 5
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  		Fig. 5. Wnt signaling-mediated cell fate switching is partially dependent on Gli3, but not on Gli2. (A-D) Removal of endogenous Gli2 did not significantly change the number of Isl+ MNs or Chx10+ V2 interneurons. Note that when Gli2 is removed, some of the MNs tend to scatter around the ventral spinal cord. (K) Quantification of Isl1/2+ MNs and Chx10+ V2 interneurons. (E,F) Upregulation of Gli3 transcription (red arrowheads) in mouse embryos with stabilized β-catenin. (G-J) Wnt signaling antagonizes Shh signaling partly through activation of Gli3. Further removal of Gli3 partially releases the inhibition on pMN and pV3. Note the different levels of Nkx2.2 in J. (L,M) Quantification of different cell types. Embryos were analyzed at E10.5. Bars represent average number ± s.e.m. *, P<0.05; **, P<0.01; NS, not significant. Scale bar: 50 µm.

 

Figure 6
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  		Fig. 6. Disruption of canonical Wnt signaling in the ventral spinal cord promotes ventral cell fates. (A-J) Although β-catenin was depleted by E9.5 in both pMN and pV3 domains in mutants (white brackets in D,E), the level of β-catenin in the floor plate (arrows in D,E,I,J) and the expression of Shh protein (C,H) were not significantly altered. (K,L) No drastic changes were seen in the expression of N-cadherin in mutants. (M-R) Expansion of ventral markers in Olig1-Cre;Ctnnb1lof mutants at E10.5. Loss of β-catenin results in a dorsal shift of the Pax6 domain (M,P, white brackets indicate weak Pax6 domains) and in an increase in the numbers of Nkx2.2+ and FoxA2+ cells (O,R, white brackets indicate Nkx2.2+ domains), although the numbers of Isl+ MNs or Chx10+ V2 interneurons were not affected (N,Q, white brackets indicate Isl+ MN domains). (V) Quantification of the changes in cell number as a result of disruption of Wnt signaling using Olig1-Cre. **, P<0.01; NS, not significant (P>0.05). (S-U) Disruption of β-catenin using Gli1-CreER resulted in expansion of Isl+ (T), Nkx2.2+ or FoxA2+ cells (U), and a reduction of Chx10+ V2 interneurons (T). TM was given at 18:00 h on E7.5. Embryos were analyzed at E10.5. Scale bar: 70 µm in A-E; 50 µm in F-U.

 

Figure 7
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  		Fig. 7. A model for how Wnt signaling influences the specification of cell fate in the mouse ventral neural tube. At E8.5, Wnt signaling is active throughout the neural tube when Shh signaling is initiating in the floor plate. As development proceeds, there is a shift of Wnt signaling in the ventral spinal cord. From ventral to dorsal, progenitor cells are released from Wnt signaling gradually. The release from Wnt signaling creates a permissive environment for cell fates to be specified. Wnt signaling promotes dorsal cell fates through the activation of dorsal genes (in particular Msx1/2) and of Gli3, which encodes a major repressor of Shh signaling. Shh signaling, in turn, may induce Wnt inhibitors, such as sFRPs (secreted frizzled-related proteins), to antagonize Wnt signaling. Hh, Hedgehog.

 

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