DEV034330 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Loss of β-catenin reduces the level of α-E-catenin expression in ventral midbrain. (A,B) Representative cross sections of ventral midbrain in E12.5 control and Shh-Cre;β-Ctn^fl/fl embryos highlighting the loss of α-E-catenin expression in the conditional mutants (arrowheads, B). Scale bar: 40 µm.

• Supplemental Figure S2 -

  Fig. S2. Increased cell death in the neuroepithelium of Shh-Cre;β-Ctn^fl/fl mutants. (A,B) Compared with control, there is a significant increase in cell death in the ventral midbarin (vMB) of Shh-Cre;β-Ctn^fl/fl mutants at E12.5. This is supported by the increased number of activated caspase 3-positive cells (red, arrowheads). Many of the dying cells in Shh-Cre;β-Ctn^fl/fl mutants are embedded within the disorganized neuroepithelium (B). Scale bar in B: 100 µm. The white lines in A and B define the region of the vMB where recombination by Shh-Cre occurs (cf. Fig. 2C). (C) Quantification of the number of activated caspase 3-positive cells confirms the increase in cell death in Shh-Cre;β-Ctn^fl/fl mutants at E10.5 and E12.5 (Student’s t-test, n=3 for control and conditional mutants).

• Supplemental Figure S3 -

  Fig. S3. Reduced number of Pitx3-positive cells in Th-IRES-Cre;βCtn^fl/fl mutants during development. (A,B) Immunohistochemistry for Pitx3 expression shows a reduced number of Pitx3⁺ cells in the vMB of Th-IRES-Cre;β-Ctn^fl/fl mutants at E12.5 and P0. Scale bar: 200 µm. (C) Quantification of Pitx3⁺ cells using stereology confirms the reduction in Th-IRES-Cre;β-Ctn^fl/fl mutants. Similar to the previously published results (Maxwell et al., 2005), the numbers of Pitx3⁺ cells in Th-IRES-Cre;β-Ctn^fl/fl mutants at E12.5 and P0 are slightly lower than those of Th⁺ cells in Fig. 3S (Student’s t-test, n=3 for control and conditional mutants).

  Maxwell, S. L., Ho, H. Y., Kuehner, E., Zhao, S. and Li, M. (2005). Pitx3 regulates tyrosine hydroxylase expression in the substantia nigra and identifies a subgroup of mesencephalic dopaminergic progenitor neurons during mouse development. Dev. Biol. 282, 467-479.

• Supplemental Figure S4 -

  Fig. S4. Reduced cell death in Th-IRES-Cre;β-Ctn^fl/fl mutants during period of programmed cell death. (A,B) In contrast to Shh-Cre;β-Ctn^fl/fl mutants, the number of activated caspase 3-positive cells is reduced in Th-IRES-Cre;β-Ctn^fl/fl mutants at the period of programmed cell death at P0. Arrowheads in A and B highlight dying cells in control and Th-IRES-Cre;β-Ctn^fl/fl mutants. Scale bar: 40 µm. (C) Quantification of the number of activated caspase 3-positive cells confirms the decrease in cell death in Th-IRES-Cre;β-Ctn^fl/fl mutants (Student’s t-test, n=4 for control and n=5 for conditional mutants.

• Supplemental Figure S5 -

  Fig. S5. No detectable reduction in Ngn2⁺ progenitors in Th-IRES-Cre;β-Ctn^fl/fl mutants. (A-D) Loss of β-catenin in DA neurons does not affect the number of Ngn2⁺ (red) cells in the ventral zone of the vMB at E11.5 or E12.5. The majority of Ngn2⁺ cells do not express Th (green), but a very small number of Ngn2⁺ progenitors, especially those further away from the ventricular zone, co-express Th. Scale bar: 200 µm. (E,F) Quantification shows no significant reduction in the number of Ngn2⁺;Th⁺ or Ngn2⁺;Th⁻ cells in Th-IRES-Cre;β-Ctn^fl/fl mutants at E11.5 and E12.5 (Student’s t-test, n=3 for control and conditional mutants at each embryonic stage; ns, not significant).

• Supplemental Figure S6 -

  Fig. S6. No detectable reduction in BrdU⁺ progenitors in the ventricular zone of Th-IRES-Cre;β-Ctn^fl/fl mutants. (A-D) A short 2-hour pulse injection of BrdU (50 mg/kg) is used to label the progenitors in S-phase in the ventricular zone at E10.5 and E12.5. Loss of β-catenin in DA neurons does not affect the number of BrdU⁺ (red) cells in the ventral zone of the vMB at E10.5 or E12.5. Scale bars: 50 µm in B; 100 µm in D. (E-G) No significant reduction in the number of BrdU⁺ cells in Th-IRES-Cre;β-Ctn^fl/fl mutants was observed at E10.5 and E12.5, either at 2 hours (E,F), or 24 or 48 hours after BrdU injection (G; Student’s t-test, n=3 for control and conditional mutants at each embryonic stage; ns, not significant).

• Supplemental Figure S7 -

  Fig. S7. No evidence of cell fate alteration in Th-IRES-Cre;β-Ctn^fl/fl mutants. (A-D) Using the R26R allele as a fate-mapping tool, we generated control and Th-IRES-Cre;β-Ctn^fl/fl mutants that carry one copy of the R26R allele (i.e. Th-IRES-Cre;β-Ctn^fl/+;R26R and Th-IRES-Cre;β-Ctn^fl/fl;R26R mice, respectively). We reasoned that, if the reduced DA neuron number in Th-IRES-Cre;β-Ctn^fl/fl mutants was due to a change in cell fate, there would be a significant increase in β-Gal⁺ and Th⁻ cells in Th-IRES-Cre;β-Ctn^fl/fl;R26R mice. Using confocal microscopy, we examined DA and non-DA neurons in the SNpc and VTA in control and Th-IRES-Cre;β-Ctn^fl/fl;R26R mice at P0, and found no detectable increase in β-Gal+ and Th⁻ cells in Th-IRES-Cre;β-Ctn^fl/fl;R26R mice.

• Supplemental Figure S8 -

  Fig. S8. A working model for the multiple roles of β-catenin in the development of midbrain dopamine neurons. β-catenin controls the adherent junctions and cell polarity of progenitors, and the integrity of radial glia processes within the neurogenic niche of dopamine neurons at early stages. At later stages, β-catenin continues to regulate the progression from committed progenitors to differentiated DA neurons.