DEV013276 Supplementary Material

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• Supplemental Table S1-2 -

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• Supplemental Figure S1 -

  Fig. S1. Cux2 and P27^Kip1 protein levels in the developing spinal cord at E11.5. (A-C) Cux2 levels (A), P27^Kip1 levels (B), and the merged image of Cux2/P27^Kip1 (yellow; C) in transverse sections of the ventral neural tube at the forelimb bud level of E11.5 embryos. (A) Little or no Cux2 levels were detected in the motoneuron domain in ventrolateral mz. (C) Cux2 and P27^Kip1 are co-expressed in the iz and fp, but not in post-mitotic motoneurons in the mz, which expresses p27^Kip1 (red) but not Cux2 (green). cn, commissural neurons; fp, floorplate; iz, intermediate zone; mz, marginal zone; vz, ventricular zone.

• Supplemental Figure S2 -

  Fig. S2. The growth of the dorsal root ganglia (drg) is dependent on Cux2 function. (A-C) Immunohistochemistry of NeuN in the drg of E11.5 wild-type (A), Cux2^neo/neo mutant (B) and Nestin-Cux2-ires-EGFP transgenic (C) embryos. (D-F) Immunohistochemistry of P27^Kip1 in wild-type (D), Cux2 mutant (E) and transgenic (F) drg. (G-I) Immunohistochemistry of Neurod showing greatly reduced staining in a Cux2 mutant (H), and enhanced staining in Cux2 transgenics (I) relative to controls (G). All three postmitotic neuronal markers show robust expression in Cux2 mutants and transgenics, but the drg is smaller in the mutants and larger in the transgenics.

• Supplemental Figure 3 -

  Fig. S3. Modulation of Cux2 protein expression in gain-of-function and loss-of-function experiments in the chick and mouse embryonic spinal cord. (A-C) Electroporation of HH12 chick neural tubes in ovo with Cux2-IRES-nucEGFP bicistronic vector (A) and analysis of Cux2 expression using a mouse-specific polyclonal antibody 48 hours later (B) reveals GFP-positive cells express mCux2 protein (C). (D-F) Cux2 protein expression in a Nestin-Cux2-ires-EGFP transgenic neural tube at E11.5. High GFP expression (D) co-labels with ectopic Cux2 protein expression (E,F) in the vz. (G-H) Cux2 protein levels in the spinal cord at the forelimb level of control wild-type (G) and Cux2^neo/neo mutant (H) embryos at E10.5. (H) The Cux2 mutation is hypomorphic and leads to a near complete loss of Cux2 expression in the ventral spinal cord at E10.5. fp, floorplate.

• Supplemental Figure S4 -

  Fig. S4. Dependence of expression of P27^Kip1 and Neurod on Cux2 function in the developing spinal cord. (A,B) P27^Kip1 (red) and Neurod (green) immunohistochemistry on E9.5 trunk-level neural tubes from littermate control (A) and Cux2^neo/neo mutants (B). P27^Kip1 is localized in the maturing populations in the ventrolateral neural tube, whereas Neurod is localized in neuroblasts at the lateral half of the vz. The occasional nucleus is positive for both P27^Kip1 and Neurod (arrow), but most expression remains mutually exclusive. (B) Cux2 mutants showed a strong reduction of P27^Kip1 levels at E9.5 and a less severe loss of Neurod-positive nuclei at E9.5. (C,D) P27^Kip1 (red) and Neurod (green) levels in forelimb bud level spinal cords at E10.5 in a littermate control (C) and a Cux2^neo/neo mutant (D). At E10.5, P27^Kip1 and Neurod levels are largely non-overlapping except for a two- to four-cell layer thick zone in the ventral half of the neural tube (arrow in C,D), which identifies the iz, and in the drg. (D) Cux2 mutants show a loss of Neurod expression in the neural tube at E10.5 (but not in the drg). P27^Kip1 levels were reduced in the mz of the medial neural tube, but enhanced in the mns, reflecting an increase in postmitotic mns at the expense of in (see Fig. 7). mns, motoneurons; in, interneuron.