DEV005520 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. No detectable defect in neural crest formation or early migration in Wnt1-Cre⁺ Rbpsuh^fl/fl mice relative to littermate controls. Wholemount immunohistochemistry was performed with anti-E-box Sox antibody (recognizes Sox 8/9/10) to visualize neural crest cells at E9.5 (embryos had 22-23 somites). Photographs of one control (A,C,E) and three Wnt1-Cre⁺ Rbpsuh^fl/fl embryos (B,D,F) were taken at different somite levels to quantify the number of Sox8/9/10⁺ cells emerging from the dorsal neural tube and in the migration staging area between somites 15 and 21 (box in A,B). The control embryo had 90 Sox8/9/10⁺ cells per somite length, and the Wnt1-Cre⁺ Rbpsuh^fl/fl embryos had 81±10 Sox8/9/10⁺ cells per somite length. In both the Wnt1-Cre⁺ Rbpsuh^fl/fl and control embryos, neural crest migration (C,D, arrow) occurred four to seven somites rostral to the last somite formed. The emergence of Sox8/9/10⁺ cells in the neuroepithelial region prior to somite formation (caudal to the last somite formed; arrow in E and F) was similar in mutant and control embryos. A-F represent montages of multiple non-overlapping images of the same section that provide a higher resolution image than could be obtained with a single lower-magnification photo.

• Supplemental Figure S2 -

  Fig. S2. Sensory ganglia from E13.5 Wnt1-Cre⁺ Rbpsuh^fl/fl embryos exhibit little staining with the glial marker S100β. Transverse sections of E13.5 dorsal root ganglia from control (A,C) and Wnt1-Cre⁺ Rbpsuh^fl/fl (B,D) embryos were stained for the glial marker S100β and the early neuronal marker TuJ1 by immunohistochemistry (n=5 embryos per treatment). S100β expression was detected in control dorsal root ganglia (arrowheads in A,C) but not in mutant dorsal root ganglia (B,D). At later stages of development, low levels of S100β staining were seen in some Wnt1-Cre⁺ Rbpsuh^fl/fl dorsal root ganglia, but not with the same intensity or density of filamentous staining as observed in controls (data not shown).

• Supplemental Figure S3 -

  Fig. S3. Sympathetic chain of E14.5 Wnt1-Cre⁺ Rbpsuh^fl/fl embryos showed reduced S100β staining relative to controls. Transverse sections of sympathetic chain from E14.5 control (A-D and I-L) and Wnt1-Cre⁺ Rbpsuh^fl/fl (E-H and M-P) embryos were stained by immunohistochemistry for p75, S100β, and TuJ1. Wnt1-Cre⁺ Rbpsuh^fl/fl sympathetic ganglia exhibited less S100β staining, and the S100β⁺ cells that were present co-labeled with the NCSC marker p75 (arrowheads, E-F and M-N). By contrast, S100β⁺ cells in control sympathetic chains (arrows, A-B, I-J) rarely (arrowhead, I-J) co-labeled with p75. These data suggest that undifferentiated neural crest progenitors persisted in the sympathetic ganglia of Wnt1-Cre⁺ Rbpsuh^fl/fl embryos but failed to undergo gliogenesis. Scale bars: 20 μm.

• Supplemental Figure S4 -

  Fig. S4. The neural tubes of E11.5 Nestin-Cre⁺ Rbpsuh^fl/fl embryos contained similar numbers of Chx10⁺ cells, Olig2⁺ cells, HB9⁺ cells and Ngn2⁺ cells, but significantly decreased numbers of Gata2⁺ cells relative to control embryos. Transverse sections of the neural tubes of E11.5 control (A,C,E,G,I) and Nestin-Cre⁺ Rbpsuh^fl/fl (B,D,F,H,J) embryos (at least four embryos of each genotype) were stained for Chx10 and Gata2 (markers of V2 interneurons derived from p2 domain progenitors) and Olig2 (pMN domain progenitors) and HB9 (a marker for motoneurons derived from the pMN domain). Note that sections from cervical, thoracic and lumbar levels were all stained for Olig2 and no axial level showed a difference in the number of Olig2⁺ or activated caspase-3⁺ cells per section in Nestin-Cre⁺ Rbpsuh^fl/fl versus control embryos (data not shown). A-J represent montages of multiple non-overlapping images of the same section that provide a higher resolution image than could be obtained with a single lower-magnification photo. Scale bars: 50 μm. (K) Summary of numbers of cells per section (*, P<0.05).

• Supplemental Figure S5 -

  Fig. S5. The neural tubes of E14.5 Nestin-Cre⁺ Rbpsuh^fl/fl embryos contained significantly more Chx10⁺ cells and significantly fewer Gata2⁺ cells relative to control embryos, but no difference in HB9⁺ cells. These differences were observed at all axial levels (at least three embryos per condition). Scale bars are 50 μm. The number of cells in an entire cross section of spinal cord is shown in panel G (*, P<0.05). RBP/J might regulate the choice between a Chx10⁺ V2a interneuron fate and a Gata-2⁺ V2b interneuron fate by neuronal progenitors from the p2 domain, as suggested by the increase in the number of Chx10⁺ neurons (A versus B) and decrease in GATA2⁺ neurons (C versus D) in the Nestin-Cre+ Rbpsuh^fl/fl spinal cord.

• Supplemental Figure S6 -

  Fig. S6: Rbpsuh deficiency reduces the number of astrocytes and increases the number of Olig2⁺ oligodendrocytes in the E19.5 diencephalon. (A-F) Numbers of astrocytes were significantly reduced in the Nestin-Cre⁺ Rbpsuh^fl/fl (D,E) diencephalon relative to control embryos based on staining for GLAST (A versus D) and S100β (B versus E). Images are of the medial diencephalon (Di), near where the thalamic adhesion bridges the third ventricle (v). (G-L) There were also more Olig2⁺ cells (J versus G) and fewer Sox9⁺ cells (K versus H) in the Nestin-Cre⁺ Rbpsuh^fl/fl diencephalon relative to control embryos. (M) These effects were quantified as number of cells per 40× visual field (*, P<0.05; at least three mice per genotype). This demonstrates that the effects associated with Rbpsuh deletion in the developing spinal cord were also observed in at least certain regions of the brains of Nestin-Cre⁺ Rbpsuh^fl/fl embryos. A detailed comparison of other regions of these brains was difficult because Rbpsuh deletion lead to an expansion of the ventricles and some hemorrhaging, changing brain morphology and making it difficult to compare homologous brain regions between mutant and control mice. Scale bars: 50 μm.