DEV02694 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Tbx20 is expressed in post-mitotic BM/VM neurons and is absent in BM/VM progenitors and SM neurons. (A-D) Immunohistochemical detection of Tbx20, Phox2b, Is1l and Hb9 in transverse sections at r4 (A,B) and r5 (C,D) hindbrain levels at E10.5. (E-H) Expression of Tbx20, Phox2b, Isl1, Hb9 and Lhx3 in transverse sections at r8 detected by in situ hybridization (E) or immunohistochemistry (F-H). Tbx20 expression overlaps with Phox2b and Isl1, but does not co-localize with the somatic markers Hb9 and Lhx3. Scale bars: in D, 40 μm for A-D; in H, 80 μm for E-H.

• Supplemental Figure 2 -

  Fig. S2. Tbx20 mutants lack Tbx20 protein and exon2 transcript. (A-D) Expression of Tbx20 and Isl1 in transverse sections of hindbrains from wild-type or Tbx20 mutant mice at E11.5. A and B show only the Tbx20 labeling from C and D, respectively. (E,F) Detection of exon 2-containing Tbx20 transcripts. Labeling was detected in control embryos (E) but not in Tbx20 conditional mutant embryos (F). Scale bars: in C, 30 μm for A,C; in D, 50 μm for B,D; in F, 250 μm for E,F.

• Supplemental Figure 3 -

  Fig. S3. SE1::gfp transgenic mice label cranial motor neurons. (A) Flat-mounted hindbrain preparation of SE1::gfp transgenic mice at E11.5. All Isl1-expressing cranial neurons express GFP. (B) Isl1 expression in flat-mounted hindbrain preparation of E11.5 embryo. Isl1 and GFP produce a similar labeling pattern (compare A with B); although GFP labels axonal processes and is easier to visualize. (C,D) Co-expression of GFP and Isl1 in transverse sections of SE1::gfp mice at the r4 level. C is the GFP-only channel from D. Scale bars: in B, 350 μm for A,B; in D, 80 μm for C,D.

• Supplemental Figure 4 -

  Fig. S4. Facial motor neuron migration is normal in Ret mutant mice. (A-F) Isl1 expression in flat-mounted hindbrain preparations from Ret-deficient mice and littermate controls at E11.5, E12.5 and E13.5 revealed normal migration pattern of facial cells. Scale bar: 350 μm.

• Supplemental Figure 5 -

  Fig. S5. Tbx20 mis-expression induces ectopic floor plate cells in chick embryos. (A-D) In ovo electroporation of HA-tagged wild-type Tbx20 results in ectopic generation of Isl1-, MNR2- and HNF3β-expressing cells. Because Tbx20 induces HNF3β<^b>⁺<^/b> floor plate cells, it is possible that motor neuron differentiation is a non-autonomous result of creating ectopic Shh-expressing cells.

• Supplemental Figure 6 -

  Fig. S6. Gene expression analysis using in situ hybridization. E11.5 embryos from wild type and Tbx20 cKO mutants were sectioned and adjacent sections from each embryo were hybridized with the indicated probes. Hoxb1 served as a marker to determine the r4 level. These low-power images indicate that the changes in gene expression found in Tbx20 mutants appear to be confined to areas of the hindbrain where Tbx20<^b>⁺<^/b> cells normally develop, while the gene expression in the surrounding areas (marked with blue dashed line) was found to be unchanged. Scale bar: 200 μm