DEV02352 Supplementary Material

Files in this Data Supplement:

• Supplemental Table 1 - Adobe PDF

• Supplemental Figure 1 -

  Fig. S1. Invariability in position of the Otx2 posterior expression boundary. Double detection of otx2 and hoxb1b transcripts in wild-type embryos at 60% (A), 80% (B) and bud (C) stages. hoxb1b anterior limit defines the r3/r4 boundary. The gap between the two expression domains remains constant, arguing against an anterior shift in otx2 expression during late gastrulation. (D-I) Rostral expansion of cerebellum in OtH embryos. Lateral (D-G) or dorsal (H,I) views of cerebellar areas from wild-type (D,F,H) and OtxH (E,G,I) embryos, showing expansion of pax6 (D,E) and reelin (F,G) expression in granule cells, and zebrin (H,I) in Purkinje cells in OtxH embryos. (J-O) Rescue of granular cells in OtxH embryos lacking Fgf signal. (J-K) Lateral views, anterior towards the left, of 12-somite stage brains showing fgf3 expression, which is never detectable in the isthmus of fgf8^–/– mutant embryos (arrows indicate the presumptive isthmic area). (L-O) Lateral views of wild-type (L), OtxH (M), wild-type treated with SU5402 (N) and OtxH treated with SU5402 (O) whole-mount embryos, showing atoh1a expression. Granule cell precursors in the upper rhombic lip in wild type (arrow on dorsal view in inset L) are lost after inhibition of Fgf signal (arrow on dorsal view in inset N). The residual expression in lateral and lower rhombic lip is not giving rise to granule precursors. The upper rhombic lip population increases in OtxH (arrow on dorsal view in inset M), and is kept in OtxH+SU5402 (arrow on dorsal view in inset O).

• Supplemental Figure 2 -

  Fig. S2. Absence of Fgf targets in fgf8^–/–and fgf8^–/–; OtxH embryos. (A-P) sprouty 4 (A-D,I-L) and pea3 (E-H,M-P) expression pattern in the mid/hindbrain region of the genetic context studied at bud (A-H), 20-somite (M-P) and prim-5 stages (I-L). These Fgf transcriptional targets are never induced in fgf8^–/– or fgf8^–/–; OtxH embryos (see D,H,L,P)

• Supplemental Figure 3 -

  Fig. S3. Expression profile of iro2a and iro1b. All prim-5 brains are lateral view, anterior to the left. Expression of iro2a (A-D) and iro1b (E-H) in wild type (A,E), OtxH (B,F), fgf8^–/– (C,G) and fgf8^–/–; OtxH (D,H) embryos. Both genes are expressed in diencephalon subdomains, midbrain (mb), r1 and hindbrain in wild type. Diencephalic and midbrain domains are, respectively, reduced or lacking in OtxH embryos. A fainter midbrain domain is expanded in fgf8^–/– embryos. Prospective cerebellar territories are iros-positive in wild type, OtxH and fgf8^–/–; OtxH embryos (arrows), while OtxH embryos might have a general slight decrease in mid/hinbrain staining.