DEV026583 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Timing of neural crest emigration from the midbrain and forebrain. (A) Immunohistochemical detection of the HNK-1 (brown staining) was used to visualize neural crest cells. At HH9−, HNK-1-positive cells (black arrows) have begun migration from the prosencephalon (pr) and mesencephalon (mes). (B) By HH9, no HNK-1-positive cells were observed above the dorsal midline of the neural tube, and the nerual crest-derived cells continued to migrate laterally and rostrally (arrows). (C) By HH10, the HNK-1-positive cells reached the lateral and rostral edges of the embryo (arrows). (D) Scale bar: 250 µm in A-C; 500 µm in D.

• Supplemental Figure S2 -

  Fig. S2. Apoptosis in the brain alter activating SHH signaling is not altered. (A,B) In control embryos at 24 hours after bead implantation, cell death is apparent in the ventral forebrain (A), and this pattern is observed in treated embryos at this time (B). (C-E) This same pattern was observed at 48 hours in control (C) and treated (D) embryos, and again in control (E) and treated (F) embryos 60 hours after bead implantation. Scale bar: 250 µm.

• Supplemental Figure S3 -

  Fig. S3. In severe cases, a medial cleft in the FNP was apparent in treated embryos. (A) A control embryo 96 hours after bead implantation. (B) At this time, these embryos had a medial cleft of the FNP. Growth was restricted to lateral regions of the FNP. The black line in A and B are the same size and indicate that each half of the FNP in treated embryos is similar in size to the equivalent region of the normal embryo. (C) A normal embryo at day 13. (D) Embryos with median clefts exhibit severe structural malformations at later time points. In this embryo, the lateral aspects of the upper jaw grew as independent right and left structures, and formed left and right sides of the upper jaw that were not integrated. Scale bar: 1 mm in A, B; 2.5 mm in C,D.

• Supplemental Figure S4 -

  Fig. S4. Expression of Bmp2, Bmp4 and Noggin 72 hours after treatment. (A-D) In control (A,B) and treated (C,D) embryos Bmp4 was expressed in the medial region of the FNP, but not adjacent to the nasal pit. No differences between treated and control embryos were detected. (E-H) In control (E,F) and treated (G,H) embryos, Bmp2 expression was not altered. Bmp2 transcripts were detected near the nasal pit. (I-K) Controls (I,J) and treated (K,L) embryos did not exhibit noticeable Noggin expression in the mesenchyme of the FNP. Scale bar: 250 µm.

• Supplemental Figure S5 -

  Fig. S5. Apoptosis 24 hours after implantation of SU5402 beads. (A-D) In normal (A,B) and treated (C,D) embryos, there was little evidence of cell death in mesenchyme. Positive cells in the brain (arrows) exhibit small, punctate and intense fluorescence compared with the background fluorescence near the bead. The position of the beads (B) is shown. There was considerable background fluorescence associated with debris created by sectioning through the beads. Scale bar: 250 µm.