DEV017350 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Syn4 is required for the development of NC derivatives. (A-K) Lateral (A,B,D,E,F,H,I,J) and ventral (C,G,K) views of zebrafish embryos showing the expression of GFP in the cartilage of the transgenic line sox10:egfp (B,C,F,G,J,K) and the development of melanocytes (D,H,L). (A-D) Embryos injected with control MO. (E-H) Embryos injected with 6 ng of syn4 MO2. (I-K) Embryos injected with 6 ng of syn4 MO1. Note that both syn4 MOs produced an equivalent inhibition in cartilage development (45% inhibition for MO2, n=60; 52% inhibition for MO1, n=104). (D,H,L) syn4 MO2 produced a clear inhibition in melanocyte development, similar to the one produced by syn4 MO1 (see Fig. 2F,I).

• Supplemental Figure S2 -

  Fig. S2. Inhibition of convergent extension by Has2 MO does not inhibit NC migration. (A) sox10 expression in embryo injected with control MO. (B) sox10 expression in embryo injected with 1 nl of 0.25 mM Has2 MO. No effect on NC migration is observed.

• Supplemental Figure S3 -

  Fig. S3. Syn4 is required for NC migration. Migration of NC was examined in vivo using the transgenic zebrafish line sox10:egfp. (A,B) Embryo injected with control MO, showing normal migration of cephalic NC at 24 hpf. (C,D) Embryo injected with syn4 MO2 showing inhibition of NC migration at 24 hpf. The inhibition of NC migration is similar to that produced by syn4 MO1 (see Fig. 5F-J). (E) Dose-dependent effect of syn4 MO1 or MO2 on neural crest migration. (F) The mix of syn4 MO1 + syn4 MO2 has an additive effect on NC migration compared with injection of each single morpholino (n=79).

• Movie 1 -

  Movie 1. Late migration. Low-power view of two Sox10GFP zebrafish embryos at 24 hpf. The embryo to the right is injected with 8 ng control MO; dorsal is to the left. Note the migration of individual cells. The embryo to the left was injected with 8 ng Syn4 MO; dorsal is to the right. Note that the migration of individual cells is visible, but many groups of NC cells are formed. In both embryos, anterior is to the top. Images captured every 3 minutes over a 6-hour period using a DM5500, 20× water immersions.

• Movie 2 -

  Movie 2. Control. Close up of mandibular NC migrating around the eye. Sox10GFP zebrafish embryo at 20 somites, injected with 8 ng control MO. Anterior is to the left and dorsal to the top. Note that the migrating NC cells are elongated along the axis of migration and some cell protrusions are visible. Images captured every 2 minutes over a period of 30 minutes using a LeicaSP1-DMRE confocal microscope with 40× plan apochromat NA 1.25 Ph3 oil objective. Arrow indicates the anteroposterior axis and the expected direction of migration; e, eye; white line indicates the border of the eye

• Movie 3 -

  Movie 3. Syn4 MO. Close up of mandibular NC migrating around the eye. Sox10GFP zebrafish embryo at 20 somites, injected with 8 ng Syn4 MO. Anterior is to the left and dorsal to the top. Note that the NC cells have a round shape and many protrusions are generated in all directions. Images captured every 2 minutes over a period of 30 minutes using a LeicaSP1-DMRE confocal microscope with 40× plan apochromat NA 1.25 Ph3 oil objective. Arrow indicates the anteroposterior axis and the expected direction of migration; e, eye; white line indicates the border of the eye