DEV034736 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. lfng exon1-intron1 MO blocks the splicing of lfng pre-mRNA. (A) Diagram of lfng transcript organization between exon 1 and exon 2. Red line indicates the area in which lfng^E1I1 MO binds. Blue arrows indicate the position and direction of the primers used (see Materials and methods). (B) The combinations of P1 with P2 and P1 with P3 produce two bands with different sizes that correspond to spliced and unspliced lfng pre-mRNA. Microinjection of two different doses of lfng^E1I1 MO blocks splicing of lfng pre-mRNA, whereas the mismatch control MO injected at the same doses does not block splicing.

• Supplemental Figure S2 -

  Fig. S2. Knockdown of lfng does not affect hindbrain boundary formation or segmentation. Dorsal views of control MO (A-D) and lfng^E1I1 MO (E-H) injected embryos at 16 hpf (C,D,G,H) and 24 hpf (A,B,E,F), showing rfng (A,E), foxb1.2 (B,F), krox20 (C,G) and ephrin B3 (D,H) mRNA expression in the hindbrain. The arrows in A, B, E and F indicate boundary expression. Scale bars: 100 µm in A for A,B,E,F; 100 µm in C for C,D,G,H.

• Supplemental Figure S3 -

  Fig. S3. The early expression of proneural, neurogenic and neural progenitor markers following knockdown of lfng. (A-J) Dorsal views of control MO (A-E) and lfng^E1I1 MO (F-J) injected embryos at 18 hpf, showing ngn1 (A,F), ascl1a (B,G), ascl1b (C,H), deltaA (D,I), and deltaB (E,J) mRNA expression in the hindbrain. (K-X) Dorsal views of control MO (K-Q′) and lfng^E1I1 MO (R-X′) injected embryos at 28 hpf, showing ngn1 (K,K′,R,R′), ascl1a (L,L′,S,S′), ascl1b (M,M′,T,T′), deltaA (N,N′,U,U′), deltaB (O,O′,V,V′), her4 (P,P′,W,W′) and sox3 (Q,Q′,X,X′) mRNA expression in the hindbrain. K′-X′ are higher magnifications of the indicated areas in K-X. Scale bars: 100 µm.

• Supplemental Figure S4 -

  Fig. S4. Many neuronal subtypes are affected following knockdown of lfng. (A-T) Dorsal views of the hindbrain of control MO (A-E,K-O) and lfng^E1I1 MO (F-J,P-T) injected embryos at 26 hpf (D,E,I,J,L,Q), 30 hpf (K,M,N,O,P,R,S,T) and 48 hpf (A-C,F-H) following immunostaining for neurofilament (A,F) or Zn-5 (B,C,G,H), or detection of isl1 (D,I), tbx20 (E,J), pax2.1 (K,P), gad67 (L,Q), evx1 (M,R), lxh2 (N,S) or lhx9 (O,T) mRNA. There is an increase in the number of somatic motor neurons (arrowheads in B and G), dorsal hindbrain neurons (G,H), branchiomotor neurons (D,E,I,J), various interneuronal populations (K,L,M,P,Q,R) and commissural neurons (M,N,O,R,S,T). Note that reticulospinal neurons are not affected (arrows in A and F). The outline in E and J indicates the expression of tbx20 in the developing heart. The arrows in K and P indicate the mid-hindbrain boundary. The open arrows and arrowheads in M and R show the expression of evx1 in putative interneurons and commissural neurons, respectively. Cb, cerebellum; OTO, otocyst. Scale bars: 50 µm in A and B for A-C,F-H; in (B); 100 µm in D for D,E, I-T.

• Supplemental Figure S5 -

  Fig. S5. Expression patterns of Notch genes. (A-I) Dorsal views of wild-type embryos at 18 hpf (A,D,G), 24 hpf (B,E,H) and 34 hpf (C,F,I), showing notch1a (A-C), notch1b (D-F) and notch3 (G-I) mRNA expression in the hindbrain. The arrows in B and C indicate the expression of notch1a in the neurogenic zones. (J, K) Single overlay confocal images showing the dorsal view of wild-type embryos at 40 hpf following detection of notch1a (green) and either deltaA (J) or deltaB (K) (red) mRNA. The expression of notch1a is outlined and compared to that of the Delta genes. Dotted lines indicate the boundary regions. Scale bars: 100 µm in A for A-I; 25 µm in K for J,K.

• Supplemental Figure S6 -

  Fig. S6. Proneural genes are required for lfng expression. (A-G) Dorsal views of control MO (A,E), ngn1 MO (B,F), ascl1b MO (C,G) and triple ascl1a, ascl1b and ngn1 MO (D,H) injected embryos at 16 hpf, showing lfng (A-D) and deltaA (E-H) mRNA expression in the hindbrain. Arrowheads in A-C and E-G indicate the expression of lfng and deltaA in the middle of each rhombomere. Scale bar: 100 µm.