DEV02665 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. olt and ord genes do not affect overall brain patterning. (A-C) tag-1 mRNA expression in the wild-type (A), olt^rw689 (B) and ord^rw71 (C) embryos at 24 hpf. Although the nVII motor neurons showed abnormal behavior and ectopic localization in the olt and ord embryos, they expressed tag-1 mRNA, a marker for nVII motor neurons. All or most of the tag-1-positive cells were located in r4 in the olt and ord embryos (arrows in B and C). Some of the tag-1-positive cells were located in r5 in the ord embryos (arrowheads in C). (D-F) Isl1-GFP expression in the wild-type (D), olt (E) and ord (F) embryos at 5 days post-fertilization. The trajectories of facial motor axons were normal in the mutant embryos (arrows) and the axons reached the target muscles. (D′,E′,F′) Higher magnifications of the boxed regions in D-F. (G-O) Expression pattern of the rhombomere-specific genes in the wild-type (G,J,M), olt (H,K,N) and ord (I,L,O) embryos at 20 hpf. Expression patterns of hoxb1a (G-I), krox20 (J-L) and val/mafB (M-O) mRNA were unaffected in the mutant embryos. (P-R) Commissural axons labeled with zn-5 antibody (red) in the wild-type (P), olt (Q) and ord (R) Isl1-GFP embryos at 36 hpf. (S-U) Reticulospinal neurons retrogradely labeled (red) in the wild-type (S), olt (T) and ord (U) Isl1-GFP embryos at 5 hpf. (S′,T′,U′) Single-channel images for the labeled neurons. Dorsal views of the embryos are shown; the anterior of the embryo is at the top of the figure. Va, anterior trigeminal nuclei; Vp, posterior trigeminal nuclei; VII, facial nucleus; X, vagus nucleus; Allg, anterior lateral line ganglion; Pllg, posterior lateral line ganglion; M, Mauthner's cell. Asterisks indicate the r6-derived putative octavolateralis efferent (OLe) neurons (Wada et al., 2005). Otic vesicles (ov) are indicated by broken lines. Scale bars: 50 μm.

• Supplemental Figure 2 -

  Fig. S2. Analyses of the fz3a and celsr genes. (A) Comparison of DNA-sequencing output for the region of the olt mutation and the same region on the wild-type allele. (B) Subcellular localization of Fz3a-Venus and Xdsh-GFP proteins at the blastula stages. Fz3a-Venus, but not Fz3a^rw689-Venus, was associated with the plasma membrane. Overexpressed Xdsh-GFP localized in the cytoplasmic regions. Co-injection with the fz3a mRNA, but not the mutated fz3a^rw689 mRNA, resulted in recruitment of Xdsh-GFP to the plasma membrane. Scale bar: 20 μm. (C) Lateral views of wild-type embryos reacted with the fz3a RNA probe at the indicated embryonic stages. fz3a mRNA was slightly expressed in the brain at 12 hpf. (D) The position of fz3a-MO was designed to disrupt the splicing of the first intron of the fz3a transcript, giving rise to a protein containing the truncated CRD with 19 irrelevant amino acids. (E) RT-PCR analyses were performed as described (Goutel et al., 2000). The analyses showed a significant reduction of the normal fz3a mRNA in the fz3a-MO injected embryos. The primers used to detect the mRNA (positions are indicated in D) were shown in Table1. Control primers were used to amplify the ef1a gene (Goutel et al., 2000). (F) Comparison of DNA sequencing output for the regions of the ord mutations and the same regions on the wild-type allele. (G-H) Expression patterns of celsr2 (G) and celsr3 (E) genes at the indicated embryonic stages. celsr2 was detected throughout the embryo in the gastrula stages and became restricted to the CNS region thereafter. celsr3 mRNA was barely expressed in the hindbrain. Lateral views of the embryos are shown. (I) The positions of celsr1a-MO, celsr1b-MO and celsr2-MO were designed to disrupt the second intron of each gene transcript, giving rise to truncated proteins containing the cadherin repeats domain with 39 irrelevant amino acids (celsr2-MO) or 17 irrelevant amino acids (celsr1a-MO and celsr1b-MO). (E) RT-PCR analyses showed significant reductions in the normal celsr mRNA in embryos injected with each MO. The primers used to detect mRNA (positions are indicated in I) were shown in Table1. Scale bars: 50 μm.

• Supplemental Figure 3 -

  Fig. S3. Behavior of the nVII motor neurons compared with the neighboring neuroepithelial cells. (A,B) To compare the behavior of the nVII motor neurons with the neighboring neuroepithelial cells, we observed mosaic embryos obtained by the transplantation of WT>WT (A) and ord>ord (B) Isl1-GFP embryos. The donor cells labeled with rhodamine-conjugated dextran (red) were incorporated into the neuroepithelium. Behavior of the nVII motor neurons (green) was shown at the time indicated in each panel (hpf). In the wild-type embryo, the nVII motor neurons migrated caudally relative to the neighboring cells and they were associated with the pial surface of the brain (A). By contrast, in the ord embryo, one of the nVII motor neurons (indicated by arrows) became detached from the pial surface of the brain (B). Single optical planes are shown in dorsal view. Pial surfaces of the brain are indicated by lines. Double-headed arrows indicate midlines of the embryos. (A′,B′) Schematic drawings of each panel. (C,C′) Single optical plane of a cross section at r5 from a mosaic embryo obtained by the transplantations of WT>WT Isl1-GFP embryos at 24 hpf. The donor cells labeled with rhodamine-conjugated dextran (red) were incorporated into the neuroepithelium. Most of the labeled neuroepithelial cells were round in shape (C). However, when the images were obtained at a higher amplified gain (C’), long processes associated with ventricular and pial surfaces were detected (arrows in C′). These results suggest that the rhodamine-conjugated dextran strongly stained cell bodies around the nuclei, but weakly stained cellular processes in the neuroepithelial cells. Pial and ventricular surfaces are indicated by broken lines. Because most of the mosaic embryos were observed at lower gains to reduce the noise, the long neuroepithelial processes were not imaged.

• Supplemental Figure 4 -

  Fig. S4. Extension of the nVII motor axons in an olt Isl1-GFP embryo. Time-lapse observations of the extension of an nVII motor axon (arrows) in a hindbrain explant from an olt Isl1-GFP embryo at the time indicated in each panel (hpf). Such axons extended dorsolaterally from the cell bodies of the nVII motor neurons and exited the hindbrain. Frontal views at r4. Hindbrain regions are outlined by broken lines.

• Supplemental Figure 5 -

  Fig. S5. Examples of mosaic embryos containing many donor cells. Additional examples of the mosaic embryos obtained by the transplantations of olt>WT (A), WT>olt (B), ord>WT (C) and WT>ord (D) Isl1-GFP embryos. The donor cells were labeled with rhodamine-conjugated dextran (red). In these examples, many donor cells (arrows) were incorporated into the host embryos. Images are composite stacks of serial optical sections, shown in dorsal view. See also Fig. 6C-F.

• Supplemental Figure 6 -

  Fig. S6. Examples of other embryos showing mosaicism in the neuroepithelium. Two additional examples of mosaic embryos obtained by transplantations of WT>olt Isl1-GFP embryos. The donor cells were labeled with rhodamine-conjugated dextran (red). (A) In this mosaic embryo, the wild-type donor-cell cluster (red) was located in the lateral part of the neuroepithelium (asterisk) and the olt-derived nVII motor neurons (green) failed to invade this region (arrowheads in A). (B) In this mosaic embryo, the wild-type donor-cell cluster (red) was located in the medial part of the neuroepithelium (asterisk), and the olt-derived nVII motor neurons (green) were also excluded from this wild-type donor-cell cluster (arrowhead in B). The yellow signals in the merged panels are technical artifacts caused by superimposition of the red signals of the donor cells and the green signals of the nVII motor neurons. As red signals were not detected in the axons of the nVII motor neurons, all of the nVII motor neurons were derived from the olt host embryos. Images are composite stacks of serial optical sections, shown in dorsal view. See also Fig. 6H.

• Supplemental Figure 7 -

  Fig. S7. OLe neurons fail to migrate caudally in the ord Isl1-GFP embryos. The lateral line was retrogradely labeled with DiI (red) in ord Isl1-GFP embryos at 30 hpf. In the ord embryos, some of the nVII motor neurons migrated caudally to r5 (arrow). DiI labeling demonstrated that the OLe neurons failed to migrate caudally in the ord embryos (arrowhead). Sites of the DiI application are indicated by triangles. Composite stacks of serial optical sections are shown in dorsal view. A single-channel image of the labeled neuron is shown in the right panel. Scale bars: 50 μm.

• Movie 1
• Movie 2
• Movie 3 -

  Movies S1, S2 and S3. Time-lapse movies showing the migration of the nVII motor neurons in hindbrain explants from the wild-type (S1), olt^rw689 (S2) and ord^rw71 (S3) Isl1-GFP embryos. The composite stacks of serial optical images were made every 10 minutes at 18-33 hpf (see Materials and methods). Lateral views are shown, with the anterior of the embryo facing left. In the wild-type embryo, all of the migrating neurons were located near the pial surface of the hindbrain. By contrast, in the olt embryo, none of the nVII motor neurons migrated caudally. In the ord embryo, some of the nVII motor neurons became detached from the pial surface, but they still migrated caudally in the dorsal part of the hindbrain. See also Fig. 5A,B,D in which several frames of these movies are shown. The inner lumen of the otic vesicle (ov) is indicated by dotted lines. nV, trigeminal motor neurons; nX, vagus motor neurons; nVII, facial motor neurons; VIIn, facial motor axons. Asterisks indicate the r6-derived putative OLe neurons.

• Movie 4
• Movie 5 -

  Movies S4 and S5. Time-lapse movies showing migration of the nVII motor neurons in hindbrain explants from the wild-type (S4) and olt^rw689 (S5) Isl1-GFP embryos. The composite stacks of serial optical images were made every 10 minutes at 20-22 hpf. Frontal views are shown, with the dorsal part of the embryo at the top of the frame. The nVII motor neurons migrated radially towards the ventricular surface by extending and retracting aberrant processes in the olt embryo. See also Fig. 5C, in which several frames of the movie S5 are shown.