QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 1 November 2006
doi: 10.1242/dev.02665

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wada, H. Articles by Okamoto, H. Search for Related Content PubMed PubMed Citation Articles by Wada, H. Articles by Okamoto, H. Social Bookmarking                         What's this?

Frizzled3a and Celsr2 function in the neuroepithelium to regulate migration of facial motor neurons in the developing zebrafish hindbrain

¹ Laboratory for Developmental Gene Regulation, Brain Science Institute, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
² Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST), 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan.

View larger version (97K): [in a new window]   Fig. 1. olt and ord genes are required for migration of nVII motor neurons. (A-I) Morphology and Isl1-GFP expression of wild-type (A,D,G), MZ-oltrw689 (B,E,H) and MZ-ordrw71 (C,F,I) Isl1-GFP embryos at 48 hpf (A-F) and 30 hpf (G-I). In wild-type embryos (D,G), the nVII motor neurons are located in r6 (arrows). By contrast, in olt (E,H) and ord (F,I) embryos, most or all of the neurons are located in r4 (arrows). In ord embryos (F,I), some neurons migrate into r5 (arrowheads). (A-C,G-I) Lateral views; anterior facing left. (D-F) Dorsal views; anterior at the top. (A-F) Images obtained using a dissecting microscope. (G-I) Composite images of serial optical sections obtained by confocal microscopy. Va, anterior trigeminal nuclei; Vp, posterior trigeminal nuclei; VII, facial nucleus; X, vagus nucleus. Scale bars: 50 µm. The images in A,C,D,F,G,I were previously published in Wada et al. (Wada et al., 2005).

View larger version (48K): [in a new window]   Fig. 2. Identification of the olt gene as zebrafish frizzled3a gene. (A) Genetic map of the olt locus. (B) Genomic structure of the zebrafish fz3a gene. The nucleotide substitution resulting from the oltrw689 mutation is indicated. (C) Schematic drawings for the mouse Fz3 and zebrafish Fz3a proteins. Amino acid sequence similarity (%) is shown for each domain. The mis-sense amino acid substitution in the Fz3arw689 protein is indicated. (D) The phylogenetic tree for fz3-family genes. (E) Lateral view of a wild-type embryo reacted with the fz3a RNA probe at 24 hpf. (E') Cross section of E at r5. (F-H) Wild-type Isl1-GFP embryo injected with fz3a-MO (H) showing the same neuronal migration defects as those observed in an olt embryo (G). The embryos are shown in dorsal view and the images are composite stacks of serial optical sections. (I,I') Wild-type Isl1-GFP embryo injected with fz3a-C mRNA. Migration of the nVII motor neurons was specifically impaired (arrow in I'). Lateral views. Scale bars: 50 µm.

View larger version (44K): [in a new window]   Fig. 3. Identification of the ord gene as zebrafish celsr2 gene. (A) Genetic map of the ord locus. (B) Genomic structure of the zebrafish celsr2 gene. The nucleotide substitution resulting from each mutation is indicated. (C) Schematic drawings of the mouse and zebrafish Celsr2 proteins. Amino acid sequence similarity (%) is shown for each domain. The amino acid substitution resulting from each mutation is indicated. (D) The phylogenetic tree for celsrfamily genes. (E-G) Lateral views of wild-type embryos reacted with RNA probes for celsr2 (E), celsr1a (F) and celsr1b (G) at 24 hpf. (E',F',G') Cross sections at r5 for each embryo, E-G, respectively. (H-K) A wild-type Isl1-GFP embryo injected with celsr2-MO (J) shows incomplete disruption of the neuronal migration as observed in an ord embryo (I). (K) An ord mutant embryo injected with celsr1a/1b-MOs shows complete loss of migration of the nVII motor neurons. The embryos are shown in dorsal view and the images are composite stacks of serial optical sections. (L) The neuronal migration phenotype in each experiment was scored as follows: r4, complete loss of migration, as shown in K; r5, partial disruption of migration, as shown in I; and r6, normal migration, as shown in H. Scale bars: 50 µm.

View larger version (57K): [in a new window]   Fig. 4. Defective migration of the nVII motor neurons in mutant embryos. (A-K) Wild-type (A,D,G,J), oltrw689 (B,E,H) and ordrw71 (C,F,I,K) Isl1-GFP embryos were stained with anti-acetylated -tubulin antibody. (A-C) Composite stacks of serial optical sections, shown in dorsal view. (D-K) Images are single focal planes of cross sections at the rhombomeric regions indicated by broken lines in A-C. Hindbrain regions are outlined by broken lines. (H,J,K) Higher magnifications of E, G, I, respectively. In the mutant embryos, some of the neurons reach the ventricular surface (arrowheads in H and K). However, these mismigrated neurons extend axons normally (shown by arrows in H and J). GFP-expression in single axons was barely detectable (H). Therefore, the yellow signals of the axons of the nVII motor neurons in the wild-type embryos (J) are technical artifacts caused by superimposition of the red signals of the axons and the green signals of the cell bodies of the overlapping neurons. (L-P) Aberrant radial processes in the mutant embryos (arrowheads in M). Frontal views of the live wild-type (L) and olt (M) Isl1-GFP embryos at r4, and a dorsal view of the olt Isl1-GFP embryo (N) at 24 hpf. Higher magnification of the boxed region is shown in the inset. (L-N) Images are composite stacks of serial optical sections. (O) Scoring of aberrant processes in the wild-type (WT), olt and ord embryos injected with celsr1a/1b-MOs (ord+MOs). Bars represent S.D. (P) Direction of the aberrant processes was quantified in the olt embryos. The angle of each process was measured as the deviation from the right angle to the midline. Scale bars: 50 µm.

View larger version (106K): [in a new window]   Fig. 5. Time-lapse analyses of defective migration of the nVII motor neurons. (A-D) Time-lapse observations of migrating nVII motor neurons in hindbrain explants of the wild-type (A), olt (B,C) and ord (D) Isl1-GFP embryos at the time (hpf) indicated in each panel. (Also see Movies S1-S5 in the supplementary material.) Images are composite stacks of serial optical sections. (A,B,D) Lateral views with anterior to the left; the inner lumen of the otic vesicle (ov) is indicated by broken lines. (C) Frontal views at r4. In the olt embryos, all neurons failed to migrate caudally (B) but mismigrated towards the ventricle (arrows in C) by extending aberrant radial processes (arrowheads in C). In the ord embryo, some of the late-born nVII motor neurons (indicated by arrows in D) migrated caudally in the dorsal part of the hindbrain. nV, trigeminal motor neurons; VIIn, facial motor axons. Asterisks indicate the r6-derived putative octavolateralis efferent (Ole) neurons (Wada et al., 2005). Scale bar: 50 µm.

View larger version (97K): [in a new window]   Fig. 6. Functional fz3a and celsr2 genes in neuroepithelial cells are required for preventing integration of nVII motor neurons into the neuroepithelium. (A,B) Single focal-plane images of cross sections at r4 in olt Isl1-GFP embryos stained with anti-ß-catenin antibody (red) at 24 (A) and 33 (B) hpf. Orientation of the neuroepithelial cells is shown by double-headed arrows. Hindbrain regions are outlined by broken lines. (C-F) Mosaic experiments were performed to determine the cell autonomy of the olt and ord genes. The donor cells were labeled with rhodamine-conjugated dextran (red). The nVII motor neurons (arrows) derived from the oltrw689 (C) and ordrw71 (E) Isl1-GFP embryos migrated caudally in the wild-type host embryos, although some were still located in r4 at the time of observation (arrowheads in C). By contrast, none of the wild-type-derived nVII motor neurons (arrows) reached r6 in the olt (D) and ord (F) host embryos. Red puncta signals may be the debris of the dead transplanted cells. However, as we observed the growth of the peripheral axons of the nVII motor neurons in each mosaic embryo (see Materials and methods), it is unlikely that such debris had serious adverse effects on the development of these embryos. Dorsal views of the embryos are shown. (G-J) Embryos showing mosaicism in the neuroepithelium at the r4 region. When the olt-derived cells were incorporated into the neuroepithelium of the wild-type host embryos at r4, the nVII motor neurons migrated aberrantly into the mutant neuroepithelium (arrows in G and I'). By contrast, when wild-type-derived cells were incorporated into the r4 region of the olt host embryos, the nVII motor neurons failed to invade the wild-type neuroepithelium (arrowheads in H and J'). (C-H) Images are composite stacks of serial optical sections. (I,J) Computationally reconstructed 3D images of the embryos shown in G and H. Dorsal and ventral views of the embryos are shown. In I, mismigrated nVII motor neurons (green) are hidden by the surrounding olt embryo-derived neuroepithelial cells (red) as no transparency was given to the images of the donor-cell clusters. (I',J') Computationally reconstructed cross sections at r4 indicated by the broken line in I and J. (I',J'') Schematic cross sections at r4 of the embryos shown in G and H, respectively. The yellow signals in the merged panels of G and H are technical artifacts caused by the superimposition of the red signals of the donor cells and the green signals of the motor neurons. As red signals were not detected in the axons of the motor neurons (G,H, middle panels), all of the nVII motor neurons were derived from the host embryos. Scale bars: 50 µm in A for A,B; and 50 µm in C for C-H.

View larger version (24K): [in a new window]   Fig. 7. Schematic drawing of nVII motor-neuron migration in the wild-type and olt embryos. In the wild-type embryos, the nVII motor neurons (green) migrated caudally near the pial surface of the hindbrain. By contrast, in the olt embryos, because the neuroepithelial cells (red) had lost their ability to prevent integration of the nVII motor neurons, the motor neurons migrated towards the ventricle by extending aberrant processes radially to the ventricle. Directions of migration of the nVII motor neurons are indicated by arrows. See text for details.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter