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doi: 10.1242/10.1242/dev.00465

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N-cadherin mediates retinal lamination, maintenance of forebrain compartments and patterning of retinal neurites

¹ Masai Initiative Research Unit, The Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan
² Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK
³ Max-Planck-Institute for Immunobiology, Stuebeweg 51, D-79108 Freiburg, Germany
⁴ Laboratory for Developmental Gene Regulation, RIKEN Brain Science Institute and CREST, Japan Science and Technology Corporation (JST), Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan

View larger version (61K): [in a new window]   Fig. 1. lyr is a new allele of the Ncad mutant, pac. (A,B) Lateral views of 3 dpf wild-type (A) and lyr–/– (B) eyes labeled with anti-acetylated -tubulin antibody. Arrowheads indicate disruption of the IPL. (C) Map of microsatellite markers used to map lyr to the same position on LG20 as ncad. Recombination rate is indicated by parenthesis. (D,E) Lateral views of wild-type (D) and mutant (E) embryos produced by crossing lyr and pacfr7 carriers. Morphological defects are evident in the tectum and hindbrain of the mutant (E, arrowheads). (F) Mutation sites in the two pac alleles used in this study. A nonsense mutation present in pac fr7 (Lele et al., 2002) and a mis-sense mutation (TyrCys) found in lyr–/– embryos both occur in EC4 domain of Ncad. (G,H) Confocal images of aggregates of cells in which animal caps labeled with Alexa-488 conjugated dextran (green) and expressing either wild-type Ncad RNA (G) or lyr-mutated Ncad RNA (H) were juxtaposed to non-injected animal caps labeled with rhodamine-conjugated dextran (red). The sharp boundary between the Ncad-expressing and non-expressing cell masses is disrupted by the lyr mutation. (I) Quantification of cell intermingling. The bars indicate the average number of cells isolated within the apposing animal cap. Numbers of animal caps examined are indicated under the columns. h, hindbrain; inl, inner nuclear layer; ipl, inner plexiform layer; MHB, midbrain-hindbrain boundary; onl, outer nuclear layer; rgl, retinal ganglion cell layer; Tc, optic tectum.

View larger version (126K): [in a new window]   Fig. 2. Ncad is required for lamination and structural integrity of retinal cells. (A-D) Plastic sections of 3 dpf wild-type (A), pacrw95 (B), pacrw95/fr7 (C) and pacfr7 (D) retinae. The partial fusion of the RGC and amacrine cell layers is indicated in the pacrw95 retina (B, red asterisks). (E-H) High magnification of the INL in wild type (E), pacrw95 (F), pacrw95/fr7 (G) and pacfr7 (H). A patch of the IPL adjacent to the bipolar cell layer is indicated in the pacrw95 retina (F, broken red square). (I-L) High magnification of the ONL in wild type (I), pacrw95 (J), pacrw95/fr7 (K) and pacfr7 (L). Some photoreceptors show irregular shape and abnormally aggregate in pacrw95 (J, red broken parentheses). The photoreceptor layer is wavy and forms rosette structures in pacrw95/fr7 (K, broken red square). In pacfr7, cells adjacent to the pigmented epithelium do not display photoreceptor-specific columnar shape (L, red arrowheads). (M-P) BODIPY-ceramide labeled living 48 hpf wild-type (M), pacrw95 (N), pacrw95/fr7 (O) and pacfr7 (P) retinae. In wild type, the IPL forms at the interface between the RGC layer and INL. By contrast, patches of IPL neuropil are observed in the amacrine cell layer of pacrw95 eye. In the pacrw95/fr7 retina (O), patches of INL (white arrowheads) are more irregular and the OPL is also wavy (open white arrow). White asterisks show the CMZ. a, amacrine cell layer; b, bipolar cell layer; CMZ, ciliary marginal zone, h, horizontal cell layer; inl, inner nuclear layer; ipl, inner plexiform layer; pe, pigmented epithelium; pl, photoreceptor layer; rgl, retinal ganglion cell layer.

View larger version (99K): [in a new window]   Fig. 3. Neuronal patterning is affected in pac mutants. (A-D) Two dpf wild-type (A), pacrw95 (B), pacrw95/fr7 (C) and pacfr7 (D) retinae labeled with zn5 antibody (red), which stains axons and cytoplasm of RGCs. Nuclei of retinal cells are counter-stained green with Sytox Green Nucleic Acid Stain. RGCs differentiate in all mutants (white arrowheads) but their localization is progressively more disrupted in pacrw95, pacrw95/fr7 and pacfr7 eyes. (E-H) Four dpf wild-type (E), pacrw95 (F), pacrw95/fr7 (G) and pacfr7 (H) retinae labeled with anti-Pax6 antibody, which stains amacrine cells (red). (I-L) Four dpf wild-type (I), pacrw95 (J), pacrw95/fr7 (K) and pacfr7 (L) retinae labeled with zpr-1 antibody, which stains double-cone photoreceptors (red). In pacrw95, the photoreceptor cell layer is normal. In pacrw95/fr7, the photoreceptor cell layer is locally disrupted and in the pacfr7 retina, zpr-1-positive photoreceptors do not form an outer layer, but instead are located sparsely near the scleral region of the eye (white arrowheads). a, amacrine cell layer; pl, photoreceptor layer; rgl, retinal ganglion cell layer.

View larger version (83K): [in a new window]   Fig. 4. Ncad is required for maintenance of adherens junctions in the retina. (A,B) Twenty-eight hpf wild-type (A) and pacfr7 (B) retinae labeled with rhodamine-conjugated phalloidin, which stains actin microfilaments (red). In wild type, strongly stained actin foci are visible in the apical termini of retinal cells, closely associated with the pigmented epithelium (A, white arrowheads). In the pacfr7 eye, actin foci are normally localized in the dorsal retina (white arrowheads), while the line of actin foci (white arrows) is detached from the pigmented epithelium (broken lines) in the ventral retina and localization of actin foci is completely disrupted in the most ventral cells (B, white asterisk). (C,D) Twenty-eight hpf wild-type (C) and pacfr7 (D) retinae labeled with anti--tubulin antibody, which stains centrosomes. Sections shown in B,D are serial sections. In the wild-type retina, the centrosomes are localized in the apical termini of retinal neuroepithelial cells (white arrowheads). In pacfr7, the centrosomes fail to be localized apically in the ventral retina (white arrows) and their localization is completely random in the most ventral region (D, white asterisk). In the dorsal region, position of the centrosomes seems to be normal (white arrowheads). (E,F) Plastic sections of 24 hpf wild-type (E) and pacfr7 (F) retinae labeled with anti-phosphorylated histone H3 antibody, which stains proliferating cells in late G2 and M-phase. In the wild-type retina, dividing cells are localized to the apical surface of the neural retina (white arrows). In pacfr7, proliferating cells are positioned far from the apical surface in the ventral retina (F, red arrows), but normal in the dorsal retina (F, white arrows). (G,H) Forty-eight hpf wild-type (G) and pacfr7 (H) retinae labeled with anti-phosphorylated histone H3 antibody. The location of dividing cells is random throughout the pacfr7 retina. dr, dorsal retina; vr, ventral retina.

View larger version (87K): [in a new window]   Fig. 5. Ncad regulates outgrowth of amacrine cell dendrites. (A-D) Confocal images of GFP+ cell columns in wild-type (A,C) and pacrw95 (B,D) retinae. White arrows and arrowheads show retinal axons and amacrine cell dendrites, respectively. Irregularly branched neurites extend from the INL in pacrw95 (B,D, red arrowheads). (E,F) GFP (green) and anti-Pax6 antibody (red) labeling of wild-type (E) and pacrw95 (F) retinae. In the wild-type retinae, amacrine cell dendrites spread horizontally at the interface between RGCs and amacrine cells (white arrowheads). By contrast, Pax6-positive amacrine cells extend abnormally arborized neurites in the amacrine and RGC layers in pacrw95 (white arrowheads). There is no equivalent arborization in the bipolar cell layer. (G,H) Confocal images showing morphology of bipolar cells (G) and a RGC (H) in 72 hpf pacrw95 eyes. White arrows indicate axons of RGCs. (I,J) Anti-PKC labeling (red) of 4 dpf wild-type (I) and pacrw95 retinae (J). (K) Section of a 3 dpf pacrw95 eye in which wild-type cells have incorporated into the retina. The wild-type column of cells (brown) forms a normal IPL. The red arrow indicates a wild-type cell positioned within the mixed RGC/amacrine cell layer. (L) Section of a 3 dpf wild-type eye incorporating pacrw95 cells. The red arrow shows disruption of IPL formation by mutant cells. The red arrowheads indicate isolated mutant amacrine cells showing normal neurite morphology. a, amacrine cells; b, bipolar cells/cell layer; inl, inner nuclear layer; ipl, inner plexiform layer; le, lens; pe, pigmented epthelium; pl, photoreceptor layer; rgc, retinal ganglion cells; rgl, retinal ganglion cell layer.

View larger version (91K): [in a new window]   Fig. 6. Ncad is required for guidance of RGC axons. (A) Dorsal view of 4 dpf wild-type embryos labeled with diI (red) and diO (green) in the left and right eyes, respectively. RGC axons project to contralateral optic tectum. (B) Trajectories of diI/diO labeled RGC axons in pacfr7. Retinal axons project to both ipsilateral and contralateral tecta. White arrowheads indicate mis-routed retinal axons. (C,D) Ventral view of 60 hpf diI/diO-labeled wild-type (C) and pacfr7/rw95embryo (D). In pacfr7/rw95, RGC axons change outgrowth direction by following retinal axons that come from the opposite eye (D, white arrowheads). (E,F) Expression of ath5:GFP in 3 dpf wild-type (E) and pacfr7 embryos (F). In pacfr7, retinal cells have migrated out from the optic cups and invaded the ventral brain (white arrowheads). There is variability in the phenotype and this embryo is more severe than most. (G-I) Three dpf chimaeric eyes in which pacfr7 cells (green) were transplanted into wild-type embryos. Asterisks indicate retinal cells that escape from the eye cups. (H) Red indicates diI-labeled wild-type axons that bifurcate at ectopically positioned retinal cells (white arrowheads). (I) pac (green, GFP) and wild-type (red, diI) retinal axons (white arrowheads) turn in an inappropriate direction at the interface with retinal cells (white asterisk) and project to the telencephalon. (J) Three dpf wild-type embryos, in which pacfr7 cells were transplanted to the retinae at 24 hpf. pac retinal axons adopt a normal contralateral trajectory. (K,L) Ventral view of 30 hpf wild-type (K) and pacfr7 (L) forebrains labeled with a slit3 RNA probe. Arrows indicate three domains of slit3 expression, which bound the anterior commissure, post-optic commissure and the optic chiasm. AC, tract of anterior commissure; OC, position of optic chiasm; POC, post-optic commissure

View larger version (51K): [in a new window]   Fig. 7. pac mutants exhibit severe coloboma. (A,B) Lateral views of 4 dpf wild-type (A) and pacfr7 (B) heads. The choroid fissure does not close (red arrowheads) in pacfr7. (C,D) High magnification of the optic nerve exit point/choroid fissure in 4 dpf wild-type (C) and pacfr7 (D). Retinal cells spill out of the open fissure (D, red arrowheads) and spread to the diencephalon in pacfr7. (E-J) pax2.1 labeling of optic stalks in wild-type and pac mutants. (E,F) Dorsal views of 19.5 hpf embryos showing pax2.1 (blue) and pax6 expression (red). Broader expression of pax2.1 in the optic vesicles of the pac mutant is indicated (arrows/lines) (G,H) Lateral views of 24 hpf optic cups showing expanded pax2.1 expression in the ventral retina of the pac mutant. (I,J) Ventral view of 48 hpf heads. pax2.1 expression is present in prospective astrocytes of the optic nerve. Additionally in the pac mutant, pax2.1-expressing cells are present in the ventronasal and ventrotemporal retina and spread from the optic nerves into the brain (arrowheads). (K) Lateral views of the choroid fissure in wild-type and pacfr7 eyes. Light blue indicates the neural retina and dark blue shows pax2.1+ prospective astrocytes. a, amacrine cell layer; b, bipolar cell layer; di, diencephalon; r.ax, retinal axon; pl, photoreceptor layer.

View larger version (65K): [in a new window]   Fig. 8. Lens differentiation is perturbed in pac–/– embryos. (A) The developing lens. Proliferating apical epithelial cells (yellow) move to the equatorial zone (orange) where they start to differentiate as lens fiber cells. The apical and basal ends of the elongating lens fiber cells (green) migrate medially along the apical surface of proliferative epithelium (yellow) and basal lens capsule, respectively. The thinning fiber cells consequently cover the core of older lens fibers (blue) like the layers of an onion. The arrow indicates the progression of lens fiber cell maturation. (B,C) Plastic sections of 3-day-old wild-type (B) and pacfr7 lens (C). In pac the apical proliferating cells are irregularly shaped and cells on the basal side of the lens do not display the elongate morphology of differentiating lens fibers (red arrows). Red asterisk indicates degenerated lens fibers. (D-G) Wild-type (D,F) and pacfr7 (E,G) lenses labeled with BODIPY-ceramide. (D,E) Thirty-one hpf lens. Cuboidal proliferating cells form a superficial single layer on the lens surface. Differentiating cells are forming at the equatorial zone and elongating (red circles) in both wild-type and pacfr7 lenses. (F,G) Forty-eight hpf lens. In wild type, lens fibers have differentiated (red circles), whereas they fail to elongate (red arrows) and remain on the basal side of the lens in pac mutants (white asterisk).

View larger version (106K): [in a new window]   Fig. 9. SU5402 treatment induces abnormal RGC axon trajectories but does not affect retinal lamination. (A-C,E-G) diI/diO-labeled RGC axons in SU5402-treated (A-C,E,F) and wild-type (G) embryos at 2 dpf (C) or 4 dpf (A,B,E-G). (A,B) Dorsal view showing ipsilateral projections (A) and mixed ipsi- and contralateral projections (B) (10 µM SU5402, 8-96 hpf). (C) Frontal view showing RGC axons extending ipsilaterally before reaching the optic chiasm (10µM SU5402, 8-48 hpf). (D) slit3 expression in SU5402-treated 30 hpf embryos. The distance between the anterior commissure and retinal axons is narrow (arrows/lines). White asterisks indicate ectopic slit3 expression at positions where retinal axons enter the midline area (compare with Fig. 6K). (E) Dorsal view showing retinal axons which fail to enter target areas in the tectum (white asterisks) (10 µM SU5402, 30-96 hpf). (F,G) Lateral views. Wild-type retinal axons project and arborize in the optic tectum, but SU5402-treated axon fails to enter the tectum (white asterisk). (H) SU5402-treated 60 hpf retinae (10 µM, soaked from 8 hpf) labeled with anti-phosphorylated-histone H3 antibody. The locations of dividing cells (arrowheads) resemble wild-type (compare with Fig. 4G). (I) SU5402-treated 3 dpf retinae (10 µM, soaked from 8 hpf) labeled with anti-Pax6 antibody. (J) SU5402-treated 60 hpf retinae (10 µM, soaked from 8 hpf) labeled with BODIPY-ceramide. Arrowheads indicate the IPL (compare with Fig. 2M). (K) SU5402-treated 60 hpf retinae (10 µM, soaked from 30 hpf) labeled with BODIPY-ceramide. (L) Lateral view of a 48 hpf living SU5402-treated eye (10 µM SU5402, 8-48 hpf). (M) Plastic section through the closed choroid fissure (red arrowheads) of a 4 dpf SU5402-treated retina. (N) Plastic section of 4 dpf SU5402-treated retina (10 µM, soaked from 8 hpf). Retinal lamination is globally normal, but some dying cells are observed as dark-stained nuclei (red arrowheads). (O) SU5402-treated 60 hpf retinae labeled with BODIPY-ceramide. Embryos were soaked with 1mM SU5402 from 30 hpf. No patches of IPL neuropil are observed, but instead large areas of cell degeneration are evident (red asterisks). Such severe cell death was not observed with treatment of 100 µM SU5402 (data not shown).

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