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First published online 20 September 2006
doi: 10.1242/dev.02566

Development 133, 4085-4096 (2006)
Published by The Company of Biologists 2006
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Cadherin is required for dendritic morphogenesis and synaptic terminal organization of retinal horizontal cells

Koji Tanabe1,2,3, Yoshiko Takahashi1,4, Yuki Sato1,4, Koichi Kawakami5, Masatoshi Takeichi1,2 and Shinichi Nakagawa1,3,*

1 RIKEN Center for Developmental Biology, 2-2-3 Minatojima Minamimachi, Chuoku, Kobe 650-0047, Japan.
2 Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan.
3 Nakagawa Initiative Research Unit, RIKEN Frontier Research Program, 2-1 Hirosawa, Wako 351-0198, Japan.
4 Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, NARA, 630-0192, Japan.
5 Division of Molecular and Developmental Biology, National Institute of Genetics, Mishima, Shizuoka, Japan.


Figure 1
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  		Fig. 1. A transposon-mediated gene transfer system that enables conditional gene expression in the developing chicken retina. (A) Schematic drawing of conditional gene expression. The gene cassettes flanked by the Tol2 cis-sequence (Tol2) are integrated into the host genome by the transient expression of Tol2 transposase (T2TP). Only the cells that stably incorporated both CAG-rtTA-M2 and TRE-mEGFP cassettes express mEGFP upon the addition of doxycycline (DOX), which enhances the binding of rtTA-M2 to its cis-element TRE. (B,C) Exogenous mEGFP expression (green) in horizontal cells expressing Prox1 (B) and Pax6 (C) (magenta). Scale bars: 20 µm.

 

Figure 2
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  		Fig. 2. Chicken horizontal cells are classified into three subtypes based on their morphology. (A-C) Confocal images of three types of horizontal cells. Projection images and optical vertical sections are shown in upper and lower panels, respectively. (A) Type I horizontal cell with a small, bushy dendritic field and a single long axon (arrow). (B) Type II horizontal cell with large and highly branched dendritic arbors. (C) Type III horizontal cell with simple, less branched dendrites that contains bulge-like terminals (arrows). (D) The three subtypes of horizontal cell are shown in the same scale. (E) Expression of N-cadherin mRNA in the E16 retina. Chicken N-cadherin (cNcad, arrowheads) is expressed in all the horizontal cells that strongly express Prox1 (green, arrows). Scale bars: 10 µm in A-D; 20 µm in E.

 

Figure 3
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  		Fig. 3. Developmental changes in horizontal cell morphology. (A) A single optical section at the level of the horizontal cell layer. Horizontal cells are identified by their expression of Prox1. (B-G) Projection images (upper panels) and optical vertical sections (lower panels) of mEGFP-expressing horizontal cells at E9 (B), E12 (C,D) and E14 (E-G). Vertical processes are shown by arrowheads in B-D. (C) At E12, some horizontal cells have a long process reminiscent of an axon (arrow). (D) Other horizontal cells have less branched dendrites extending in one direction. (E-G) At E14, the three subtypes of horizontal cells have become apparent. Arrows in G indicate the dendrite terminals with growth cone-like morphology. Scale bar: 10 µm.

 

Figure 4
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  		Fig. 4. Subtype-specific projections of dendrite terminals. (A) Serial confocal images from vitreal (left) to scleral (right) side of the outer nuclear layer. PNAL and N-cadherin expression are shown in magenta and green, respectively. The PNAL-expressing cells at the pedicle levels can be traced back to the double-cones at the level of the outer nuclear layer (asterisks). The principal- and accessory-cone pedicles are indicated by arrowheads and arrows, respectively. (B,E,H) Projection images (upper panels) and optical vertical sections (lower panels) of three types of horizontal cells double-stained with PNA and EGFP. (C,D,F,G,I,J) Higher magnification view of single optical sections taken at the level of double-cone pedicle, double-stained for EGFP and PNAL (C,F,I) or GluR4 (D,G,J). Note that the dendrite terminals of Type I horizontal cells project to both the principal- and accessory-cone pedicles (arrowheads and arrows in C and I, respectively), whereas type III horizontal cells project solely to the accessory-cone pedicles (arrows in I). (K) Schematic drawing of the projection patterns of type I and type III horizontal cell dendrites. Scale bars: 5 µm in A,C,D,F,G,I,J; 10 µm in B,E,H.

 

Figure 5
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  		Fig. 5. Simultaneous expression of dominant-negative cadherins and mEGFP in the horizontal cells. (A) The three vectors used for the conditional expression. The bidirectional promoter enables simultaneous expression of the dominant-negative cadherin and mEGFP marker. (B) Schematic drawing of domain structures of the dominant-negative molecules. (C-E) Expression patterns of full-length N-cadherin (C), cN390{Delta} (D) and cN390{Delta} CBR (-) (E) in mEGFP-expressing horizontal cells. Scale bar: 10 µm (C-E).

 

Figure 6
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  		Fig. 6. cN390{Delta} decreases the dendritic field size of horizontal cells. (A-J) Morphology of horizontal cells expressing full-length N-cadherin (A-D) or cN390{Delta} (E-J) visualized by mEGFP expression at E16, 4 days after the induction of the exogenous genes. Projection images and optical vertical sections are shown in upper and lower panels, respectively. (K,L) Statistical analysis of the effect of the dominant-negative cadherin on the area of the dendritic field (K) or axonal length (L). Numbers inside the bars represent the number of cells examined. ***P<0.0001, n.s. P>0.8. Error bars represent s.d. All figures are shown at the same scale. Scale bar: 10 µm.

 

Figure 7
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  		Fig. 7. cN390{Delta} inhibits an early phase of dendrite extension. (A-G) Morphology of horizontal cells expressing full-length N-cadherin (A-C) or cN390{Delta} (D-G) visualized by mEGFP expression at E14, 2 days after the induction of the exogenous genes. Projection images and optical vertical sections are shown in upper and lower panels, respectively. (H,I) Statistical analysis of the effect of the dominant-negative cadherin on the area of the dendritic field (H) and axonal length (I). Numbers inside the bars represent the number of cells examined. **P<0.001, ***P<0.0001, n.s. P>0.8. Error bars represent s.d. All figures are shown in the same scale. Scale bar: 10 µm.

 

Figure 8
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  		Fig. 8. Cadherin regulates synaptic contacts. (A-C) Morphology of horizontal cells expressing cN390{Delta}. Projection images and optical vertical sections are shown in upper and lower panels, respectively. The dendrite terminals of type I/III and type II horizontal cells correctly target the PNAL-positive and PNAL-negative sublamina, respectively. (D-K) Higher magnification views of single optical sections at the level of double-cone pedicles. The dendrite terminals expressing full-length N-cadherin (D-G) and the cN390{Delta} (H-K) are shown in green and the synaptic markers PNAL (D,H,F,J) and GluR4 (E,G,I,K) in magenta. Many of the dendrite terminals of type I (H) and type III (J) horizontal cells covered smaller areas of the double-cone and accessory-cone pedicles, respectively (arrows). GluR4 expression is either absent (arrows) or suppressed (arrowheads) in the regions that have received projections from horizontal cells expressing the cN390{Delta}, shown as a broken line (I,K). Scale bars: 10 µm A-C; 5 µm D-K.

 

Figure 9
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  		Fig. 9. Statistical analysis of the effect of cN390{Delta} on dendrite terminals. (A) Number of dendritic terminals projecting to the double-cone pedicles from a single neuron. (B) Number of dendritic terminals projecting from a single horizontal cell divided by the dendritic field area. (C) Number of dendritic terminals with mature morphology. (D) Numbers of dendrite terminals with different intensities of GluR4 signals. Numbers in the bars represent the number of cells and dendritic terminals examined, respectively. ***P<0.0001, n.s. P>0.08. Error bars represent s.d.

 

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© The Company of Biologists Ltd 2006