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Math3 and NeuroD regulate amacrine cell fate specification in the retina

Tomoyuki Inoue1,2, Masato Hojo1,*, Yasumasa Bessho1, Yasuo Tano2, Jacqueline E. Lee3 and Ryoichiro Kageyama1,{dagger}

1 Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan
2 Department of Ophthalmology, Osaka University Medical School, Suita 565-0871, Japan
3 Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309, USA
* Present address: Department of Neurosurgery, Kurashiki Central Hospital, Okayama 710-8602, Japan



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  		Fig. 1. Expression of Math3 in the developing retina. In situ hybridization of Math3 in mouse retina. (A) At P1, the retina consists of the ganglion cell layer (GCL) and ventricular zone (VZ). Math3 is expressed in the ventricular zone. (B,C) At P4 and P7, the retina consists of three cellular layers. Math3 is expressed broadly in the inner nuclear layer (INL). (D,E) At P14 and adult, Math3 is expressed in the outer region of the INL. (F) The sense strand was used as a probe. No signal is observed. (G-I) At P7, Math3 is expressed by calbindin+ amacrine and horizontal cells. (J-L) At P7, Math3 is expressed by HPC1+ amacrine cells. (M-O) At P7, Math3 is expressed by NF+ horizontal cells. (P-R) At P14, Math3 is not expressed by HPC1+ amacrine cells. (S-U) At P14, Math3 is still expressed by NF+ horizontal cells. Scale bar: 25 µm.

 


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  		Fig. 2. The lack of amacrine cells and concomitant increase of ganglion and Müller glial cells in Math3/NeuroD double-mutant retina. The retinal explants were prepared from wild-type (a), Math3–/– (b), NeuroD–/– (c) and Math3–/–/NeuroD–/– embryos (d) at E17.5 and cultured for 2 weeks. (A) HE staining. The GCL, INL and ONL are formed in all retinal explants. (B) Immunohistochemistry for rhodopsin. There are fewer rods (rhodopsin+) in NeuroD–/– (c) and Math3–/–/NeuroD–/– retina (d). (C) PKC-positive cells (bipolar cells) are normally generated in all retinal explants. (D) Calbindin-positive amacrine (black arrowheads) and horizontal cells (white arrowhead) are normally generated in wild-type (a), Math3–/– (b) and NeuroD–/– retina (c). By contrast, in Math3–/–/NeuroD–/– retina (d), amacrine cells are completely missing (asterisks), whereas calbindin-positive horizontal cells are normally generated (white arrowhead). (E) Amacrine cells (HPC1+) are missing in Math3–/–/NeuroD–/– retina (d). (F,G) Ganglion cells (Thy1.2+, p75+) are significantly increased in Math3–/–/NeuroD–/– retina (d). There are ectopic ganglion cells in the inner region of the INL of Math3–/–/NeuroD–/– retina (d). (H,I) The number of Müller glial cells (glutamine synthetase+, vimentin+) is slightly increased in Math3–/–/NeuroD–/– retina (d). Scale bar: 25 µm.

 


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  		Fig. 3. Quantification of retinal cells of the mutant retina. (A) The cell number was counted in a section (16 µm thick, 500 µm wide) of the central region of at least three independent samples from each genotype. The number of the ONL cells is reduced in NeuroD–/– and Math3–/–/NeuroD–/– retina. The absolute numbers of the GCL and INL cells are not significantly affected by mutations. (B) Ratios of ganglion and amacrine cells in the GCL. The percentage of calbindin-positive and Thy1.2-positive cells per the total GCL cells was calculated. About a half of the GCL cells are amacrine cells in wild-type, Math3–/– and NeuroD–/– retina. By contrast, there are no amacrine cells (asterisk) in the GCL of Math3–/–/NeuroD–/– retina. As the absolute number of the GCL cells is not affected (A), these data indicate that the absolute number of ganglion cells is increased in Math3–/–/NeuroD–/– retina. (C) Ratios of cells in the INL. The cell number was counted as above, and the percentage of each retinal cell type per the total INL cells was calculated. There are ectopic ganglion cells but no amacrine cells (asterisk) in Math3–/–/NeuroD–/– INL.

 


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  		Fig. 4. The optic nerves are thickened in the double-mutant mice. (A,B) Section of the wild-type (A) and Math3–/–/NeuroD–/– optic nerve (B) at E17.5. (C,D) NF staining of the wild-type (C) and Math3–/–/NeuroD–/– optic nerve (D) at E17.5. Scale bar: 50 µm. (E) The NF+ region of the optic nerve sections was quantified (n=3). The section of the double-mutant optic nerves is about 1.7-fold wider than that of the wild type.

 


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  		Fig. 5. The fate switch from amacrine cells to ganglion and Müller glial cells in Math3–/–/NeuroD–/– retina. (A) X-gal staining of the retinal explants that were prepared at E17.5 and cultured for 14 days. (a) In NeuroD+/– retina, X-gal staining is observed in NeuroD-expressing cells: rods in the ONL and amacrine cells in the GCL and the inner region of the INL. (b) In Math3–/–/NeuroD–/– retina, X-gal-positive cells are not missing but present in the GCL and INL as well as in the ONL. The majority of them are small in size, while others display a Müller glia-like morphology (arrowhead). Scale bar: 25 µm. (B) Retinal explants were prepared at E17.5, cultured for seven days, dissociated and subjected to immunocytochemistry (n=3). (a-d) In NeuroD+/– retina, some (2.7±0.3%) of the lacZ+ cells express the amacrine cell marker HPC1 (arrows). (e-h) In Math3–/–/NeuroD–/– retina, no lacZ+ cells express HPC1. (i-l) In NeuroD+/– retina, no lacZ+ cells express the ganglion cell marker Thy1.2. (m-p) In Math3–/–/NeuroD–/– retina, some (4.3±0.6%) of the lacZ+ cells express Thy1.2 (arrows). (q-t) In NeuroD+/– retina, no lacZ+ cells express the Müller glial marker vimentin. (u-x) In Math3–/–/NeuroD–/– retina, some (0.9±0.2%) of the lacZ+ cells express vimentin (arrow).

 


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  		Fig. 6. bHLH gene expression in the mutant retina. In situ hybridization was performed with E17.5 retina. (A,B) Math3 expression is not changed in NeuroD–/– retina. (C,D) NeuroD expression is not changed in Math3–/– retina. (E,F) Math5 expression is upregulated in Math3–/–/NeuroD–/– retina, suggesting that this upregulation leads to the increase of ganglion cells in the double-mutant retina. Scale bar: 15 µm.

 


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  		Fig. 7. Misexpression of Math3 and NeuroD with retrovirus. (A) Retroviral vectors. GFP, green fluorescent protein; IRES, internal ribosomal entry site; LTR, long terminal repeat. (B) Retinal explants were prepared from E17.5 mouse embryos and infected with CLIG, CLIG-Math3, and CLIG-NeuroD. After 2 weeks, the explants were subjected to immunohistochemistry using anti-GFP antibody. (C) Ratios of retinal cell types infected with CLIG, CLIG-Math3 and CLIG-NeuroD. Rod genesis is increased while gliogenesis is inhibited by Math3 and NeuroD.

 


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  		Fig. 8. Co-expression of Pax6 and bHLH genes. (A) Retroviral vectors. The bHLH genes (Math3, NeuroD and Mash1) were fused with GFP, and Pax6 has three repeats of Myc tag. (B-E) Retinal explants were infected with CLIG-Pax6 (B), CLIG-Pax6-Math3 (C), CLIG-Pax6-NeuroD (D) and CLIG-Pax6-Mash1 (E). After 2 weeks of culture, the explants were sectioned and subjected to immunohistochemistry for Myc only (a) or Myc and either calbindin (b), HPC1 (c), PKC (d) or GS (e). (B) Misexpression of Pax6 alone generates INL cells, but they are negative for the markers. (C) Co-expression of Math3 and Pax6 significantly increases the population of amacrine and horizontal cells (b,c, arrowheads) but not bipolar (d) or Müller glial cells (e). (D) Co-expression of NeuroD and Pax6 significantly increases the population of amacrine cells (b,c, arrowheads) but not horizontal (b), bipolar (d) or Müller glial cells (e). (E) Co-expression of Mash1 and Pax6 does not induce mature INL cells. Scale bar: 25 µm.

 


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  		Fig. 9. Ratios of retinal cell types induced by misexpression of bHLH genes and Pax6. Ratios with s.e. of amacrine cells (A, calbindin+), bipolar cells (B, PKC+), horizontal cells (C, calbindin+), rods (D, rhodopsin+) and Müller glial cells (E, GS+) are the average of at least three independent experiments. Co-expression of Pax6 and Math3 significantly increases the population of amacrine and horizontal cells while that of Pax6 and NeuroD only increases the population of amacrine cells. By contrast, co-expression of Pax6 and Mash1 does not increase the number of any mature cells.

 


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  		Fig. 10. Co-expression of Six3 and bHLH genes. (A) Schematic structure of retroviral vectors. The bHLH genes (Math3, NeuroD) were fused with GFP, and Six3 has three repeats of Myc tag. (B) Retinal explants were infected with CLIG-Six3 (a), CLIG-Six3-Math3 (b) and CLIG-Six3-NeuroD (c). After 2 weeks of culture, the explants were sectioned and subjected to immunohistochemistry for Myc and calbindin. Misexpression of Six3 alone generates some INL cells, but they are negative for calbindin expression (a). Co-expression of Six3 and Math3/NeuroD increases the population of calbindin-positive cells (b,c, arrowheads). Scale bar: 25 µm. (C) Ratios of amacrine (a) and horizontal cells (b). Co-expression of Six3 and NeuroD efficiently generates amacrine cells while co-expression of Six3 and Math3 efficiently generates horizontal cells.

 


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  		Fig. 11. Transcription factors for retinal cell fate specification. (A) Model for fate switches. The time course of ganglion, amacrine, bipolar and Müller glial cell genesis (Young, 1985Go) and the bHLH genes that regulate generation of these cells are shown. Because ganglion cell genesis is overlapped with amacrine cell genesis, the cells that are blocked from differentiation into ganglion cells may adopt the amacrine cell fate. By contrast, as amacrine cell genesis is overlapped with ganglion and Müller glial cell genesis, the cells that are blocked from differentiation into amacrine cells may adopt the ganglion and Müller glial cell fates. Because bipolar and Müller glial cells are the last cell types to be generated, the cells that are blocked from differentiation into bipolar cells may adopt Müller glial cell fate. (B) The transcription factor code for retinal cell fate specification. Proper combinations of bHLH and homeobox genes are important for retinal cell fate specification.

 

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