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First published online 21 April 2004
doi: 10.1242/dev.01099

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The homeobox gene Xbh1 cooperates with proneural genes to specify ganglion cell fate within the Xenopus neural retina

¹ Dipartimento di Fisiologia e Biochimica, Laboratorio di Biologia Cellulare e dello Sviluppo, Università di Pisa, 56010 Ghezzano, Pisa, Italy
² Developmental Biology Programme, EMBL Heidelberg, Meyerhofstrasse 1, 69012 Heidelberg, Germany
³ Centro di Eccellenza AmbiSEN, Università di Pisa, Pisa, Italy

View larger version (94K): [in a new window]   Fig. 1. (A-L) Xbh1 expression during retinal development. In situ hybridization with either a Xbh1 (A-H) or a Xath5 (H-L) probe on Xenopus embryos at stage 28 (A,E,I), 33 (B,F,J), 38 (C,G,K) and 42 (D,H,L), respectively. (A-D) Lateral views of embryos after whole-mount in situ hybridization (anterior is on the left). (E-L) In situ hybridization on transversal retinal sections. The red hatch (G) delimitates the ciliary marginal zone (CMZ) and the three main retinal layers (ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer). (H) Cross section of a retina from a stage 42 embryo analyzed by double in situ hybridization, showing Xbh1 (purple) and Xath5 (red) expression. (L) The same section as in H showing Xath5 expression alone. The yellow bracket indicates the region where Xath5 and Xbh1 expression overlaps in the CMZ; the yellow arrow indicates co-expression of Xbh1 and Xath5 in INL cells. (M-O) High magnification of CMZ following double in situ hybridization shows expression of Xbh1 (M), Xath5 (N), or both (O). (P,Q) Combined BrdU staining (green) and in situ hybridization (blue) performed on adjacent sections of a stage 42 retina with a Xbh1 (P) or a Xath5 probe (Q). Arrowheads show two identical nuclei stained on adjacent sections.

View larger version (25K): [in a new window]   Fig. 2. (A) Representation of the domain of Xbh1 expression in comparison with those of other genes used as morphogenetic landmarks of different regions of the CMZ and central retina. (B) Xbh1 is positively regulated by Xath3 and Xath5, but not by XneuroD. RT-PCR was performed on animal caps injected with 1 ng of either Xath3 or Xath5 mRNA, or with 500 pg of XneuroD mRNA. Uninjected animal caps (uninj. caps) and stage 28 whole embryos (WE st. 28) were used as negative and positive controls, respectively; Xbrn3.0 and Xbrn3d were used as positive control markers. (C) Xbh1 mRNA injection (1 ng) is able to activate Xbrn3 genes and Xath3, but not Xath5.

View larger version (62K): [in a new window]   Fig. 3. (A-D) Xbh1 promotes ganglion cell differentiation in lipofection experiments. Retinal precursors lipofected with GFP+vector DNA alone (B), or with GFP+Xbh1 (C,D). Retinal layers are separated by dashed lines (B,C) and are indicated in B as follows: GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; l, lens. (A) Distribution of retinal cell types in lipofected clones. Ph, photoreceptors; H, horizontal; B, bipolars; Am, amacrine; G, ganglion; Mü, Müller. The proportion of each cell type is represented as average±s.e.m. The experiment has been repeated at least three times for both GFP and Xbh1. In the experiment represented here, n=3907 cells from 26 retinae for GFP, and n=2799 cells from 25 retinae for Xbh1. Asterisks represent significant differences between Xbh1 and GFP, as calculated by Student's t-test (*P<0.05, **P<0.01, ***P<0.001). (D) Lower magnification image of section shown in C; GFP-positive axons extend into the optic nerve (arrows).

View larger version (57K): [in a new window]   Fig. 4. Xbh1 enhances the RGC-inducing ability of atonal-related genes. (A,B) Retinas lipofected with Xath3+GFP or Xath3+Xbh1+GFP. (C,D) Percentage of RGCs (C) or photoreceptors (D) in retinae lipofected with GFP+pCS2 vector, or with GFP in combination with Xbh1, Xath5, Xath5+Xbh1, Xath3, or Xath3+Xbh1. The percent representation of each cell type was calculated as the average±s.e.m. Counted cells were: n=2625 cells from six retinae for GFP; n=2908 cells from six retinae for Xbh1; n=1087 cells from 10 retinae for Xath5; n=1375 cells from 11 retinae for Xath5+Xbh1; n=2018 from seven retinae for Xath3; and n=1406 from eight retinae for Xath3+Xbh1. Significant differences have been determined by one-way analysis of variance (ANOVA) with the Tukey-Kramer Multiple Comparisons Test as a post-test. Black asterisks represent significant differences (**P<0.01, ***P<0.001).

View larger version (55K): [in a new window]   Fig. 6. (A-D) Co-lipofection of Xath5 with Xbh1 and Xbh1Vp16 constructs. Xath5 was lipofected either with GFP+pCS2 (A), or with GFP+Xbh1 (B) or GFP+Xbh1Vp16 (C), and their effect analyzed on cell-type frequencies in transfected clones (D). Counted cells were: n=2625 cells from six retinae for GFP; n=2908 cells from six retinae for Xbh1; n=3707 cells from 19 retinae for Xbh1Vp16; n=885 from eight retina for Xath5; n=1368 cells from eight retinae for Xath5+Xbh1; n=1081 from five retinae for Xath5+Xbh1Vp16. Error bars represent s.e.m.; significant differences have been calculated by one-way analysis of variance with the Tukey-Kramer Multiple Comparisons Test as a post test (***P<0.001). Symbols are as in Fig. 3. (E) RT-PCR analysis of animal caps injected with different combinations of mRNA, as indicated. Caps injected with either Xath5 (500 pg) or Xbh1 (500 pg) show activation of Xbrn3d. By contrast, animal caps injected with Xath5 (500 pg)+Xbh1Vp16 (500 pg), or with Xbh1Vp16 (500 pg) alone show no activation of Xbrn3d expression.

View larger version (46K): [in a new window]   Fig. 5. (A) Comparison of the ability of Xbh1, Xbh1Vp16 and Xbh1EngR mRNA to activate the ganglion cell marker Xbrn3d in animal caps. RT-PCR analysis on animal caps injected with RNA from different Xbh1 wild-type and fusion constructs, as indicated. Injected amounts of RNA were 500 pg for each construct, either alone or in combination. (B-E) Lipofection experiments with Xbh1, Xbh1EngR and Xbh1Vp16 constructs. Embryos were lipofected with GFP+pCS2 vector (C), GFP+Xbh1 (D), GFP+Xbh1Vp16 (E) or GFP+Xbh1EngR (not shown), and retinae were analyzed at stage 42. The percent representation of each cell type was calculated as a weighted average±s.e.m. Counted cells were: n=2625 cells from six retinae for GFP; n=2908 cells from six retinae for Xbh1; n=2382 from 15 retinae for Xbh1EngR; and n=3707 cells from 19 retinae for Xbh1Vp16. Asterisks represent significant differences as determined by one-way analysis of variance with the Tukey-Kramer Multiple Comparisons Test as a post-test (*P<0.05, ***P<0.001).

View larger version (73K): [in a new window]   Fig. 7. (A) Lipofection of Xbh1 at late stages promotes ganglion cell fate. Counted cells were: n=401 cells from 23 retinae for GFP (orange) (sample section shown in B); n=564 cells from 36 retinae for Xbh1 (green) (C). The percent representation of each cell type was calculated as a weighted average±s.e.m. Asterisks represent significant differences between Xbh1 and GFP, as calculated by Student's t-test (**P<0.01).