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First published online 12 November 2008
doi: 10.1242/dev.023572

Development 135, 4081-4089 (2008)
Published by The Company of Biologists 2008
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Foxg1 regulates retinal axon pathfinding by repressing an ipsilateral program in nasal retina and by causing optic chiasm cells to exert a net axonal growth-promoting activity

Natasha M. Tian, Thomas Pratt and David J. Price*

Centre for Integrative Physiology, The University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, UK.


Figure 1
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  		Fig. 1. Transcriptional activation of Foxg1 in the retina at E14.5. X-gal staining (blue) of Foxg1LacZ/+ embryos is used to show where Foxg1 is active. (A) Schematic of a mouse embryo head showing the horizontal plane of section; broken lines indicate the limits between which sections were taken. (B) Schematic of retina showing the locations of sections in E-K; blue circles indicate Foxg1-expressing RGCs. (C) Diagram of a flat-mounted retina, with blue shading indicating the area of transcriptional activation of Foxg1; boxed areas indicate regions from which DN (Foxg1-positive) and VT (Foxg1-negative) retinal explants were prepared for the co-culture experiments. (D) A Foxg1LacZ/+ embryo showing X-gal staining mainly in nasal retinae. (E-K) Dorsal to ventral series of sections (locations marked in B) through a retina shown in D counterstained with Nuclear Fast Red (pink). (E) At the dorsal pole, X-gal staining is found throughout all layers of nasal and temporal retina. (F-H) Moving from dorsal to central sections, X-gal staining is present throughout nasal retina and occupies progressively less of temporal retina. (I-K) Moving through ventral sections, the X-gal-stained part of nasal retina becomes smaller and increasingly restricted to the anterior-most region of nasal retina until it disappears in the most ventral section. Scale bars: 500 µm in D; 200 µm in E-K. Abbreviations: D, dorsal; N, nasal; T, temporal; V, ventral; l, lens; rpe, retinal pigment epithelium. Broken lines in E-K indicate nasal-temporal boundary.

 

Figure 2
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  		Fig. 2. Zic2 is expressed ectopically in nasal retina of Foxg1-/- embryos. Zic2 (green) and Brn3a (red) immunohistochemistry in (A-H) wild-type and (I-P) Foxg1-/- retinas at E14.5. (E-H) Higher magnifications of boxed areas in A-D, respectively. (M-P) Higher magnifications of boxed areas in I-L, respectively. (A,B,E,F) In wild-type dorsal retina, there are few Zic2-expressing cells in the inner retinal (RGC) layer; some are seen in other layers and there is strong expression in CMZ (marked in F), as reported previously (Herrera et al., 2003Go). (C,D,G,H), Zic2-expressing inner retinal layer cells are found predominantly in VT retina clustered adjacent to the CMZ (in boxed area in C). (I,J,M,N) In Foxg1-/- dorsal retina; numerous Zic2-expressing cells are seen nasally, adjacent to the CMZ in the inner retinal layer (boxed area in J). (K,L,O,P) In Foxg1-/- ventral retina; Zic2-expressing inner retinal layer cells are seen both nasally and temporally (boxed areas in K and L). Abbreviations: N, nasal; T, temporal; cmz, ciliary marginal zone. Scale bars: 200 µm in A-D,I-L; 100 µm in E-H,M-P.

 

Figure 3
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  		Fig. 3. Increased proportion of Zic2-expressing cells in nasal retina of Foxg1-/- embryos at E14.5 and E16.5. (A) For each retina, numbers of nasal and temporal Zic2-positive and Brn3a-positive cells were counted in six sections spaced at 80-100 µm intervals through the retina from dorsal (blue horizontal lines) to ventral (green horizontal lines). (B,C) Means (±s.e.m.) are counts of Zic2-positive cells expressed as a proportion of numbers of Brn3a-positive cells in the four retinal quadrants. At E14.5 and E16.5, Foxg1-/- nasal quadrants showed large significant increases in the proportion of Zic2-positive cells compared with equivalent wild-type quadrants. No significant differences were found between proportions of Zic2-positive cells in Foxg1-/- and wild-type VT retina at both ages. Brackets indicate significant differences, with P values indicated above each bracket. Numbers of retinae: Foxg1+/+, n=4; Foxg1-/-, n=3. Abbreviations: DN, dorsonasal; VN, ventronasal; DT, dorsotemporal; VT, ventrotemporal.

 

Figure 4
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  		Fig. 4. Ephb1 and Foxd1 are expressed ectopically in the nasal retina of Foxg1-/- embryos. (A-H) Ephb1 in situ hybridization in (A-C) wild-type and (D-H) Foxg1-/- retinas at E16.5; C,G,H show higher magnifications of boxed areas in B,E,F, respectively. In wild-type and in Foxg1-/- retinas, Ephb1 is expressed in peripheral VT RGCs (B,C,F,H). In Foxg1-/- retinas, Ephb1 is expressed ectopically in the nasal retina in (D) dorsal, (E,G) medial and (F,H) ventral sections (indicated by arrows in D, F and boxed area in E). (I-N) Foxd1 in situ hybridization in (I-K) wild-type and (L-N) Foxg1-/- retinas at E16.5. In Foxg1-/- retinas, Foxd1 is expressed ectopically in DN retina. Temporal is towards the top of all panels. Scale bars: 200 µm in A,B,D-F; 100 µm in C,G,H; 400 µm in I-N.

 

Figure 5
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  		Fig. 5. Foxg1 is not required for retinal outgrowth in culture. (A) DN Foxg1+/±, (B) VT Foxg1+/±, (C) DN Foxg1-/- and (D) VT Foxg1-/- retinal explants cultured in collagen without chiasm cells. (A-D) Neurofilament (NF) immunohistochemistry revealed neurite outgrowth from all explants. (A'-D') Brn3a expression in co-cultures A-D respectively revealed healthy RGCs. (E) Mean percentage axon coverage (±s.e.m.) for Foxg1+/± and Foxg1-/- retinal explants from DN and VT retina 45 µm from the retinal explant (measured as in Fig. S2 in the supplementary material). (F) Mean length of the longest neurites (±s.e.m.) for each set of explants. (E,F) Numbers of explants are indicated in parentheses. (E) One-way ANOVA showed a significant effect of explant type; significant differences are marked by brackets with P values indicated. (F) One-way ANOVA revealed no significant differences in the lengths of outgrowth among retinal explants. Abbreviations: DN, dorsonasal; VT, ventrotemporal. Scale bars: 100 µm in A-D; 10 µm in A'-D'.

 

Figure 6
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  		Fig. 6. Loss of Foxg1 from retina or chiasm impairs outgrowth of dorsonasal retinal axons on chiasm cells in culture. (A-D) Co-cultures of Foxg1+/± or Foxg1-/- dorsonasal (DN) retinal explants with dissociated Foxg1+/± or Foxg1-/- chiasm cells; immunohistochemistry is for the axonal marker neurofilament (NF; green) and the RGC marker Brn3a (red); the nuclear counterstain TO-PRO-3 (blue) reveals cells in the retinal explant and surrounding dissociated chiasm cells. Cultures in A and C are shown at half the magnification of those in B and D. (A'-D') Brn3a expression in co-cultures A-D, respectively. (E) Mean percentage axon coverage (±s.e.m.) 45 µm from the retinal explant, showing significant differences among the four combinations. (F) Mean lengths of the five longest neurites (± s.e.m.). (E,F) Numbers of explants are in parentheses. One-way ANOVA revealed significant differences in outgrowth among retinal explants. Significant differences with P values are indicated above each bracket. Scale bars: 200 µm in A,C; 100 µm in B,D; 10 µm in A'-D'.

 

Figure 7
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  		Fig. 7. Loss of Foxg1 from retina or chiasm has no significant effect on outgrowth of ventrotemporal retinal axons on chiasm cells in culture. (A-D) Co-cultures of Foxg1+/± or Foxg1-/- ventrotemporal (VT) retinal explants with dissociated Foxg1+/± or Foxg1-/- chiasm cells; immunohistochemistry and counterstaining are as in Fig. 6. (A'-D') Brn3a expression in co-cultures A-D, respectively. (E,F) Mean percentage of axon coverage (±s.e.m.) 45 µm from the retinal explant (E) and mean lengths of the five longest neurites (±s.e.m.) (F). One-way ANOVA revealed no significant differences in outgrowth among retinal explants. (E,F) Numbers of explants are in parentheses. Scale bars: 100 µm in A-D; 10 µm in A'-D'.

 

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