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First published online October 27, 2004
doi: 10.1242/10.1242/dev.01431

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Foxd1 is required for proper formation of the optic chiasm

Eloísa Herrera^1,*, Riva Marcus¹, Suzanne Li^2,, Scott E. Williams¹, Lynda Erskine³, Eseng Lai^2, and Carol Mason^1,

¹ Departments of Pathology, Anatomy and Cell Biology and Center for Neurobiology and Behavior, Columbia University, College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032, USA
² Cell Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA
³ Departments of Visual Science and Molecular Genetics, Institute of Ophthalmology, University College London, London EC1V 9EL, UK

View larger version (61K): [in a new window]   Fig. 1. The optic chiasm is perturbed in Foxd1 deficient embryos. (A) Anterograde DiI labeling in E12.5, E14.5 and E17.5 Foxd1+/+ (a,c,e) and Foxd1lacZ/lacZ (b,d,f) embryos reveals that the characteristic X-shape of the optic chiasm becomes elongated and splayed, and that the ipsi- to contralateral ratio of retinal axons increases. Contra, contralateral projection; ipsi, ipsilateral projection; ON, optic nerve; asterisk, midline. (B) P0 embryos lacking Foxd1 (Foxd1lacZ/lacZ, b-e) display four aberrant phenotypes; compare with Foxd1+/+ embryos (a,f). (b) Most retinal axons are arrested at the midline and end in `nodules'. (c) Retinal axons reach the midline in a more caudal position than in Foxd1+/+ littermates and project contra- or ipsilaterally. Axons are arrested as the optic nerves converge (asterisk), adding to the increased breadth of the chiasmatic crossing point; some axons are routed into the optic tracts and grow in an aberrant ipsi-versus contralateral ratio. (d) The position of the crossing site is shifted caudally and the RGC project ipsi- or contralaterally with a greatly increased uncrossed component. (e) Retinal axons take four different routes (arrows) after leaving the optic chiasm, forming two optic tracts on each side rather than one. (f,g) Diagrams summarizing the chiasm phenotypes in Foxd1+/+ and Foxd1lacZ/lacZ mice. Scale bars: 200 µm.

View larger version (57K): [in a new window]   Fig. 2. In Foxd1 (BF-2 in figure) deficient embryos, RGCs that project ipsilaterally arise from the entire retina, rather than exclusively from the ventrotemporal quadrant. (A) Schematic indicates that after retrograde labeling from one optic tract at E17.5, RGCs in Foxd1+/+ embryos are labeled over the entire contralateral retina (left), whereas in the ipsilateral eye, RGCs are labeled only in the peripheral ventrotemporal retina (right). (B) Wholemounts of retina after retrograde labeling from the contralateral optic tract at E17.5. (a) In Foxd1+/+ retina, contralateral-projecting RGCs come from all retinal quadrants except the VT crescent. (b) In Foxd1 deficient retina contralateral RGCs also arise from all the quadrants, including the VT crescent (asterisks). Overall, there are fewer contralateral labeled cells in Foxd1lacZ/lacZ retina than in Foxd1+/+ retina, probably because many axons stall at the optic chiasm. (C) Retinal wholemounts after retrograde labeling from the ipsilateral optic tract at E17.5. In Foxd1 deficient embryos (b), RGCs that project ipsilaterally arise from all retinal quadrants, not only from the VT crescent (a). Red lines in the contralateral retina delimit the VT crescent, where contralateral RGCs are not found in wild-type retina. Red lines in the ipsilateral retina mark the VT crescent, where ipisilateral RGCs are located. Note that both sectors are not equivalent in size; this is because at E17.5, when the backfill labeling is performed, both populations (ipsi- and contralaterally-projecting RGCs) partially intermingle, and the boundary segregating both populations is more peripheral at this time than at E14.5-E16.5. Scale bar: 500 µm.

View larger version (55K): [in a new window]   Fig. 3. Neurite growth from Foxd1 (BF-2 in figure) deficient VT explants is not inhibited by chiasm cells. (A) Retinal explants from E14.5 Foxd1+/+ (a,c) or Foxd1lacZ/lacZ (b,d) embryos co-cultured with dissociated chiasm cells from Foxd1+/+ (a, b) or Foxd1lacZ/lacZ (c,d) embryos. Drawings at the top indicate the two different combinations: (left) RGC axons from Foxd1+/+ VT but not DT retina are inhibited by Foxd1+/+ or Foxd1lacZ/lacZ chiasm cells, whereas (right) RGC axons from the VT retina of Foxd1lacZ/lacZ embryos are not inhibited by Foxd1+/+ or Foxd1lacZ/lacZ chiasm cells. (B) Quantification of mix-and-match experiments shows that RGC axons from Foxd1+/+ VT retina are inhibited by chiasm cells from either Foxd1lacZ/lacZ or Foxd1+/+ embryos. However, there is a loss of significant difference between VT and DT axon behaviors when retina from Foxd1lacZ/lacZ embryos are co-cultured with Foxd1+/+ or Foxd1lacZ/lacZ chiasm cells. **P<0.005. Scale bars: 500 µm.

View larger version (110K): [in a new window]   Fig. 4. Zic2 and EphB1 are not expressed in VT retina in Foxd1 (BF-2 in figure) deficient embryos. (A,B) Cryosections of E16.5 Foxd1+/+ (A) and Foxd1lacZ/lacZ (B) embryos showing similar Islet1/2 expression in both wild type and mutants. (C-F) Zic2, detected by anti-Zic2 antibodies, in frontal sections from wild-type and Foxd1lacZ/lacZ embryos. (C,D) At E14.5, Zic2 is not detected in Foxd1lacZ/lacZ VT retina (elongated arrows, D; compare wild type in C), but it is detected in the ciliary margin zone (short arrows). (E,F) At E16.5, Zic2 expression seen in wild-type VT retina (E) is absent in the mutant (F). (G,H) In situ hybridization for EphB1 in frontal cryosections of E15.5 Foxd1+/+ (G) and Foxd1lacZ/lacZ (H) embryos reveals that EphB1, like Zic2, is absent in embryos lacking Foxd1. (I,J) mRNA for Islet2 is similarly expressed Foxd1lacZ/lacZ and Foxd1+/+ retina. Scale bars: 100 µm.

View larger version (79K): [in a new window]   Fig. 5. Foxd1 (BF-2 in figure) is expressed in VT retina and its absence leads to expansion of the Foxg1 (BF-1) and ephrin-A domains. (A) X-gal staining (blue) of retinal wholemounts from Foxd1+/lacZ embryos at E14.5 (a) and at E16.5 (e) indicates that Foxd1 is highly expressed in VT retina at these ages. Flattened retinal wholemounts at E14.5 (b) and E16.5 (f) summarize the relationship between Foxd1 expression domain and Zic2-positive cells over the entire retina. Blue indicates Xgal-Foxd1 staining and dark brown speckles represent Zic2-positive cells. The cartoon shows that the borders of Zic2 and Foxd1 expression overlap, although the Foxd1 expression domain extends more centrally than that of Zic2. Moreover, some Zic2-positive cells are found outside the most ventral lateral border of the Foxd1 expression domain. Higher magnification views of the two boxed regions in b and f are shown in c and d, and g and h, respectively. These pictures show that most Zic2-positive cells (dark brown speckles) are located in the Foxd1-positive domain (blue area) in the VT retina (d,h). c and g show that the peripheral temporal borders of the Zic2 and Foxd1 domains overlap. Scale bars: 100 µm. (B) (a-f) In situ hybridization for Foxg1 in serial horizontal sections from the middle of the retina (a,d) and through the most ventral retina (c,f) in E15.5 Foxd1+/+ and Foxd1lacZ/lacZ embryos, indicating that Foxg1 expands into VT retina in the absence of Foxd1. Arrows in b-f indicate that Foxg1 is expressed in the ventrotemporal quandrant of Foxd1 deficient retina but not in normal retina. (g,h) Antibody fusion protein localization in horizontal sections of E12.5 embryos shows that in the absence of Foxd1, ephrin-A is also expressed in the temporal retina (h, arrow), which is in contrast to the normal high-nasal-low-temporal expression in wild-type embryos (g, arrow). Scale bars: 100 µm. (C) Schematic showing that Zic2 and EphB1, thought to regulate the uncrossed projection, are missing in the retina of Foxd1 deficient embryos, and that Foxg1 and ephrin-A proteins expand their territory into VT retina. Top, wild type; bottom, mutant.

View larger version (75K): [in a new window]   Fig. 6. Foxg1 (BF-1) expression is expanded, and Zic2 and Islet1 zones are reduced in the Foxd1 (BF-2)-deficient ventral diencephalon. (A) Top left panel is a dorsal view of an E15.5 embryo head with dashed red lines (1, 2) indicating the plane and level of section shown in panels a-h. Top right panel is a drawing of a sagittal section; red-dashed square indicates the area shown in panels a-h. (a-h) Sagittal cryosections of E15.5 Foxd1lacZ/lacZ (b,d,f,h) and Foxd1+/+ embryos (a,c,e,g), hybridized for Foxg1 (a-d), or immunostained (e-h) against Zic2 (green) and Islet1 (red). Dashed yellow lines indicate where the axons are located, in different positions in the Foxd1+/+ embryo compared with in the Foxd1 deficient embryo. The light blue-dashed lines represent the posterior boundary of Foxg1 domain in Foxd1+/+ embryos. Note that in the most lateral sections (level 1) of Foxd1 deficient embryos, the Foxg1 expression domain is expanded posteriorly behind the optic recess, the Zic2 domain is reduced, and Islet1 appears to be normal. In more medial sections (level 2), the Foxg1 expression domain is expanded caudally, there is no Zic2 expression, and Islet1 expression is restricted to the most posterior aspect of the chiasmatic region. Bottom panels, schematics comparing the expression patterns of Foxd1, Foxg1, Zic2 and Islet1 in Foxd1+/+ (left) and Foxd1lacZ/lacZ (right) embryos, as viewed in sagittal sections. OC, optic chiasm; OR, optic recess; SOA, supraoptic area; HYP, hypothalamus; III, third ventricle. Scale bars: 200 µm. (B) Top left panel is a lateral view of an E15.5 embryo head, and the red-dashed line indicates the plane and level of section showed in panels a-d. Top right panel is a drawing of a frontal section; red-dashed square indicates the area shown in panels a-d. (a,b) In situ hybridization for Foxg1 in frontal cryosections of E15.5 Foxd1lacZ/lacZ (b) and Foxd1+/+ (a) embryos. (c,d) Immunostaining against Zic2 (green) in the same sections as in a and b, respectively. Bottom panels are summary cartoons comparing the expression patterns of Foxd1, Foxg1, Zic2 and Islet1 in Foxd1+/+ (left) and Foxd1lacZ/lacZ (right) embryos, in frontal view. Note that Foxg1 expression is expanded ventrally and Zic2 expression is restricted to a few cells in the most lateral aspect of the chiasm; however, Zic2 expression is normal more dorsally. Scale bars: 200 µm.

View larger version (83K): [in a new window]   Fig. 7. Axon guidance factors are relatively unchanged in the optic chiasm of Foxd1 (BF-2) deficient embryos. (A) In situ hybridization for ephrin-B2 in frontal cryosections of E15.5 Foxd1+/+ (a) and Foxd1 mutant (b) embryos, revealing similar expression pattern in cell bodies at the floor of the third ventricle, thought to belong to the midline radial glia. (B) In situ hybridization for Slit2 in horizontal cryosections at E13.5 (a-d) and E15.5 (e-h). Comparison of Foxd1+/+ (left panels) and Foxd1lacZ/lacZ (right panels) chiasms at E13.5 show that although Slit2 expression appears normal in more dorsal sections (a,b), Slit2 levels are increased in the region ventrocaudal (c,d) to the chiasm in Foxd1 deficient embryos (red arrows, d). At E15.5, the same pattern is maintained (e-h). Green dashed lines indicate the position of retinal axons in each section. (C) Immunostaining with anti-CD44/SSEA antibodies (red) and anti-neurofilament (green) in horizontal cryosections at E13.5 (a,b) and E15.5 (c,d). Comparison of Foxd1+/+ (left panels) and Foxd1lacZ/lacZ (right panels) chiasms indicates that although CD44/SSEA expression appears normal in Foxd1 deficient embryos, retinal axons enter the CD44/SSEA-positive area (d), in contrast to the wild type in which axons never transgress the CD44/SSEA zone (c). Green indicates retinal axons labeled with neurofilament antibodies. Red arrows indicate that retinal axons invade the CD44/SSEA expression zone. Scale bars: 200 µm. (D) Schematic indicating that in the chiasm, the Foxg1 territory expands in the absence of Foxd1, as in retina, and retinal axons aberrantly course through the CD44/SSEA zone. Islet1 and Zic2 expression territories are reduced around the chiasmatic midline, and Slit2 expression is expanded ventrocaudally. Ephrin-B2 and CD44/SSEA expression appear unaltered in the Foxd1 deficient chiasm; however, in contrast to wild-type axons, Foxd1 deficient RGCs trespass the CD44/SSEA domain.

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