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First published online 7 July 2004
doi: 10.1242/dev.01246

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The winged helix transcription factor Foxg1 facilitates retinal ganglion cell axon crossing of the ventral midline in the mouse

Genes and Development Group, Biomedical Sciences, George Square, The University of Edinburgh, Edinburgh EH8 9XD, UK

View larger version (73K): [in a new window]   Fig. 1. Transcriptional activation of the Foxg1 gene in tissues involved in RGC axon guidance in E14.5 embryos with and without Foxg1. Foxg1lacZ/+ embryos have one functional copy of Foxg1 and are phenotypically normal. Foxg1lacZ/Cre embryos are unable to produce Foxg1 protein. The Foxg1lacZ allele reports transcriptional activation of the Foxg1 gene by expression of lacZ protein. (A,F,I,N) X-gal stained brains. (A,F) Foxg1lacZ/+ brain viewed (A) dorsolaterally to show staining of the superior colliculus (sc), dorsal midline and telencephalon (tel). (F) Ventral view shows staining in the anterior part of the ventral hypothalamus (vh; circled in red). (I,N) Foxg1lacZ/Cre brain showing (I) staining in the superior colliculus, along the dorsal midline and in the hypoplastic telencephalon (tel*). (N) A ventral view shows that, as in Foxg1lacZ/+ embryos, staining is restricted to anterior ventral hypothalamus. The yellow oval encloses the mutant elongated retina (er), which has remained attached to the brain. The retinal pigment epithelium (rpe) has been removed to show that lacZ staining is far stronger in the anterior (nasal) retina. (B-E,G,H,J-M,O,P) X-gal stained cryostat sections with nuclei stained red. (B-E,J-M) Sections through the eye. Both (B-E) Foxg1lacZ/+ and (J-M) Foxg1lacZ/Cre retinas exhibit predominantly nasal (n) lacZ staining, although in both cases a few stained cells are also detected in temporal (t) retina. J is dorsal to K. (K) Nasal staining in the medial elongation of the mutant retina. B and J are dorsal to D and K, respectively. (D,E,L,M) Higher magnifications of Foxg1lacZ/+ and Foxg1lacZ/Cre (D,L) temporal and (E,M) nasal retina, showing pronounced staining of the nasal RGC layer. (G,H,O,P) Sections through the ventral hypothalamus with paired arrows demarcating its midline. A population of midline cells overlying bundled axons at the optic chiasm (oc) stains strongly in both (G) Foxg1lacZ/+ and (O) Foxg1lacZ/Cre embryos. This dense midline staining is absent more posteriorly in (H) Foxg1lacZ/+ and (P) Foxg1lacZ/Cre embryos. (Q) The relationship between Foxg1 expression and the retinofugal tract. Regions expressing Foxg1 are shaded blue. Ovals represent cell bodies. The retinofugal tract comprises RGCs (red) projecting axons (black lines) towards the optic chasm and into the brain. l, lens; ot, optic tract; r, retina; avh, anterior ventral hypothalamus; on, optic nerve. Wholemounts (A,F,I,N) are shown with anterior facing downwards. Cryostat sections (B-D,J-L) are horizontal with anterior (nasal) at the bottom; (E,G,H,M,O,P) coronal sections. Scale bars: 200 µm in B,C,J,K; 50 µm in D,EG,H,L,M,O,P.

View larger version (61K): [in a new window]   Fig. 4. Mapping ipsilateral and contralateral projecting RGCs in Foxg1+/+ and Foxg1Cre/Cre embryos by retrograde tract tracing from the optic tract growing over the thalamus at E15.5. (A-H) Wild type. (A,B,G) DiI and DiA double-label experiment showing predominance of contralateral (con) projections (A,B) at the optic chiasm (oc) and (G) in the retina; an ipsilaterally (ips) labelled cell (red) is marked with arrow in G. (C-F,H) Single DiI-labelling experiments. (C,E) At the optic chiasm, the majority of labelling is in the contralateral optic nerve (on). (D,F) In the contralateral retina (F) a large number of labelled cells are distributed both nasally and temporally. (D) In the ipsilateral retina, far fewer cells are labelled. (I-O) Mutants. (I,M,N) Double-label experiment showing (I) the optic chiasm and (M,N) ipsilaterally (red) and contralaterally (green) projecting RGCs in the retina (note the absence of double labelled, yellow, RGCs). (J-L,O) Single label experiment showing increased ipsilateral labelling (compared with wild type) in (K,K') both nasal (n) and temporal (t) parts of the elongated retina (er) and (J-L) their axons in the mutant correlate of the optic nerve (on*). (H,O) Higher magnification showing the disposition of RGCs in (H) wild-type and (O) mutant retina. All sections are horizontal with anterior (nasal) at the bottom. Each asterisk indicates which optic tract was injected with DiI (red) and DiA (green). Broken lines indicate the ventral midline. Scale bars: 500 µm in A; 100 µm in B,C,G,I,J,M; 200 µm in D F,K,L; 50 µm in H,N,O.

View larger version (74K): [in a new window]   Fig. 3. RGC axon navigation in wild-type and Foxg1-/- embryos visualised by anterograde tract tracing at (A-E) E13.5 and (F-L) E15.5. For each age, progressively more caudal coronal sections of an individual embryo show injections of DiI (red asterisks) (A,F) in wild-type eye (e) and (D,I) in mutant eye; (B,G,D,I) labelling of optic nerve (on) and optic chiasm (oc); and (C,H,E,J) labelling of optic tracts (ot). (C,H) In the wild-type optic tracts, contralateral labelling is stronger than the ipsilateral labelling (white arrows). (E,J) In the mutant optic tracts, ipsilateral labelling appears stronger (white arrows). (K,L) Horizontal sections of embryos in which DiI was injected into one eye and DiA into the other eye of (K) an E15.5 wild-type and (L) an E15.5 Foxg1-/- embryo (ventral midline is marked by broken white line). (K') Horizontal sections of the wild-type optic tract, growing over the diencephalon, from the same embryo as in K showing that each optic tract is predominantly labelled with dye from the contralateral (con) eye, although some ipsilateral (ips) label is also detectable (appears yellow where dyes overlap). (L') Horizontal sections of the mutant optic tract from the same embryo as in L, showing abnormally high levels of red label from the ipsilateral eye. Scale bars: 500 µm in A-E; 650 µm in F-J; 100 µm in K,K',L,L'.

View larger version (50K): [in a new window]   Fig. 2. Lineage tracing of Foxg1-expressing cells in E14.5 Foxg1Cre/+; R26RS embryos. (A) Thick horizontal section of X-gal-stained facial whole mount (the eye on the right is sectioned more ventrally). lacZ staining is uniformly intense throughout the lens (l) and nasal (n) retina but appears patchy in the temporal (t) retina. (B,C) Thick horizontal sections of the eye. (B) Medial section; (C) ventral retina. (D) Thin horizontal section through the eye showing the distribution of lacZ-stained cells. Broken lines demarcate the position occupied by nasal RGCs (nRGCs) and temporal RGCs (tRGCs). In the nasal retina, lacZ staining is uniformly strong. In the temporal retina radial columns of lacZ-expressing cells are embedded in larger areas of non-expressing cells. In B and D, these columns are viewed side-on and in C they are viewed from above. Arrows in B-D indicate examples of lacZ-stained radial stripes. on, optic nerve. Scale bars: 500 µm in A; 200 µm in B-D.

View larger version (85K): [in a new window]   Fig. 5. The distributions of Ephb2 and ephrin B2 proteins in the developing retina and its axons are not dramatically altered in the Foxg1-/- mutant. Ephrin B2 protein forms a dorsolateral[High] to ventrolateral[Low] gradient in the developing retina at E13.5 in (A) the wild-type and (C) the mutant. This gradient remains at E16.5 in both (B) wild-type and (D) mutant. Ephb2 protein forms a complementary ventrolateral[High] to dorsolateral[Low] gradient in the developing retina at E13.5 in both (E) wild-type and (G) mutant retina. At E16.5, these gradients are shallower than at E13.5 in both (F) wild type and (H) mutant. The optic nerve (on) stains strongly for Ephb2 protein consistent with Ephb2 expression by navigating RGC axons. (I,J) In the wild type, Ephb2 is present in the optic nerve as it leaves the retina (r) and approaches the optic chiasm (oc) to emerge into the optic tract (ot) (boxed area in I). (K,L) In the mutant, Ephb2 is expressed in the elongated retina (er), at the optic chiasm and in the mutant optic tract as it emerges from the chiasm (boxed area in K). (A-H) Coronal sections (dorsal is towards the top); (I-L) horizontal sections (posterior is towards the top). Scale bars: 100 µm in A-H; 200 µm I-L.

View larger version (83K): [in a new window]   Fig. 7. Patterning of the chiasm at E13.5. Sections are horizontal, anterior is upwards. Immunohistochemistry was performed using antibodies for SSEA-1 (A-F) and Nkx2.2 (G,H). (A) Wild-type section through the optic chiasm showing SSEA-1-positive neurons arrayed in an inverted `V'-shape. Arrow indicates the tip of the `V'. SSEA-1 expression is also evident in the optic nerves (on). (B) A Foxg1-/- section equivalent to (A) showing SSEA-1 expression in the mutant correlate of the optic nerve (on*). Note that the plane of section is slightly tilted such that the left side is ventral to the right. The SSEA-1-positive `arm' of the `V' is visible on the right side, but is less clear on the left, which is further ventral. (C,D) Immediately dorsal to the optic chiasm, wild-type and Foxg1-/- embryos show bilaterally symmetrical SSEA-1 expression posterior to the third ventricle. SSEA-1 expression extends posteriorly and laterally in both C and D. (E,F) High magnification showing the morphology of SSEA-1 neurons in the wild type and Foxg1-/- mutant. (G,H) In the wild type and Foxg1-/- mutant, Nkx2.2 is expressed at the posterior optic chiasm, where it overlaps partly with SSEA-1 expression. Broken lines indicate the position of the midline. Scale bar: 200 µm in A,B; 100 µm in C,D,G,H; 50 µm in E,F.

View larger version (51K): [in a new window]   Fig. 6. Ephrin B2 protein is expressed at the ventral hypothalamic midline in both the Foxg1-/- mutant and the wild type. (A,B,E,F) Wild type. (A,B) At E13.5, ephrin B2 expression is most widespread in the more dorsal structures in the developing forebrain and eye (e). Ventrally ephrin B2 is detected at the ventral hypothalamic (ht) midline (B shows area boxed in A at higher power). (E,F) At E16.5, ephrin B2 protein is detected at the optic chiasm (oc) where expressing cells contact the optic nerve (on). (F) Higher magnification of the optic chiasm. (C,D,G,H) Mutant. (C,D) At E13.5, ephrin B2 is expressed in the hypoplastic telencephalon (tel*) and in the developing eye (see Fig. 5). Ephrin B2 is also detected at the hypothalamic midline (D shows area boxed in C at higher power). (G,H) At E16.5 ephrin B2 expression persists at the mutant optic chiasm where it contacts Ephb2-expressing axons (section in H is adjacent to the Ephb2 stained section shown in Fig. 5L). Paired arrows indicate the ventral hypothalamic midline. (A-D,F,G) Coronal sections with dorsal towards the top. (E,H) Horizontal sections with posterior towards the top. cc, cerebral cortex; vtel, ventral telencephalon. Scale bars: 200 µm in A,C,E; 50 µm in B,D,F-H.

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