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First published online May 16, 2007
doi: 10.1242/10.1242/dev.002345

Development 134, 2137-2146 (2007)
Published by The Company of Biologists 2007
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Ena/VASP function in retinal axons is required for terminal arborization but not pathway navigation

Asha Dwivedy1, Frank B. Gertler2, Jeffrey Miller3, Christine E. Holt1 and Cecile Lebrand4,*

1 Department of Anatomy, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK.
2 Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
3 Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA.
4 Département de Biologie Cellulaire et de Morphologie, University of Lausanne, Rue de Bugnon, 9, 1005 Lausanne, Switzerland.


Figure 1
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  		Fig. 1. Sequestration of Xena after the expression of mitochondria-binding Ena/VASP proteins and its effects on growth cone morphology. (A-C) FP4-Mito-GFP (green, A) colocalized with the mitotracker (red, B) in retinal axons (arrowheads, C). (D-F) Xena-GFP (green, D) colocalized with FP4-Mito-RFP (red, E) in retinal cells co-expressing both constructs (F) in cryostat sections of stage 42 eyes. Arrowheads show the regions in the inserts. Inserts highlight the sequestration of Xena by the Mito construct. (G,H) In a stage 39 retinal explant, Xena-GFP was enriched along the retinal axon shaft and at the tips of growth cone filopodia (arrows). (H) Phase-contrast picture of G. (I) Phase-contrast picture of J-L. (J-L) Expression of the FP4-Mito construct (red, K) selectively depleted Xena-GFP (green, J) from sites of normal localization and sequestered it on the mitochondrial surface (L). (K) RFP-labeled mitochondria in a retinal axon expressing FP4-Mito-RFP. (M-O) Growth cone morphology of control GAP-GFP-labeled (M), control RFP-labeled (N) or FP4-Mito-GFP+RFP-labeled inactivated (O) retinal ganglion cells (RGCs) in explant culture. RGC, retinal ganglion cells. Scale bar: 56 µm in M for D-F; 8.5 µm in M for A-C,G-O.

 

Figure 2
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  		Fig. 2. Depletion of Xena/XVASP function affects growth cone dynamics in vivo. (A,B) In vivo time-lapse sequences over 1 hour (sequential pictures taken at 15-minute intervals) of growth cones growing along the optic tract of control neurons expressing GAP-GFP (A) or FP4-Mito-RFP+GAP-GFP-expressing neurons (B). Arrows point to filopodia and arrowheads to lamellipodia; dashed lines indicate growth cone progression between sequential pictures. (C,D) The number of filopodia (C) and the length of filopodia (D) of retinal axon growth cones expressing either GAP-GFP or FP4-Mito (mean±s.e.m. from a sample of 15 retinal axons). Measurements were performed along the visual pathway: from the ventral to the medial optic tract (VOT-MOT), from the medial to the dorsal optic tract (MOT-DOT), at the tectum border (DOT-TECT) and inside the tectum (TECT). *P<0.05; ***P <0.001, FP4-Mito-expressing growth cones compared with control growth cones in corresponding segments of the visual pathway. Scale bar: 22 µm in B for A,B.

 

Figure 3
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  		Fig. 3. Depletion of Xena/XVASP function interferes with the elongation but not with the guidance of retinal ganglion cell axons. Retinal ganglion cell (RGC) axon growth and guidance were analyzed in serial coronal cryostat sections at stage 39 (A-H) and stage 42 (J,K). RGCs expressed the control constructs GAP-GFP (A-D) or RFP (J), or the combined FP4-Mito-GFP+RFP constructs (E-H,K). (I) Drawing representing the stereotypical route followed by RGC axons during development and indicating the regions illustrated in A-H,J,K. (A-H) Arrowheads outline individual axons. (A,B) Axons of control RGCs exit the eye through the optic nerve head (onh; arrow in A). (B) Close-up of A. (C,D) The fibers navigate contralaterally across the optic chiasm, and cross the ventral (VOT), the medial (MOT) and the dorsal (DOT) optic tract to reach the tectum. (E-H) At stage 39, axons of RGCs expressing FP4-Mito initiate (arrowheads in E) and develop normally inside the optic nerve head (F and arrow in E), but only very few axons were detected at the level of the optic chiasm (G) and of the optic tract (H). (J,K) At stage 42, as in controls, the majority of RGC axon terminals of neurons expressing FP4-Mito had reached the tectum. on, optic nerve; onh, optic nerve head; RGC, retinal ganglion cell; DOT/MOT/VOT, dorsal/medial/ventral optic tract, respectively. Scale bar: 120 µm in A,D,E,J,K; 60 µm in B,C; 40 µm in F-H.

 

Figure 4
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  		Fig. 4. Function of the Xena/XVASP proteins is not required for axon pathfinding in the optic tract. (A-E) Lateral views of stage 39 whole-mount brains illustrating the axons of control GAP-GFP-expressing (A,B) and FP4-Mito-RFP+GAP-GFP-expressing (C-E) RGC neurons. The solid line approximates the location of the rostral border of the tectum. (A,B) Control axons labeled with GAP-GFP grew in the medial and dorsal optic tracts (MOT and DOT, respectively), turned caudally in the mid-diencephalon (Di) and penetrated into the tectum (Tect), where they arborized. (C) Axons of neurons expressing FP4-Mito navigated correctly. However, only very few retinal axons with impaired function of the Xena/XVASP proteins were detected in the optic tract or in the tectum. (D,E) We confirmed that the GAP-GFP axons in D expressed the Mito construct by detecting the presence of FP4-Mito-RFP on the mitochondria (E; arrows). CH, optic chiasm; Di; mid-diencephalon; DOT/MOT/VOT, dorsal/medial/ventral optic tract, respectively; Tect, tectum; Tel, telencephalon. Scale bar: 84 µm in A; 42 µm in A for B,C; 30 µm in A for D-E.

 

Figure 5
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  		Fig. 5. Retinal ganglion cell axonal outgrowth is slower after the depletion of the Xena/XVASP proteins. (A) The percentage of retinal axons, lipofected with control RFP or with FP4-Mito-GFP+RFP, with terminals in the optic nerve head, the brain entry point, the optic chiasm or the optic tract and tectum at stage 39 (left) and with terminals in the optic tract and tectum at stage 42 (right) [mean±s.e.m. from samples of 120 retinal ganglion cells (RGCs)]. ***P<0.001, FP4-Mito-expressing growth cones compared with control growth cones in the corresponding region. (B) The average rate of elongation, basal rate of extension, frequency and duration of growth cone pauses from the ventral to the dorsal optic tract (VOT-DOT), as well as at the tectum border, are shown (mean±s.e.m. from samples of 15 RGCs). *P<0.05, ***P<0.001; FP4-Mito-expressing growth cones compared with control growth cones in similar portions of the optic tract. °P<0.05, °°P<0.01, °°°P<0.001, compared axons in the optic tract and at the tectum border. OT, optic tract; RGCs, retinal ganglion cells.

 

Figure 6
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  		Fig. 6. Retinal axons with impaired activity of the Xena/XVASP proteins generated a limited number of filopodia-like processes and only rarely a few branches. (A,B) In vivo time-lapse sequences of terminal arborizations of control GAP-GFP-expressing (A) and of FP4-Mito-RFP+GAP-GFP-expressing (B) retinal ganglion cell (RGC) neurons in the tectum. Growth cones were photographed every hour during the 3 hours following the entry of retinal axons in the tectum. White arrowheads point to growth cone filopodia and black arrowheads to branches. (C,D) Diagrams of control GAP-GFP-expressing (C) and FP4-Mito-RFP+GAP-GFP-expressing (D) axonal terminal arborizations. Primary (blue), secondary (green) and tertiary (red) branch segments are illustrated. Scale bar: 32 µm in B for A,B.

 

Figure 7
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  		Fig. 7. Activity of the Xena/XVASP proteins controls the formation of branches at retinal axon terminals. Branching parameters in control GAP-GFP-expressing and FP4-Mito-RFP+GAP-GFP-expressing neurons. The total number of branches, the total number of nodes, the number of primary and secondary branches, the length of primary branch segments, and the length of the total arborization are shown at the time of tectum entry (t0h), or at 1, 2 and 3 hours (t1h, t2h and t3h, respectively) after the retinal ganglion cell (RGC) axons have invaded the tectum (mean±s.e.m. from a sample of 20 retinal axons). *P<0.05, **P<0.01 and ***P<0.001, FP4-Mito-expressing axons compared with corresponding control axons. °P<0.05 and °°P<0.01, axons at 1, 2 and 3 hours compared with axons at 0 hours.

 

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© The Company of Biologists Ltd 2007