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doi: 10.1242/10.1242/dev.00299

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Invariant Sema5A inhibition serves an ensheathing function during optic nerve development

Departments of Ophthalmology and Physiology, Medical Scientist Training Program, Biomedical Sciences Program, Neuroscience Program, University of California San Francisco, CA94143, USA

View larger version (23K): [in a new window]   Fig. 4. Sema5A inhibition of post-optic disc retinal axons. (A) Diagram of the retina-optic nerve preparation. (B) Retinal axons and growth cones extending from the cut optic nerve of a retina-optic nerve preparation (boxed region in A), stained with TexasRed-Phalloidin. (C) The response rate of growth cones from retina-optic nerve preparations and standard retinal explants to oligomerized ECD-Fc. Growth cone responses were observed with time-lapse microscopy. Scale bar, (B) 50 µm.

View larger version (89K): [in a new window]   Fig. 1. Expression of Sema5A mRNA. (A) Sema5A in situ hybridization in the E14 mouse retinal pathway. Sema5A was expressed at the optic disc and along the developing optic nerve to the ventral diencephalon. The boxed areas are shown at higher magnification in B and E. (B) Sema5A mRNA expression at the optic disc. Hybridization signal using an antisense probe was present in neuroepithelial cells that flank the exiting retinal axons (*). As a group, these Sema5A-positive neuroepithelial cells formed a collar around the retinal axon bundle. (C) Hybridization at the optic disc using the Sema5A sense probe. (D) Hybridization at the optic disc using the Sema5B probe showed no signal. (E) Sema5A mRNA expression in the developing optic nerve. Note the presence of hybridization signal in neuroepithelial cells flanking the central core containing retinal axons. (F) Hybridization in the optic nerve using the Sema5A sense probe. (G) Sema5A mRNA expression in the lamina propria (LP) under the olfactory epithelium (OE). Scale bars: (A) 150 µm; (B-F) 25 µm; (G) 75 µm.

View larger version (76K): [in a new window]   Fig. 2. Recombinant Sema5A proteins inhibit RGC axons on a laminin substratum. (A) Domain structure of Sema5A, and diagrams of three Sema5A extracellular domain constructs. (B) Silver staining of the three Sema5A recombinant proteins (ECD-Fc, sema-Fc, TSP-Fc). (C) The percentage of growth cones on a laminin substratum exhibiting collapse in response to increasing amounts of oligomerized ECD-Fc. (D,E) Examples of growth cone responses to a substratum choice assay. (D) Retinal axons grew freely into regions containing heat-inactivated ECD-Fc (*), but (E) avoided entering regions containing active ECD-Fc (*). (F) The response rate of growth cones encountering a border of either heat-inactivated ECD-Fc or ECD-Fc. (G,H) Composite images showing the pattern and density of axon outgrowth for 25 explants grown on a laminin substratum in the presence of parental 293 cells (G), or Sema5A-myc expressing 293 cells (H), at 20% confluence. (I) Mean total neurite outgrowth per explant on a laminin substratum in the presence of HEK 293 cells transfected with full-length Sema5A-myc or parental cells. At both 20% and 40% confluence, explants grown in the presence of cells transfected with Sema5A-myc exhibited less neurite outgrowth. Scale bars: (D,E) 100 µm; (G,H) 250 µm.

View larger version (31K): [in a new window]   Fig. 3. Growth cone responses to Sema5A in the presence of L1 and netrin 1. (A) Timelapse sequence showing a retinal growth cone extending on an L1 substratum and exhibiting collapse in response to application of oligomerized ECD-Fc. (ECD-Fc was applied at t=0). Numbers at top left are minutes elapsed. During collapse, axons on L1 tended to show an intermediate stage characterized by the shrinkage of the growth cone into a branched structure (t=20). Growth cones eventually progressed to a fully collapsed morphology (t=40). (B) The percentage of growth cones on L1 substratum exhibiting collapse in response to increasing amounts of oligomerized ECD-Fc. (C) The percentage of growth cones on L1 substratum exhibiting collapse in response to increasing amounts of oligomerized sema-Fc or TSP-Fc. (D) The percentage of growth cones extending on laminin in the presence of netrin 1 responding to oligomerized ECD-Fc. The presence of netrin 1 did not alter the ability of ECD-Fc to mediate growth cone collapse. (E) Assay of netrin 1-dependent outgrowth. Sub-optimal levels of laminin resulted in little axon outgrowth (column 1). 100 ng/ml of netrin 1 by itself was not able to support outgrowth (column 2). The combination of sub-optimal laminin levels and 100 ng/ml netrin 1 resulted in robust outgrowth (column 3). This netrin 1-dependent outgrowth was reduced by the addition of an antibody against the netrin receptor DCC (column 4). (F) Axons dependent on netrin 1 for outgrowth remained responsive to ECD-Fc-mediated growth cone collapse. Scale bar: (A) 10 µm.

View larger version (135K): [in a new window]   Fig. 5. Anti-Sema5A antibody characterization and Sema5A localization at the optic disc. (A) In immunoblots, the Sema5A antibody recognized ECD-Fc protein (lane 1), Sema5A-myc from transfected 293 cells (lane 2), and yielded a band of the expected size for Sema5A (135 kDa) from E14 retina (lane 3). (B) Anti-Sema5A staining of live Sema5A-myc-expressing 293 cells. Top row shows Sema5A-myc-expressing cells stained with Anti-Sema5A, and the bottom row shows Sema5A-myc cells similarly treated with pre-immune serum. (C) Bright-field image of the optic disc. Arrowheads point to retinal pigment epithelium cells (* marks the center of the optic disc). (D) Same optic disc as in C. Sema5A immunoreactivity was localized to the peripheral rim of the optic disc. Image compiled from optical sections through a depth of 25 µm at the optic disc. Scale bars: (B) 25 µm; (C-D) 50 µm.

View larger version (41K): [in a new window]   Fig. 6. Sema5A function blockade and RGC axon pathfinding errors. (A) ECD-Fc-mediated retinal growth cone collapse on an L1 substratum in the presence of anti-Sema5A antibody or pre-immune serum. (B) The incidence of axon guidance errors in optic nerves treated with anti-Sema5A antibody. (C) A normal, DiI-labeled E14 optic nerve showing retinal axons contained within the retinal pathway. The retina and optic disc are towards the left. (D-F) Examples of axon guidance errors in anti-Sema5A-treated optic nerves. The retina is towards the left. (D) A mild phenotype: a pair of short axons tipped with growth cones (arrowheads) are straying from the optic nerve bundle. (E) A more severe error showing a bundle of axons (arrowhead) veering away from the main portion of the optic nerve. Note that some aberrantly projecting axons are growing parallel to the optic nerve (arrow). (F) A large fascicle of axons near the optic disc (arrowhead) have split away from the main optic nerve bundle, and terminate in a knot-like structure. (G,H) Examples of stray RGC axons within the retina (arrowheads). Unlike normal axons, stray axons fail to grow completely through the optic disc, and extend inappropriately within the retina. Scale bars: (C-H) 50 µm.