Ephrins regulate the formation of terminal axonal arbors during the development of thalamocortical projections
Fanny Mann1,*,
,
Christiane Peuckert2,
Frank Dehner2,
Renping Zhou3 and
Jürgen Bolz2
1 Institut National de la Santé et de la Recherche Médicale Unité 371, 18 avenue du Doyen Lépine, 69500 Bron, France
2 Universität Jena, Institut Allgemeine Zoologie, Erberstrasse 1, 07743 Jena, Germany
3 Laboratory for Cancer Research, Department of Chemical Biology, College of Pharmacy, Rutgers University, Piscataway, NJ 08855, USA
* Present address: University of Cambridge, Department of Anatomy, Downing Street, Cambridge CB2 3DY, UK
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Fig. 1. Comparison of thalamic axons growth on surfaces coated with membrane extracts isolated from different cortical layers. (A,B) Photomicrographs of E14 thalamic explants cultured for 2 days on membranes from cortical layer 4 and cortical layer 5. The general outgrowth of thalamic explants was reduced on membranes from layer 4 (A) (which is their target substrate) compared with membranes from layer 5 (B). (C-J) Photomicrographs and schematic drawings illustrating the branching pattern of individual growing axons on layer 4 and layer 5 membrane substrates. Thalamic axons exhibit numerous side branches on membranes from their target layer (C-F) and much less on their non-target substrate (G-J). Scale bar: 100 µm in A,B; 10 µm in C-J.
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Fig. 2. Quantitative analysis of thalamic axon growth and branching on membranes from cortical layer 4 (black bars) and cortical layer 5 (white bars). Histograms depict the number of fibers per explant (A), axon length (B) and branching density (C) on each type of membrane substrate. (D) Stripe assay with thalamic axons growing perpendicular to membrane stripes (see Fig. 3). The histogram depicts the branching density observed in each set of stripes. n, number of explants analyzed (A); n, number of fibers examined (B-D). Error bars represent the s.e.m.
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Fig. 3. Camera lucida drawings of representative axons crossing stripes with membranes from cortical layer 4 (dark gray) and membranes from cortical layer 5 (light gray). In A, thalamic axons extend first on a membrane stripe from their target layer, whereas in B the axons encounter first a membrane stripe from a non-target layer. In each case, thalamic axons branch preferentially on their appropriate target membranes. Scale bar: 50 µm.
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Fig. 4. Ephrin-A5 expression within primary somatosensory and motor cortex at P6 and EphA receptor labeling on E14 thalamic axons in vitro. (A) Serial coronal sections of the right hemisphere (medial is left) stained with Cresyl Violet, ephrin-A5 in situ hybridization and cytochrome oxidase histochemistry. The boundaries between the barrel field of the somatosensory cortex (S) and the motor cortex (M) are indicated by the arrowheads. Bars show the boundaries between cortical layers 4, 5 and 6. (B) Coronal section through the barrel field of S1 stained with Hoechst and EphA3-Fc fusion protein. (C) Phase-contrast and bright-field images of ephrin-A5-AP stained thalamic axons.
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Fig. 5. Recombinant ephrin-A5 promotes collateral formation for thalamic axons. Phase-contrast photomicrographs and schematic drawings of individual axons from thalamic neurons cultured on membrane substrates isolated from NIH3T3-vector cells (A-D) and NIH3T3-ephrin-A5 cells (E-H). In the presence of ephrin-A5 ligand, thalamic axons present a profuse branching, whereas only few branches form under control conditions. Scale bar: 10 µm.
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Fig. 6. Quantitative analysis of thalamic axon growth on membranes from NIH3T3-ephrin-A5 cells (black bars) or from control cells (white bars). (A) Number of axons extending from thalamic explants, (B) analysis of axonal length, (C) branch formation under control condition and (D) branch formation after treatment of membranes with PI-PLC enzyme. n, number of explants scored (A); n, number of fibers examined (B-D). ‘n.s.’ indicates not significant and error bars represent the s.e.m.
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Fig. 7. Histograms of thalamic axons sprouting on membranes from distinct cortical layers with PI-PLC enzyme treatment (A) and in the presence of soluble ephrin-A5-Fc (B). Bars represent the percentage of branches formed on membranes from cortical layer 4 (black bars) and cortical layer 5 (white bars). Data are presented in percentage and normalized to 100% for values obtained on membranes from layer 5. The ability of layer 4 membranes to support thalamic axons sprouting decreases after removal of GPI-anchored molecules and after exposure to ephrin-A5-Fc fusion protein. By contrast, none of these treatments influences the branching of thalamic axons on layer 5 membranes. (C) Histogram of branch formation of thalamic axons cultured on cortical membranes from layer 4 (black bar), layer 5 (white bar), and on a mixture (1:1) of both types of membranes (gray bar). On the latter substrate, thalamic axons exhibit a branching density similar to the density observed on layer 5 membranes alone, suggesting that the branch-promoting activity in membranes from layer 4 is inhibited by the membrane extracts from layer 5. n, number of fibers examined.
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Fig. 8. (A) Outgrowth of thalamic explants confronted with parallel membrane stripes from cortical layer 4 (L4) and cortical layer 5 (L5). (B) Distribution of thalamic axons on membrane stripes from cortical layer 4 (black bars) and cortical layer 5 (white bars) under control conditions and after PI-PLC treatment of one type of membrane. Bars indicate the percentage of axons growing on one type of stripe. Results demonstrate that thalamic axons preferentially extend on their target membrane stripes, and this choice can be blocked by treatment of layer 5 membranes with PI-PLC enzyme. n, number of pairs of stripes analyzed. Scale bar: 100 µm.
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Fig. 9. Recombinant ephrin-A5 ligand elicits distinct guidance effects on non-limbic and limbic thalamic axons in stripe assay. Outgrowth of thalamic fibers from non-limbic (A,B) and limbic (C,D) thalamic explants (see Materials and Methods) confronted with parallel membrane stripes from NIH3T3-ephrin-A5 cells and from untransfected control cells. Upper panels: photomicrographs of representative examples of axonal outgrowth. Lower panels: quantitative analysis of axonal preference. The histograms depict the percentage of axons growing on ephrin-A5 containing stripes (black bars) and on control stripes (white bars). Non-limbic thalamic axons grow equally on both types of stripes, whereas limbic thalamic axons avoid ephrin-A5 ligand and prefer control stripes. Scale bar: 100 µm; n, number of pair of stripes examined.
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© The Company of Biologists Ltd 2002
