Article Figures & Data
Figures
- Figure 1.
Expression of the agrin signaling system in the olfactory bulb. a, In situ hybridization for agrin shows expression in the RMS and in individual cells in the GCL, EPL, and GL (arrow). b, α3NKA transcripts are absent from the RMS but strongly present in MCL, while expression in GCL and GL was weaker. c, d, MuSK mRNA was not expressed in the OB (c), and Lrp4 was confined to the GL (d, arrow). EPL, external plexiform layer. Scale bar, 200 μm.
- Figure 2.
Agrin-deficient neuronal precursors do not integrate in the OB. a, Schematic representation of the experimental protocol. GFP-labeled WT or agrin-deficient precursors isolated at E18.5 were transplanted into the anterior SVZ of adult WT hosts. b, In the RMS, organization and distribution of mutant precursors were indistinguishable from controls (top panels). In the center of the OB, WT and agrin-deficient precursors showed the typical switch from tangential chain to radial individual migration (bottom panels, arrowheads). c, Matrigel culture of WT and agrin-deficient explants isolated at E18.5. Bottom panels are high-magnification images of the explants presented in the top panels. d, e, In both situations, explants showed the typical chain migration with no differences in the number of individual cells surrounding the explant (d) or migration distance (e). f, h, Examples for morphological categories of neurons in the GCL (f) and in the GL (h) as used for quantification. g, i, At 17 dpg, cells with mature morphologies (category 1) were significantly reduced when agrin-deficient tissue was transplanted (n = 6; control: n = 5; *p < 0.05), while simple morphologies (category 3) were over-represented in both the GCL (g) and the GL (i). j, k, At 30 dpg, integrated WT granule cells show mature morphology and dense coverage in the external plexiform layer (EPL) with dentritic spines (j). Right, High-magnification images of dendrites presented in left panels. At this time point, surviving agrin-deficient cells are rare. The few remaining cells show few signs of branching or protrusions (k). l, m, At 60 dpg, animals transplanted with WT SVZ tissue show generally large amounts of fully mature neurons in the OB (l). At this time point, mice grafted with mutant tissue were always devoid of GFP-positive cells (m). Scale bars: b, 20 μm; c, top panels, 100 μm; c, bottom panels, 40 μm; j, k, left panels, 15 μm; j, k, right panels, 5 μm; l, m, 100 μm. CC, corpus callosum; ST, striatum; ns, non-significant.
- Figure 3.
TM agrin is the active agrin isoform. a, Representation of the electroporation protocol. b, Morphological distribution of the electroporated cells in the GCL at 21 dpe. siRNA-induced knockdown of TM-agrin leads to the loss of cells with mature neuronal morphology in the GCL (category 1), while simple cells (category 3) are over-represented. This effect is entirely rescued by coelectroporation of a TM-agrin expression construct (n = 6 for control; n = 10 for siRNA; n = 10 for TM-agrin rescue). c, d, Gain-of-function of TM-agrin leads to an increasing number of dendritic spines in the EPL (17 dpe, n = 20 and 32 dendrites for control and TM; 21 dpe, n = 26 and 55 dendrites for control and TM; 28 dpe, n = 91 and 100 for control and TM. **p < 0.01, ***p < 0.001, n.s. non significant, Student's t test). Scale bar, 30 μm.
- Figure 4.
Expression of the competitive agrin antagonist C-Ag15 interferes with differentiation and spine formation in the OB. a, Electroporation of a C-Ag15 expression construct leads to the loss of cells with mature neuronal morphology and the over-representation of category 2 and 3 cells in the GCL (n = 8 for C-15Ag and for control; **p < 0.01, ***p < 0.001, Student's t test). b, c, In parallel to the shift in categories, spine density is significantly reduced in the category 1 cells that show extensions into the EPL (control: n = 69, C-Ag15: n = 59; *p < 0.05, Student's t test). Scale bar, 10 μm.
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