Fig. 5.

Atrophied terminal arborization and reduced number of synapses in Smad4-cKO sensory neurons. (A) Schematic of the alleles used here. CreERt2 was inserted into the Advillin locus to generate Advillin^CreERt2. The Advillin^CreERt2 line then was crossed with Smad4^flox and the reporter line Rosa^PLAP. (B) PLAP staining of a single trigeminal mechanosensory neuron innervating one whisker follicle at P1. PLAP and CO staining of consecutive sections of control (C) and Smad4-cKO (D) SpI nuclei at P1. Magnified view (Upper Right) of the central terminal projection highlighted (Left). 2D projection (Lower Right) of the complete 3D Neurolucida reconstruction of the same terminal arbor. Representative 2D projections of terminal arbors in SpI nucleus of control (E) and Smad4-cKO (F) mice are shown. The dots (•) represent varicosities, defined as an enlargement of the axon. Note the decreased complexity and fewer varicosities present in Smad4-cKO neurons. (G) Quantitative analysis of total length and branch and varicosity densities of Ctrl and cKO terminal arbors (Ctrl: n = 47, four mice; cKO: n = 20, four mice). (H) EM images of control and Smad4-cKO terminal boutons of PrV nuclei at P7. White arrowheads indicate asymmetrical synaptic contacts, and red arrows indicate symmetrical contacts. Note the presence of fewer synapses in Smad4-cKO boutons. (I) Average ± SEM synaptic density (number of synapses/10 μm²) and bouton area (μm²) of cKO (n = 180 boutons) and Ctrl (n = 200 boutons) mice. *P < 0.05; **P < 0.01; ***P < 0.001 by the Student's t test. cKO, Smad4-cKO; Ctrl, control. (Scale bars: A–D, 100 μm; C–F, 50 μm in enlarged views; H, 500 nm.)