Sann SB, Xu L, Nishimune H, Sanes JR, Spitzer NC.

R01MH074702 NIMH NIH HHS

	Figure 1. CaV2.2 is expressed in the developing Xenopus neural tube and is localized to the growth cone during axon outgrowth in vivo and in vitro. A, RT-PCR of a 272 base pair fragment of CaV2.2 using mRNA collected from dissections of dorsal halves of embryos from stages (St.) 12–30. B, In situ hybridization using the probe made from the same fragment reveals expression of CaV2.2 in the developing neural tube. Scale bar, 500 μm. C, Schematized dissection for immunostaining: a ventral incision was made to remove the myotomes and notochord and reveal the neural tube and skin. Staining with the neuronal marker HNK-1 reveals longitudinal tracts running anteroposteriorly, axons of sensory Rohon-Beard neurons innervating the skin, and commissural interneurons crossing the ventral midline. D, Ventral view of a spinal cord and skin of a dissected embryo stained for HNK-1 (purple) and CaV2.2 (green). Boxes outline the CaV2.2-positive growth cones pictured in E and F. Scale bar, 50 μm. E, Sensory Rohon-Beard growth cone expressing CaV2.2 (green). Scale bar, 10 μm. F, Commissural interneuron expressing CaV2.2 (green). Scale bar, 5 μm. G, Cultured neuron stained for β-tubulin (purple) and CaV2.2 (green) expressing CaV2.2 in the growth cone. Scale bar, 50 μm. H, Growth cone boxed in G. Scale bar, 10 μm.
	Figure 2. Laminin β2 is expressed in a tiled pattern in the skin. A, Schematic of Xenopus skin. B, Whole-embryo staining with the C4 antibody to laminin β2 revealed expression coextensive with hexagonal epithelial (H) cells but not ciliated (C) or intercalating nonciliated cells (INC) in stage 34 larvae. Scale bar, 25 μm.
	Figure 3. Laminin β2 specifically inhibits spinal neurite outgrowth. Xenopus neurons from stage 17 embryos were cultured on the following substrates and analyzed at 14–18 h in vitro: A, laminin (LM) with a β1 but not a β2 chain; B, a solublized 20 kDa LRE containing C-terminal fragment of laminin β2; C, a point-mutated (LRE→QRE) laminin β2 fragment. Neurons identified by immunostaining for neuron-specific β-tubulin extended significantly fewer processes on substrates containing the LRE fragment compared with QRE or nonlaminin β2 substrates. Scale bar, 50 μm. D, Incubating neurons cultured on laminin plus the β2 LRE fragment with a competing peptide corresponding to the 11th extracellular LRE-binding loop of rat CaV2.1 (91% homologous to Xenopus CaV2.2) rescued neurite extension. E, Incubation with a control peptide corresponding to the 11th extracellular loop of rat CaV1.2 (61% homologous to Xenopus CaV2.2) did not prevent inhibition by laminin β2 LRE. F, Laminin β2 LRE significantly reduces the number of neurons extending processes in vitro. This effect is rescued by incubation with the competing CaV2.1 loop peptide (**p < 0.01, *p < 0.05, comparison to LM control; n ≥ 300 neurons from ≥6 cultures). Error bars indicate SEM.
	Figure 4. Laminin (LM) β2 inhibition is calcium dependent. Stage 16 dissociated neural tubes were cultured and neurons identified by staining for β-tubulin. A, B, Neurite outgrowth on LM is inhibited by addition of the laminin β2 fragment (LRE). Scale bar, 50 μm. C, D, Incubating neurons cultured on LM plus the β2 LRE fragment in 5 μM ω-conotoxin or in calcium-free medium prevents inhibition of neurite outgrowth indicating calcium dependence of LRE inhibition. E, Incubating neurons grown on LM alone in calcium-free medium does not lead to increases in neurite extension. F, Neurite extension on LRE is rescued by preventing calcium influx through CaV2.2 (n ≥ 250 neurons from ≥7 embryos; *p < 0.05). Error bars
	Figure 5. Neurons at a border of laminin β2 generate calcium transients and stall. A, Neurons were cultured on stripes of native and UV-denatured laminin β2 LRE fragment and internal calcium concentrations were imaged using Fluo-4. Images were captured at 2 Hz. Fluo-4 is in green, denatured laminin β2 is in red, and native laminin β2 is in black. Three images were averaged to enhance visualization of the neuron. Traces show the calcium activity in each of three growth cones as they encounter the border. Axis scales apply throughout the figure. B, C, Less calcium transient activity is seen when a neuron approaches (B) or has crossed onto (C) activated point mutated laminin β2 (LRE→QRE). D, Significantly more neurons stop or turn back at an LRE border than a QRE border (*p < 0.001).
	Figure 6. Blocking signaling through CaV2.2 in vivo leads to increased innervation of the skin. A, Agarose beads soaked in 10 μM ω-conotoxin (ctx) were implanted in stage 16 embryos. After 1 d, stage 34 embryos were fixed and stained with anti-HNK-1 to reveal sensory processes and sensory nerve terminals innervating the skin. A 450 × 350 μm area centered around the bead was imaged and analyzed (box). B–D, Deconvolved and projected z-series through the skin of embryos in which no bead (B), a control bead (C), or an ω-conotoxin bead (D) were implanted. Scale bar, 50 μm. E, Enlarged image of the boxed region in D showing three sensory nerve terminal clusters, circles. Scale bar, 10 μm. F, Embryos exposed to ω-conotoxin exhibited significantly greater numbers of sensory nerve terminal clusters compared with embryos implanted with a vehicle bead or no bead (*p < 0.05). Error bars indicate SEM.
	Figure 7. Blocking CaV2.2–laminin β2 interactions in vivo leads to increased innervation of the skin. A–C, Agarose beads soaked in 10 μM CaV2.1 or CaV1.2 11th extracellular loop peptide were implanted in stage 16 embryos. After 1 d, stage 34 embryos were fixed and stained with anti-HNK-1 to reveal sensory processes and sensory nerve terminals innervating the skin as in Figure 6. Scale bar, 50 μm. D, Embryos exposed to CaV2.1 loop peptide (C) exhibited significantly greater numbers of sensory nerve terminals than embryos exposed to CaV1.2 loop peptide (B) (*p < 0.05; n ≥ 3 embryos for each condition). Error bars indicate SEM.
	Figure 8. Model of laminin β2 and CaV2.2 interactions in development of skin innervation. A, In control conditions, outgrowing sensory terminals are inhibited or slowed by laminin β2-rich regions of the skin coextensive with hexagonal cells (white). B, The number of sensory nerve terminals increases when signaling interactions between laminin β2 and CaV2.2 are blocked.

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