Panagiotaki N , Dajas-Bailador F , Amaya E , Papalopulu N , Dorey K .

Medical Research Council , Wellcome Trust , 090868 Wellcome Trust , G0900584 Medical Research Council , MRC_G0900584 Medical Research Council , WT090868 Wellcome Trust

	Fig. 2. Temporal and spatial expression pattern of spry3. (A) Total RNA was isolated from Xenopus of the indicated stages and RT-PCR performed using oligonucleotides specific for the indicated genes. E, embryo; –RT, stage 26 without reverse transcriptase. odc is a loading control. (B) Comparison of the pattern of expression of spry1, spry2, spry3 and spry4 at stage 32. (C) Analysis of the spatial expression of spry3 during Xenopus development. Embryos at the indicated stages were fixed and subjected to whole-mount in situ hybridisation using a probe specific for spry3. (D) Sections (plane indicated in C) of embryos stained with the indicated probes, showing expression in differentiated motor and sensory neurons.
	Fig. 4. spry3 expression is dependent on BDNF-TrkB. (A) Synexpression group of spry3. The pattern of expression of spry3 resembles that of bdnf and trkb. (B) Total RNA from whole embryos or isolated neural tube (stage 26) was purified from Xenopus embryos injected with morpholinos against bdnf (MO BDNF) or trkb (MO TrkB) or with a control morpholino (MOC). spry3 expression was assessed by real-time PCR using rpl8 as a reference. Knocking down bdnf and trkb results in a significant decrease in spry3 expression (*P<0.005, using a statistical ANOVA test). Error bars indicate mean ± s.e.m. (C) Xenopus embryos were injected at the 1-cell stage with MO BDNF, MO TrkB or MOC, fixed at stage 28 and processed for whole-mount in situ hybridisation using a probe specific for spry3. Embryos are scored as normal/weak when spry3 expression is detected both in the trigeminal nerve and the spinal cord, as medium when spry3 expression is only partially detected in the spinal cord and as strong when no expression of spry3 is detected. The percentage of affected embryos is indicated in each case.
	Fig. S2. FGF signalling is not necessary for spry3 expression. (A) Comparison of the pattern of expression of spry1, spry2 and spry4 with that of fgf8. (B) Xenopus embryos at stage 28 were treated with an FGFR inhibitor (SU5402), with DMSO or left untreated (UT) for 4 hours, then fixed and subjected to whole-mount in situ hybridisation with the indicated probes. The results presented are representative of two independent experiments.
	Fig. 3. Spry3 regulates axonal branching. (A)Whole-mounts (upper panels) and spinal cord sections (lower panels) of control (MOC) or spry3 (MOS3) morpholino-injected Xenopus embryos stained with anti-acetylated alpha-tubulin antibodies. For whole-mount, anterior is up, dorsal is right. For sections, dorsal is up. (B)Quantification of number of branch points per axonal tract of motoneurons, derived from the analysis of the sections shown in A. Data are expressed as the mean s.e.m. of three independent experiments (n=13 embryos for each condition). (C)Whole-mount staining of a single motoneuron expressing GFP from MOC and MOS3 embryos. Scale bars: 50um. (D)Quantification of axon length (left) and number of branch points per 100um of axon (right). The data are expressed as the mean s.e.m. of three independent experiments (n=19 neurons for MOC and n=24 neurons for MOS3). In all cases, statistical significance was determined using an unpaired Student t-test; *P=0.02, **P=0.009, ***P=0.004.
	Fig. S1. Design and efficacy of spry3 morpholinos. (A) Antisense morpholinos were designed against the splice acceptor and the splice donor site of the unique intron of spry3. The most efficient knockdown of spry3 expression was observed with a combination of MO e1i1 and MO i1e2 and therefore this combination was used for all the Spry3 knockdown experiments in this study. (B) Embryos injected with control morpholino (MOC) or a morpholino against spry3 (MOS3) were fixed at the indicated stages and stained with anti-acetylated a-tubulin antibodies. Anterior is to the right and dorsal is up.
	spry3 (sproutyhomolog 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 31/32, lateral view, anterior left, dorsal up.
	ntrk2 (neurotrophic tyrosine kinase, receptor, type 2) gene expression in Xenopus tropicalis embryo as assayed by in situ hybridization, NF stage 28, lateral view, dorsal up, anterior left.
	bdnf (brain-derived neurotrophic factor ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.
	spry1 (sprouty homolog 1, antagonist of FGF signaling) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.
	spry2 (sprouty homolog 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.
	spry4 (sprouty homolog 4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.
	spry3 (sprouty homolog 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, dorsal view, anterior up.
	spry3 (sprouty homolog 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 23, lateral view, anterior left, dorsal up.
	spry3 (sprouty homolog 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
	spry3 (sprouty homolog 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28,transverse section, mid trunk region, dorsal up.
	spry3 (sprouty homolog 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.
	spry3 (sprouty homolog 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 40, lateral view, anterior left, dorsal up.
	hox11l2 ( gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, transverse section, mid-trunk region, dorsal up.
	mnx1 (motor neuron and pancreas homeobox 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28,transverse section, mid trunk region, dorsal up.

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