Neuropilins are positive regulators of Hedgehog signal transduction

Abstract

The Hedgehog (Hh) pathway is essential for vertebrate embryogenesis, and excessive Hh target gene activation can cause cancer in humans. Here we show that Neuropilin 1 (Nrp1) and Nrp2, transmembrane proteins with roles in axon guidance and vascular endothelial growth factor (VEGF) signaling, are important positive regulators of Hh signal transduction. Nrps are expressed at times and locations of active Hh signal transduction during mouse development. Using cell lines lacking key Hh pathway components, we show that Nrps mediate Hh transduction between activated Smoothened (Smo) protein and the negative regulator Suppressor of Fused (SuFu). Nrp1 transcription is induced by Hh signaling, and Nrp1 overexpression increases maximal Hh target gene activation, indicating the existence of a positive feedback circuit. The regulation of Hh signal transduction by Nrps is conserved between mammals and bony fish, as we show that morpholinos targeting the Nrp zebrafish ortholog nrp1a produce a specific and highly penetrant Hh pathway loss-of-function phenotype. These findings enhance our knowledge of Hh pathway regulation and provide evidence for a conserved nexus between Nrps and this important developmental signaling system.

Figure 1.

Identification of Nrp1 as a regulator of mammalian Hh signaling in a cultured cell RNAi screen. (A) A library of DSPs targeting 816 genes implicated in signaling processes was screened using a luciferase-based cell culture assay for Hh signal transduction. A viability filter was applied to eliminate those DSPs that affected cell survival or growth. Of these viability-filtered DSPs, 68 significantly inhibited Shh-stimulated Gli-dependent luciferase reporter activity (Z-score <−1.5, see Supplemental Table 1). (B) The 691 viability-filtered results from the primary DSP screen are shown in Z-score rank order. A DSP targeting Nrp1 (red) significantly blocked Shh-stimulated induction of the Gli-dependent luciferase reporter. A DSP targeting Gli1 also resulted in significant inhibition of Hh signaling (black).

Figure 2.

Nrp1 and Nrp2 are partially redundant positive regulators of Hh signal transduction. (A) Gli-dependent luciferase reporter (GLuc) transcription in NIH3T3 fibroblasts treated with Nrp1, Nrp2, or Nrp1+2 RNAi. P < 0.05, two-tailed Student's t-test. Error bars indicate mean ± 1 SD. (B) GLuc transcription in NIH3T3 fibroblasts following Nrp1 3′ UTR RNAi with or without coexpression of mouse Nrp1. (**) P < 0.01, two-tailed Student's t-test. (C) Immunoblots of Gli1 and Ptc1 protein from NIH3T3 fibroblasts following Nrp1+2 RNAi and Shh treatment. The Nrp1 antibody detected Nrp1 and unrelated closely spaced nonspecific (*) bands (Supplemental Fig. S1C).

Figure 3.

Nrps are expressed at locations of active Hh signaling during mouse development. (A) Fluorescence microscopy of Nrp1 (red, Alexa-595 labeled), Smo (green, Alexa-488-labeled), and acetylated tubulin (cyan, Alexa-633-labeled) expression in the visceral endoderm (ve) and yolk sac mesoderm (ysm) of E8.5 mouse embryos. (Blue) Hoechst dye-labeled nuclei. (B) Inset from image shown in A. Bar, 6 μm. (C) Fluorescence microscopy of Nrp1 (red, Alexa-595-labeled), Smo (green, Alexa-488 labeled), and acetylated tubulin (cyan, Alexa-633-labeled) expression in neural tube (nt) and paraxial mesoderm (pm) of E8.5 mouse embryos. (Blue) Hoechst dye-labeled nuclei. (D) Inset from image shown in C. Bar, 6 μm. (E) Fluorescence microscopy of Nrp1 (red, Alexa-595-labeled) and Nrp2 (green, Alexa-488-labeled) expression in the dermal papilla and epithelium of E17.5 mouse hair follicle. (Blue) Hoechst dye-labeled nuclei. Bar, 20 μm. (F) Immunoblots of protein from Shh-stimulated primary dermal cells following infection with lentivirus expressing Nrp1+2 shRNAs.

Figure 4.

Nrp1 mediates a Hh pathway positive feedback circuit. (A) Immunoblot of Nrp1 protein abundance in NIH3T3 fibroblasts treated with Shh. (B) Quantitation of Nrp1 protein by densitometry of three independent immunoblots, normalized to p38 protein in the same lane. (**) P < 0.01, two-tailed Student's t-test. Error bars indicate mean ± 1 SD. (C) Quantitative PCR measurement of Shh-stimulated Nrp1 transcription following Smo RNAi. Units are PCR cycle thresholds normalized to those of Gapdh in the same well. Error bars indicate mean ± 1 SD. (D) Immunoblots of protein from Smo^−/− and rescued Smo^−/−;YFP-Smo MEFs following Shh or SAG stimulation. (E) Quantification of immunoblot showing Nrp1 protein expression as a function of time after addition of Shh. (F) Quantification of immunoblot showing Ptc1 protein expression as a function of time after addition of Shh. (G) GLuc transcription in NIH3T3 fibroblasts overexpressing YFP, CD4-YFP, or Nrp1-YFP. (*) P < 0.05, two-tailed Student's t-test. Measurements were normalized to a cotransfected constitutive Renilla luciferase. Error bars indicate mean ± 1 SD.

Figure 5.

Nrps regulate Hh signal transduction between Smo and SuFu. (A) Immunoblots of protein from unstimulated Ptc1^−/− MEFs treated with Nrp1+2 RNAi. (B) Immunoblots of protein from SAG-stimulated (100 nM) NIH3T3 fibroblasts treated with Nrp1+2 RNAi. (C) SAG-stimulated GLuc transcription in NIH3T3 fibroblasts overexpressing Nrp1-YFP. (**) P < 0.01, two-tailed Student's t-test. Measurements were normalized to a cotransfected constitutive Renilla luciferase. Error bars indicate mean ± 1 SD. (D) Immunoblots of protein from unstimulated SuFu^−/− MEFs treated with Nrp1+2 RNAi. Normal Shh responsiveness was restored in SuFu^−/− MEFs infected with a retrovirus expressing SuFu (“Rescued”) (Humke et al. 2010). (E) Immunoblots of protein from Shh-stimulated “Rescued” SuFu^−/− MEFs treated with Nrp1+2 RNAi.

Figure 6.

Zebrafish nrp1a morphants exhibit a Hh loss-of-function phenotype. (A) Lateral view of wild-type zebrafish embryo at ∼30 hpf. Bar, 100 μm. (B) Lateral view of chevron-shaped somites in wild-type zebrafish embryos at ∼30 hpf. Bar, 200 μm. (C) Whole-mount in situ hybridization to ptc1 (purple) demonstrates adaxial staining pattern in bud stage (10 hpf) wild-type embryos. Bar, 200 μm. Dotted line delineates somite boundary. (D) Whole-mount in situ hybridization to shha (purple) in wild-type 10 hpf zebrafish embryos demonstrates axial expression. (E) At ∼30 hpf, zebrafish embryos injected with nrp1a antisense MO1 at one- to four-cell stages exhibit ventral body curvature. (F) Zebrafish nrp1a MO1-injected embryos exhibit U-shaped somites at ∼30 hpf. Dotted line delineate somite boundary. (G) Signal from ptc1 whole-mount in situ hybridization is significantly reduced in nrp1a MO1-injected embryos. (H) Signal from shha whole-mount in situ hybridization is unchanged in nrp1a MO1-injected embryos. (I) At ∼30 hpf, zebrafish embryos injected with an orthogonal nrp1a antisense MO2 at one- to four-cell stages exhibit curved body morphology similar to nrp1a MO1 morphants. (J) Zebrafish nrp1a MO2-injected embryos exhibit U-shaped somites at ∼30 hpf, similar to nrp1a MO1 morphants. Dotted line delineates somite boundary. (K) Similar to ptc1 expression in nrp1a MO1 morphants, signal from ptc1 whole-mount in situ hybridization in nrp1a MO2-injected embryos is significantly reduced. (L) Signal from shha whole-mount in situ hybridization is unchanged in nrp1a MO2-injected embryos. Views are dorsal, with anterior toward the top.