Cellular and Molecular Life Sciences

, Volume 70, Issue 5, pp 935–950 | Cite as

Zebrafish rgs4 is essential for motility and axonogenesis mediated by Akt signaling

  • Yi-Chuan ChengEmail author
  • Paul J. Scotting
  • Li-Sung Hsu
  • Sheng-Jia Lin
  • Hung-Yu Shih
  • Fu-Yu Hsieh
  • Hui-Lan Wu
  • Chu-Li Tsao
  • Chia-Jung Shen
Research article


The schizophrenia susceptibility gene, Rgs4, is one of the most intensively studied regulators of G-protein signaling members, well known to be fundamental in regulating neurotransmission. However, little is known about its role in the developing nervous system. We have isolated zebrafish rgs4 and shown that it is transcribed in the developing nervous system. Rgs4 knockdown did not affect neuron number and patterning but resulted in locomotion defects and aberrant development of axons. This was confirmed using a selective Rgs4 inhibitor, CCG-4986. Rgs4 knockdown also attenuated the level of phosphorylated-Akt1, and injection of constitutively-activated AKT1 rescued the motility defects and axonal phenotypes in the spinal cord but not in the hindbrain and trigeminal neurons. Our in vivo analysis reveals a novel role for Rgs4 in regulating axonogenesis during embryogenesis, which is mediated by another schizophrenia-associated gene, Akt1, in a region-specific manner.


Rgs4 Axonogenesis Akt Zebrafish 



We thank Jin-Chung Chen, Hung-Li Wang and Rong-Chi Huang for discussions, Chang-Jen Huang for Tg(huC:eGFP) line, Hitoshi Okamoto for Tg(islet1:GFP) line, Min-Chi Chen for statistical analysis, Pierre Drapeau and Hey-Jen Tsay for behavior analysis, Jim-Tong Horng for western blot, Chung-Der Hsaio for ca-AKT, and David Wilkinson for neurogenin1 and pax2a constructs for riboprobes. We are also grateful to Taiwan Zebrafish Core facility at ZeTH and Zebrafish Core in Academia Sinica for providing fish. This work was supported by grants from Chang Gung Memorial Hospital (CMRPD170513 and CMRPD1B0251) and the National Science Council of Taiwan (96-2745-B-182-003-URD).

Supplementary material

18_2012_1178_MOESM1_ESM.docx (864 kb)
Supplementary material 1 (DOCX 864 kb)


  1. 1.
    Neitzel KL, Hepler JR (2006) Cellular mechanisms that determine selective RGS protein regulation of G protein-coupled receptor signaling. Semin Cell Dev Biol 17(3):383–389PubMedCrossRefGoogle Scholar
  2. 2.
    Bansal G, Druey KM, Xie Z (2007) R4 RGS proteins: regulation of G-protein signaling and beyond. Pharmacol Ther 116(3):473–495PubMedCrossRefGoogle Scholar
  3. 3.
    Abramow-Newerly M, Roy AA, Nunn C, Chidiac P (2006) RGS proteins have a signalling complex: interactions between RGS proteins and GPCRs, effectors, and auxiliary proteins. Cell Signal 18(5):579–591PubMedCrossRefGoogle Scholar
  4. 4.
    Gold SJ, Ni YG, Dohlman HG, Nestler EJ (1997) Regulators of G-protein signaling (RGS) proteins: region-specific expression of nine subtypes in rat brain. J Neurosci 17(20):8024–8037PubMedGoogle Scholar
  5. 5.
    Erdely HA, Lahti RA, Lopez MB, Myers CS, Roberts RC, Tamminga CA, Vogel MW (2004) Regional expression of RGS4 mRNA in human brain. Eur J Neurosci 19(11):3125–3128PubMedCrossRefGoogle Scholar
  6. 6.
    Grillet N, Dubreuil V, Dufour HD, Brunet JF (2003) Dynamic expression of RGS4 in the developing nervous system and regulation by the neural type-specific transcription factor Phox2b. J Neurosci 23(33):10613–10621PubMedGoogle Scholar
  7. 7.
    De Vries L, Zheng B, Fischer T, Elenko E, Farquhar MG (2000) The regulator of G protein signaling family. Annu Rev Pharmacol Toxicol 40:235–271PubMedCrossRefGoogle Scholar
  8. 8.
    Ross EM, Wilkie TM (2000) GTPase-activating proteins for heterotrimeric G proteins: regulators of G protein signaling (RGS) and RGS-like proteins. Annu Rev Biochem 69:795–827PubMedCrossRefGoogle Scholar
  9. 9.
    Levitt P, Ebert P, Mirnics K, Nimgaonkar VL, Lewis DA (2006) Making the case for a candidate vulnerability gene in schizophrenia: convergent evidence for regulator of G-protein signaling 4 (RGS4). Biol Psychiatry 60(6):534–537PubMedCrossRefGoogle Scholar
  10. 10.
    Wu C, Zeng Q, Blumer KJ, Muslin AJ (2000) RGS proteins inhibit Xwnt-8 signaling in Xenopus embryonic development. Development 127(13):2773–2784PubMedGoogle Scholar
  11. 11.
    Grillet N, Pattyn A, Contet C, Kieffer BL, Goridis C, Brunet JF (2005) Generation and characterization of Rgs4 mutant mice. Mol Cell Biol 25(10):4221–4228PubMedCrossRefGoogle Scholar
  12. 12.
    Huang KY, Chen GD, Cheng CH, Liao KY, Hung CC, Chang GD, Hwang PP, Lin SY, Tsai MC, Khoo KH, Lee MT, Huang CJ (2011) Phosphorylation of the zebrafish M6Ab at serine 263 contributes to filopodium formation in PC12 cells and neurite outgrowth in zebrafish embryos. PLoS ONE 6(10):e26461PubMedCrossRefGoogle Scholar
  13. 13.
    Higashijima S, Hotta Y, Okamoto H (2000) Visualization of cranial motor neurons in live transgenic zebrafish expressing green fluorescent protein under the control of the islet-1 promoter/enhancer. J Neurosci 20(1):206–218PubMedGoogle Scholar
  14. 14.
    Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995) Stages of embryonic development of the zebrafish. Dev Dyn 203(3):253–310PubMedCrossRefGoogle Scholar
  15. 15.
    Chung PC, Lin WS, Scotting PJ, Hsieh FY, Wu HL, Cheng YC (2011) Zebrafish Her8a is activated by Su(H)-dependent Notch signaling and is essential for the inhibition of neurogenesis. PLoS ONE 6(4):e19394PubMedCrossRefGoogle Scholar
  16. 16.
    Yeh CM, Liu YC, Chang CJ, Lai SL, Hsiao CD, Lee SJ (2011) Ptenb mediates gastrulation cell movements via Cdc42/AKT1 in zebrafish. PLoS ONE 6(4):e18702PubMedCrossRefGoogle Scholar
  17. 17.
    Roman DL, Talbot JN, Roof RA, Sunahara RK, Traynor JR, Neubig RR (2007) Identification of small-molecule inhibitors of RGS4 using a high-throughput flow cytometry protein interaction assay. Mol Pharmacol 71(1):169–175PubMedCrossRefGoogle Scholar
  18. 18.
    Kimple AJ, Willard FS, Giguere PM, Johnston CA, Mocanu V, Siderovski DP (2007) The RGS protein inhibitor CCG-4986 is a covalent modifier of the RGS4 Galpha-interaction face. Biochim Biophys Acta 1774(9):1213–1220PubMedCrossRefGoogle Scholar
  19. 19.
    Roman DL, Blazer LL, Monroy CA, Neubig RR (2010) Allosteric inhibition of the regulator of G protein signaling-Galpha protein–protein interaction by CCG-4986. Mol Pharmacol 78(3):360–365PubMedCrossRefGoogle Scholar
  20. 20.
    Drapeau P, Saint-Amant L, Buss RR, Chong M, McDearmid JR, Brustein E (2002) Development of the locomotor network in zebrafish. Prog Neurobiol 68(2):85–111PubMedCrossRefGoogle Scholar
  21. 21.
    Thisse C, Thisse B (2008) High-resolution in situ hybridization to whole-mount zebrafish embryos. Nat Protoc 3(1):59–69PubMedCrossRefGoogle Scholar
  22. 22.
    Bernstein LS, Grillo AA, Loranger SS, Linder ME (2000) RGS4 binds to membranes through an amphipathic alpha -helix. J Biol Chem 275(24):18520–18526PubMedCrossRefGoogle Scholar
  23. 23.
    Srinivasa SP, Bernstein LS, Blumer KJ, Linder ME (1998) Plasma membrane localization is required for RGS4 function in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 95(10):5584–5589PubMedCrossRefGoogle Scholar
  24. 24.
    Ding L, Mychaleckyj JC, Hegde AN (2007) Full length cloning and expression analysis of splice variants of regulator of G-protein signaling RGS4 in human and murine brain. Gene 401(1–2):46–60PubMedCrossRefGoogle Scholar
  25. 25.
    Saint-Amant L, Drapeau P (1998) Time course of the development of motor behaviors in the zebrafish embryo. J Neurobiol 37(4):622–632PubMedCrossRefGoogle Scholar
  26. 26.
    Brustein E, Saint-Amant L, Buss RR, Chong M, McDearmid JR, Drapeau P (2003) Steps during the development of the zebrafish locomotor network. J Physiol Paris 97(1):77–86PubMedCrossRefGoogle Scholar
  27. 27.
    Hjorth J, Key B (2002) Development of axon pathways in the zebrafish central nervous system. Int J Dev Biol 46(4):609–619PubMedGoogle Scholar
  28. 28.
    Lewis KE, Eisen JS (2003) From cells to circuits: development of the zebrafish spinal cord. Prog Neurobiol 69(6):419–449PubMedCrossRefGoogle Scholar
  29. 29.
    Metcalfe WK, Myers PZ, Trevarrow B, Bass MB, Kimmel CB (1990) Primary neurons that express the L2/HNK-1 carbohydrate during early development in the zebrafish. Development 110(2):491–504PubMedGoogle Scholar
  30. 30.
    Schapira G, Dreyfus JC, Joly M (1952) Changes in the flow birefringence of myosin as a result of muscular atrophy. Nature 170(4325):494–495PubMedCrossRefGoogle Scholar
  31. 31.
    Haskell RC, Carlson FD, Blank PS (1989) Form birefringence of muscle. Biophys J 56(2):401–413PubMedCrossRefGoogle Scholar
  32. 32.
    Granato M, van Eeden FJ, Schach U, Trowe T, Brand M, Furutani-Seiki M, Haffter P, Hammerschmidt M, Heisenberg CP, Jiang YJ, Kane DA, Kelsh RN, Mullins MC, Odenthal J, Nusslein-Volhard C (1996) Genes controlling and mediating locomotion behavior of the zebrafish embryo and larva. Development 123:399–413PubMedGoogle Scholar
  33. 33.
    Gahtan E, Baier H (2004) Of lasers, mutants, and see-through brains: functional neuroanatomy in zebrafish. J Neurobiol 59(1):147–161PubMedCrossRefGoogle Scholar
  34. 34.
    Zhou FQ, Snider WD (2006) Intracellular control of developmental and regenerative axon growth. Philos Trans R Soc Lond B 361(1473):1575–1592CrossRefGoogle Scholar
  35. 35.
    Rozengurt E (2007) Mitogenic signaling pathways induced by G protein-coupled receptors. J Cell Physiol 213(3):589–602PubMedCrossRefGoogle Scholar
  36. 36.
    Leone AM, Errico M, Lin SL, Cowen DS (2000) Activation of extracellular signal-regulated kinase (ERK) and Akt by human serotonin 5-HT(1B) receptors in transfected BE(2)-C neuroblastoma cells is inhibited by RGS4. J Neurochem 75(3):934–938PubMedCrossRefGoogle Scholar
  37. 37.
    He JC, Neves SR, Jordan JD, Iyengar R (2006) Role of the Go/i signaling network in the regulation of neurite outgrowth. Can J Physiol Pharmacol 84(7):687–694PubMedCrossRefGoogle Scholar
  38. 38.
    Nixon AB, Grenningloh G, Casey PJ (2002) The interaction of RGSZ1 with SCG10 attenuates the ability of SCG10 to promote microtubule disassembly. J Biol Chem 277(20):18127–18133PubMedCrossRefGoogle Scholar
  39. 39.
    Liu Z, Chatterjee TK, Fisher RA (2002) RGS6 interacts with SCG10 and promotes neuronal differentiation. Role of the G gamma subunit-like (GGL) domain of RGS6. J Biol Chem 277(40):37832–37839PubMedCrossRefGoogle Scholar
  40. 40.
    Willard FS, Willard MD, Kimple AJ, Soundararajan M, Oestreich EA, Li X, Sowa NA, Kimple RJ, Doyle DA, Der CJ, Zylka MJ, Snider WD, Siderovski DP (2009) Regulator of G-protein signaling 14 (RGS14) is a selective H-Ras effector. PLoS ONE 4(3):e4884PubMedCrossRefGoogle Scholar
  41. 41.
    Heo K, Ha SH, Chae YC, Lee S, Oh YS, Kim YH, Kim SH, Kim JH, Mizoguchi A, Itoh TJ, Kwon HM, Ryu SH, Suh PG (2006) RGS2 promotes formation of neurites by stimulating microtubule polymerization. Cell Signal 18(12):2182–2192PubMedCrossRefGoogle Scholar
  42. 42.
    Read DE, Gorman AM (2009) Involvement of Akt in neurite outgrowth. Cell Mol Life Sci 66(18):2975–2984PubMedCrossRefGoogle Scholar
  43. 43.
    Bommakanti RK, Vinayak S, Simonds WF (2000) Dual regulation of Akt/protein kinase B by heterotrimeric G protein subunits. J Biol Chem 275(49):38870–38876PubMedCrossRefGoogle Scholar
  44. 44.
    Zheng J, Shen WH, Lu TJ, Zhou Y, Chen Q, Wang Z, Xiang T, Zhu YC, Zhang C, Duan S, Xiong ZQ (2008) Clathrin-dependent endocytosis is required for TrkB-dependent Akt-mediated neuronal protection and dendritic growth. J Biol Chem 283(19):13280–13288PubMedCrossRefGoogle Scholar
  45. 45.
    Markus A, Zhong J, Snider WD (2002) Raf and akt mediate distinct aspects of sensory axon growth. Neuron 35(1):65–76PubMedCrossRefGoogle Scholar
  46. 46.
    Jaworski J, Spangler S, Seeburg DP, Hoogenraad CC, Sheng M (2005) Control of dendritic arborization by the phosphoinositide-3′-kinase-Akt-mammalian target of rapamycin pathway. J Neurosci 25(49):11300–11312PubMedCrossRefGoogle Scholar
  47. 47.
    Wang Y, Yang F, Fu Y, Huang X, Wang W, Jiang X, Gritsenko MA, Zhao R, Monore ME, Pertz OC, Purvine SO, Orton DJ, Jacobs JM, Camp DG 2nd, Smith RD, Klemke RL (2011) Spatial phosphoprotein profiling reveals a compartmentalized ERK switch governing neurite growth and retraction. J Biol Chem 286(20):18190–18201PubMedCrossRefGoogle Scholar
  48. 48.
    Soundararajan P, Fawcett JP, Rafuse VF (2010) Guidance of postural motoneurons requires MAPK/ERK signaling downstream of fibroblast growth factor receptor 1. J Neurosci 30(19):6595–6606PubMedCrossRefGoogle Scholar
  49. 49.
    Lang UE, Puls I, Muller DJ, Strutz-Seebohm N, Gallinat J (2007) Molecular mechanisms of schizophrenia. Cell Physiol Biochem 20(6):687–702PubMedCrossRefGoogle Scholar
  50. 50.
    Jaaro-Peled H, Hayashi-Takagi A, Seshadri S, Kamiya A, Brandon NJ, Sawa A (2009) Neurodevelopmental mechanisms of schizophrenia: understanding disturbed postnatal brain maturation through neuregulin-1-ErbB4 and DISC1. Trends Neurosci 32(9):485–495PubMedCrossRefGoogle Scholar
  51. 51.
    Bellon A (2007) New genes associated with schizophrenia in neurite formation: a review of cell culture experiments. Mol Psychiatry 12(7):620–629PubMedCrossRefGoogle Scholar
  52. 52.
    Bellon A, Krebs MO, Jay TM (2011) Factoring neurotrophins into a neurite-based pathophysiological model of schizophrenia. Prog Neurobiol 94(1):77–90PubMedCrossRefGoogle Scholar

Copyright information

© Springer Basel 2012

Authors and Affiliations

  • Yi-Chuan Cheng
    • 1
    Email author
  • Paul J. Scotting
    • 2
  • Li-Sung Hsu
    • 3
  • Sheng-Jia Lin
    • 1
  • Hung-Yu Shih
    • 1
  • Fu-Yu Hsieh
    • 1
  • Hui-Lan Wu
    • 1
  • Chu-Li Tsao
    • 1
  • Chia-Jung Shen
    • 1
  1. 1.Graduate Institute of Biomedical Sciences, School of MedicineChang Gung UniversityTaoyuanTaiwan
  2. 2.Children’s Brain Tumour Research Centre, Centre for Genetics and Genomics, Queen’s Medical CentreUniversity of NottinghamNottinghamUK
  3. 3.Institute of Biochemistry and BiotechnologyChung Shan Medical UniversityTaichungTaiwan

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