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Shh Induces Motor Neuron Differentiation in P19 Embryonic Carcinoma Cells

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Shh Induces Motor Neuron Differentiation in P19 Embryonic Carcinoma Cells 의의의학학학석석석사사사학학학위위위논논논문문문 Shh induces Motor Neuron differentiation in P19 Embryonic Carcinoma Cells 아아아주주주대대대학학학교교교 대대대학학학원원원 의의의학학학과과과 박박박래래래희희희 Shh induces Motor Neuron Differentiation in P19 Embryonic Carcinoma Cells by Rae Hee Park A Dissertation Submitted to The Graduate School of Ajou University in Partial Fulfillment of the Requirements for the Degree of MASTER OF MEDICAL SCIENCES Supervised by Haeyoung Suh-Kim, Ph.D. Department of Medical Sciences The Graduate School, Ajou University August, 2005 박박박래래래희희희의의의의의의학학학석석석사사사학학학위위위논논논문문문을을을인인인준준준함함함... 심심심사사사위위위원원원장장장 서서서 해해해 영영영 인인인 심심심사사사위위위원원원 이이이 영영영 돈돈돈 인인인 심심심사사사위위위원원원 조조조 은은은 혜혜혜 인인인 아아아주주주대대대학학학교교교 대대대학학학원원원 222000000555년년년666월월월222222일일일 - ABSRACT- Sonic hedgehog induces Motor Neuron Differentiation in P19 Embryonic Carcinoma Cells Sonic hedgehog (Shh) is a member of the hedgehog family of signalling molecules and secreted from two signalling centers, the notochord and the floor plate, where it functions as a morphogen to induce early dorso-ventral patterning of the cenral nervous system (CNS). More recently, multiple actions of Shh during CNS development have been discovered in additional sites, where it specifies the fates of oligodendrocytes and motor neurons as well as proliferation of neural precursors and control of axon outgrowth. To explore the roles of Shh in neuronal differentiation, we utilized embryonal carcinomal P19 cells as an in vitro model system. We overexpressed Shh in P19 cells and investigated it’s effects on proliferation and differentiation of P19 cells., P19/Shh, P19 cells overexpressing Shh, proliferated at higher rates than normal P19 cells even when normal P19 cells stopped growth and underwent differentiation. Ironically, P19/Shh also differentiated into neurons at higher rates than normal P19 cells. Upregulation of both proliferation and neuronal differentiation P19/Shh suggests that Shh may induce neuronal fates from uncommitted P19 cells and concomitantly promote the proliferation of the resulting neuronal precursor cells. As expected motor neuron markers (Hb9, i ChAT) as well as a dopaminergic neuron marker (TH) were upregulated in neuronal cells derived from P19/Shh. Collectively, these results suggest that Shh may have multiple functions in neural induction of uncommitted stem cells, proliferation of neural precursors, and dorso-ventral patterning of the CNS. Key words : Sonic hedgehog, Proliferation, Differentiation, Motor neuron, P19 cells ii TABLE OF CONTENTS ABSTACT ------------------------------------------------------------------------ i TABLE OF CONTENTS ------------------------------------------------------------- ii LIST OF FIGURES ------------------------------------------------------------------- vi LIST OF TABLE ----------------------------------------------------------------------- viii LIST OF ABBREVIATIONS ------------------------------------------------------- ix I. INTRODUCTION ----------------------------------------------------------------- 1 A. Sonic hedgehog ----------------------------------------------------------------- 1 B. Transcriptional regulation of development by Shh --------------------- 4 1. Midbrain dopaminergic neuron --------------------------------------------- 4 2. Spinal cord motor neuron ---------------------------------------------------- 5 C. P19 embryonic carcinoma cells --------------------------------------------- 6 II. MATERIALS AND METHODS ----------------------------------------------- 8 A. Materials ------------------------------------------------------------------------- 8 B. Mathods ------------------------------------------------------------------------- 9 1. P19 cell culture ---------------------------------------------------------------- 9 2. Purification & titeration of 5E1 antibody ---------------------------------- 9 2.1 Affinity chromatography ------------------------------------------------ 9 2.2 Enzyme-Linked Immunosorbent (ELISA) Assay --------------------- 10 3. Preparation of Shh conditioned medium ----------------------------------- 10 4. Concentration of secreted ShhN -------------------------------------------- 11 iii 5. Growth kinetics ---------------------------------------------------------------- 11 6. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay ------------------------------------------------------------------- 12 7. Differentiation of P19 cell --------------------------------------------------- 12 7.1 Conventional neuronal differentiation --------------------------------- 12 7.2 Current neuronal differentiation ---------------------------------------- 13 8. Reverse Transriptase-Polymerase Chain Reaction (RT-PCR) ---------- 14 9. Western blotting --------------------------------------------------------------- 15 10. Immunocytochemistry ------------------------------------------------------ 16 III. RESULTS ------------------------------------------------------------------------ 17 1. Expression of hShhN in P19 cells ------------------------------------------ 17 2. Preparation of 5E1, a neutralizing antibody against Shh ---------------- 19 3. The concentration of secreted Shh ------------------------------------------ 19 4. Shh induces proliferation of P19 cells -------------------------------------- 22 5. Shh induces neuronal differentiation of P19 cells ------------------------ 25 6. Shh promotes neuronal precursor cell/ motor neurons survival or proliferation --------------------------------------------------------------- 28 7. Shh induces motor neuron differentiation in P19 cells ------------------- 30 8. Shh induces dopaminergic and motor neuron differentiation in P19 cells ------------------------------------------------------------------------ 34 9. Shh promotes neuronal differentiation in P19 cells without treatment of RA or aggregation culture ---------------------------------------------- 37 iv IV. DISCUSSION --------------------------------------------------------------------- 39 V. CONCLUSIONS -------------------------------------------------------------------- 43 REFERENCES -------------------------------------------------------------------- 44 국문요약 ------------------------------------------------------------------------------- 54 v LIST OF FIGURES Fig. 1. Phylogentic relationship of numbers of the Hh protein family --------- 2 Fig. 2. The Shh signaling pathway ----------------------------------------------------- 3 Fig. 3. Shh-activated transcriptional pathway of spinal motor neuron generation----------------------------------------------------------------------- 6 Fig. 4. The method for neural induction of P19 cells ----------------------------- 13 Fig. 5. Generation of ShhN-Producing P19 Cell Lines --------------------------- 18 Fig. 6. Preparation of 5E1, a neutralizing antibody against Shh ----------------- 20 Fig. 7. Soluble Shh activity is inhibited by Shh neutralizing antibody, 5E1 --- 21 Fig. 8. Shh induces proliferation of P19 cells -------------------------------------- 23 Fig. 9. Cell viability of P19, P19/hShhN cells was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay ------------------------------------------------------------------------------------ 24 Fig. 10. Neuronal differentiation of P19, P19/hShhN cells ----------------------- 27 Fig. 11. eGFP-labeled neuronal precursors in P19/pNPEeGFP ------------------ 29 Fig. 12. RT-PCR of the expression of HD, bHLH and motor neuron specific markers, Nkx6.1, Olig2, Ngn2, NeuroD, Hb9 and ChAT in P19 and P19/hShhN -------------------------------------------------------------- 31 Fig. 13. Shh expression induces motor neuron differentiation of P19 cells (1) -- 32 Fig. 14. Shh expression induces motor neuron differentiation of P19 cells (2) -- 33 Fig. 15. Motor neurons and Dopamine neurons of differentiation in P19 cells vi ---------------------------------------------------------------------------------- 35 Fig. 16. Shh expression induces dopamine neurons and motor neurons differentiation of P19 cells --------------------------------------------------- 36 Fig. 17. Shh promotes neuronal differentiation in P19 cells without treatment RA or aggregation culture -------------------------------------------------- 38 vii LIST OF TABLES Table 1. Primer sequence for RT-PCR ------------------------------------------------ 14 viii LIST OF ABBREVIATION bHLH b helix-loop-helix bp base pair ChAT Choline acetyltransferase CNPase 2', 3'-cyclic nucleotide 3'-phosphodiesterase CNS Central nerve system DA Dopaminergic neuron Dhh Desert hedgehog DMEM Dulbecco's modified Eagle's medium ECL Enhanced chemiluminescence ELISA Enzyme-Linked Immunosorbent Assay FBS Fetal bovine serum FGF8 Fibroblast growth factor 8 FITC Fluorescein isothiocyanate Fsk Forskolin GFAP Glial fibrillary acidic protein GFP Green fluorescent protein HAT Hypoxanthine-Aminopterin Thymidine Hb9 Homeobox gene 9 HBSS Hank's balanced salt solution HD Homeodomain ix Hh Hedgehog HRP Horseradish peroxidase IgG Immuonglobulin G Ihh Indian hedgehog MN Motor neuron NCAM Neuronal cell adhesion molecule NF-M Neurofilament-160 kDa Ngn2 Neurogenin 2 OD Optical density Olig2 Oligodendrocyte transcription factor 2 OPD o-phenylenediamine dihydrochloride P19 cell Mouse embryonic carcinoma cell PAGE Polyacrylamide gel electrophoresis PBS Phosphate buffered saline pMN Motor neuron presursor Ptc Patched RA Retinoic acid RIPA Radioimmunoprecipitation RT-PCR Revese transcriptase - polymerase chain reaction SDS Sodium dodecyl sulfate Shh Sonic hedgehog Smo Smoothened x TBS Tris buffered saline TH Tyrosine
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