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Heterochronic Pellet Assay to Test Cell-cell Communication in the Mouse Retina

采用异时性细胞团分析法检测小鼠视网膜中的细胞间通讯

NT Nobuhiko Tachibana
DZ Dawn Zinyk
RR Randy Ringuette
VW Valerie Wallace
CS Carol Schuurmans

发布: 2017年02月05日第7卷第3期 DOI: 10.21769/BioProtoc.2117 浏览次数: 11221

评审: Oneil G. BhalalaAnonymous reviewer(s)

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Abstract

All seven retinal cell types that make up the mature retina are generated from a common, multipotent pool of retinal progenitor cells (RPCs) (Wallace, 2011). One way that RPCs know when sufficient numbers of particular cell-types have been generated is through negative feedback signals, which are emitted by differentiated cells and must reach threshold levels to block additional differentiation of that cell type. A key assay to assess whether negative feedback signals are emitted by differentiated cells is a heterochronic pellet assay in which early stage RPCs are dissociated and labeled with BrdU, then mixed with a 20-fold excess of dissociated differentiated cells. The combined cells are then re-aggregated and cultured as a pellet on a membrane for 7-10 days in vitro. During this time frame, RPCs will differentiate, and the fate of the BrdU+ RPCs can be assessed using cell type-specific markers. Investigators who developed this pellet assay initially demonstrated that neonatal RPCs give rise to rods on an accelerated schedule compared to embryonic RPCs when the two cell types are mixed together (Watanabe and Raff, 1990; Watanabe et al., 1997). We have used this assay to demonstrate that sonic hedgehog (Shh), which we found acts as a negative regulator of retinal ganglion cell (RGC) differentiation, promotes RPC proliferation (Jensen and Wallace, 1997; Ringuette et al., 2014). More recently we modified the heterochronic pellet assay to assess the role of feedback signals for retinal amacrine cells, identifying transforming growth factor β2 (Tgfβ2) as a negative feedback signal, and Pten as a modulator of the Tgfβ2 response (Ma et al., 2007; Tachibana et al., 2016). This assay can be adapted to other lineages and tissues to assess cell-cell interactions between two different cell-types (heterotypic) in either an isochronic or heterochronic manner.

Keywords: Heterochronic pellet assay (异时性细胞团分析法) search Retinal differentiation (视网膜分化) search Retinal progenitor cells (视网膜祖细胞) search Re-aggregation (重新聚合) search Amacrine cell (无长突细胞) search Negative feedback signaling (负反馈信号) search Heterotypic cell interactions (异型细胞相互作用) search

Background

Several mechanisms are employed to ensure that the correct numbers of differentiated cells are generated during organ and tissue development. For example, progenitor cells may respond to the levels of hormones or growth factors secreted by differentiated cells, progenitors may count the number of divisions they undergo, or there may be a mechanism to count the final number of differentiated cells (Lui and Baron, 2011). In the retina, negative feedback signals that are secreted by differentiated cells are sensed by progenitor cells, which stop producing that differentiated cell type when the signals reach threshold levels (Belliveau and Cepko, 1999; Reh and Tully, 1986; Waid and McLoon, 1998). We and other have demonstrated that Shh is an essential negative regulator of a RGC fate (Wang et al., 2005; Zhang and Yang, 2001). We also dissected the feedback process for retinal amacrine cells, showing that the transcription factor Zac1 acts in amacrine cells to initiate transforming growth factor b2 (Tgfb2) expression, which negatively regulates RPC proliferation and amacrine cell differentiation (Ma et al., 2007). Notably, other TGFβ family members have similar feedback functions in the olfactory epithelium (Wu et al., 2003), pancreas (Harmon et al., 2004), and skeletal muscle (Tobin and Celeste, 2005). We also used the heterochronic pellet assay to examine how amacrine cell feedback signals are themselves regulated. We found that Pten is an essential positive regulator of amacrine cell differentiation, and using the pellet assay, we demonstrated that Pten acts in RPCs to control responsiveness to Tgfβ2 signaling (Tachibana et al., 2016). Understanding how amacrine cells and RPCs interact provides important new insights into how cell number is controlled in the retina. Notably, similar interactions between Pten and Tgfβ signaling may underlie cell number control in other vertebrate organs where Tgfβ signaling is an important determinant of organ size.

Materials and Reagents

  1. Fisherbrand sterile 100 x 15 mm polystyrene Petri dish (Thermo Fisher Scientific, Fisher Scientific, catalog number: FB0875713 )
  2. 15 ml conical tubes (SARSTEDT, catalog number: 62.554.502 )
  3. Sterile individually packaged 5 ml pipettes (SARSTEDT, catalog number: 86.1253.001 )
  4. Tissue culture 24-well plates (SARSTEDT, catalog number: 83.3922.300 )
  5. SamcoTM extra long transfer pipet (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 262 )
  6. 13 mm, 0.8 μm Nuclepore Track-Etch membrane (GE Healthcare, catalog number: 110409 )
  7. Kimwipes (Kimberly-Clark Worldwide, catalog number: 34120 ) (not autoclaved, but kept clean)
  8. Superfrost Plus Micro slides (VWR, catalog number: 48311-703 )
  9. Standard microscope slide box (Heathrow Scientific, catalog number: HEA15991A )
  10. Micro cover glass (VWR, catalog number: 48404-454 )
  11. 4 in x 250 ft Parafilm roll (Bemis, catalog number: PM999 or VWR, catalog number: 52858-032 )
  12. 50 ml corning tube (SARSTEDT, catalog number: 62.547.254 )
  13. 0.22 μm sterilize filter filtropur (SARSTEDT, catalog number: 83.1826.001 )
  14. 60 ml syringe (Medtronic, catalog number: 8881560125 )
  15. Rapid-FlowTM sterile disposable bottle top filters (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 291-4520 )
  16. E15.5 CD1 mouse (Charles River Laboratories International, catalog number: 022 ) (see Note 1)
  17. P2 Ptenfl/fl; Pax6-Cre+ or Pten+/+ mouse (see Note 2)
  18. 1x Ca2+/Mg2+-free DPBS (Thermo Fisher Scientific, GibcoTM, catalog number: 14190250 )
  19. Trypan blue (Thermo Fisher Scientific, GibcoTM, catalog number: 15250061 )
  20. Aqua-Poly/Mount (Polysciences, catalog number: 18606 )
  21. Trypsin (Sigma-Aldrich, catalog number: T1005 )
  22. Heat inactivated FBS (fetal bovine serum) (Thermo Fisher Scientific, GibcoTM, catalog number: 12484028 )
  23. DMEM (Thermo Fisher Scientific, GibcoTM, catalog number: 11965092 )
  24. 1x HBSS (Thermo Fisher Scientific, GibcoTM, catalog number: 24020117 )
  25. Heat inactivated horse serum (Thermo Fisher Scientific, GibcoTM, catalog number: 26050088 )
  26. 200 mM L-glutamine (Sigma-Aldrich, catalog number: G7513 )
  27. HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 15630080 )
  28. Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  29. Amphotericin B (Thermo Fisher Scientific, GibcoTM, catalog number: 15290026 )
  30. 5-Bromouridine (BrdU) (Sigma-Aldrich, catalog number: 850187 )
  31. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
  32. Potassium chloride (KCl) (EMD Millipore, catalog number: PX1405 )
  33. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5379 )
  34. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S7907 )
  35. Crystalline PFA (Sigma-Aldrich, catalog number: P6148 )
  36. Sucrose (Sigma-Aldrich, catalog number: S9378 )
  37. 11.8 M hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 258148 )
  38. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  39. 4’,6-diamidino-2-phenylindole, dihydrochloride (DAPI) as pre-prepared working solution that is used as described by the manufacturer (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: D1306 )
  40. Antibodies against Pax6 and BrdU (see Recipes, Table 1)
    Pax6 (BioLegend, catalog number: 901301 )
    BrdU (Bio-Rad Laboratories, catalog number: OBT0030 )
    Donkey Anti-rabbit Alexa-Fluor 488 (Thermo Fisher Scientific, InvitrogenTM, catalog number: A-21206 )
    Goat Anti-rat Alexa Fluor 568 (Thermo Fisher Scientific, InvitrogenTM, catalog number: A-11077 )
  41. Optimal cutting temperature compound (VWR, catalog number: 95057-838 )
  42. 2.5% trypsin (see Recipes)
  43. 0.125% trypsin (see Recipes)
  44. Retinal explant media (REM) (see Recipes)
  45. 1 mg/ml BrdU (see Recipes)
  46. 10x phosphate-buffered saline (PBS) (see Recipes)
  47. 1x phosphate-buffered saline (PBS) (See Recipes)
  48. 20% paraformaldehyde (PFA) (see Recipes)
  49. 4% paraformaldehyde (PFA) (See Recipes)
  50. 20% sucrose (see Recipes)
  51. 2 N hydrochloric acid (see Recipes)
  52. 1x phosphate-buffered saline/0.1% Triton X-100 (PBT) (see Recipes)
  53. Blocking solution (see Recipes)

Equipment

  1. Dumont forceps #5 (Fine Science Tools, catalog number: 11252-20 )
  2. Dumont forceps #55 (Fine Science Tools, catalog number: 11255-20 )
  3. Dumont forceps AA (Fine Science Tools, catalog number: 11210-20 )
  4. Shallow form shaking water bath (Precision Scientific, catalog number: 66799 )
  5. Pipette pump (SP Scienceware - Bell-Art Products - H-B Instrument, catalog number: F37898-0000 )
  6. 37 °C, 5% CO2 water jacketed incubator (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3110 )
  7. Refrigerated tabletop centrifuge for 15 ml conical tubes (Eppendorf, model: 5810 R )
  8. Hemacytometer chamber (Hausser Scientific, catalog number: 3100 )
  9. P20 pipetmen (Gilson, catalog number: F123600 )
  10. P200 pipetmen (Gilson, catalog number: F123601 )
  11. P1000 pipetmen (Gilson, catalog number: F123602 )
  12. Cryostat (Leica Biosystems, model: CM3050 S )
  13. -20 °C freezer
  14. Upright fluorescence microscope (Leica Microsystems, model: DM RXA2 )
  15. Autoclave
  16. 1 L beaker (Corning, Pyrex®, catalog number: 1395-1L )
  17. 500 ml beaker (Corning, Pyrex®, catalog number: 1395-500 )
  18. 250 ml Erlenmeyer flask (Corning, Pyrex®, catalog number: 4450-250 )
  19. Fume hood
  20. Stereomicroscope for dissection (Leica Microsystems, model: MZ6 )
  21. Inverted light microscope (Leica Microsystems, model: DMIL LED )

Software

  1. GraphPad Prism (GraphPad Software)

Procedure

English
中文翻译

文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

  1. Tachibana, N., Zinyk, D., Ringuette, R., Wallace, V. and Schuurmans, C. (2017). Heterochronic Pellet Assay to Test Cell-cell Communication in the Mouse Retina. Bio-protocol 7(3): e2117. DOI: 10.21769/BioProtoc.2117.
  2. Tachibana, N., Cantrup, R., Dixit, R., Touahri, Y., Kaushik, G., Zinyk, D., Daftarian, N., Biernaskie, J., McFarlane, S. and Schuurmans, C. (2016). Pten regulates retinal amacrine cell number by modulating Akt, Tgfbeta, and Erk signaling. J Neurosci 36(36): 9454-9471.

    3. ris ris Download Citation in RIS Format

分类

神经科学 > 细胞机理 > 胞内信号传导

神经科学 > 感觉和运动系统 > 视网膜

细胞生物学 > 细胞信号传导 > 胞内信号传导

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