I. The zebrafish retinotectal system and RGC labeling methods
Transcript of I. The zebrafish retinotectal system and RGC labeling methods
I. The zebrafish retinotectal system�and RGC labeling methods
Rolf Karlstrom WH ZF Course 2013 Pit Talk
II. Conditional Gene Regulation �(local gene activation by heat shock)
Retinotectal lab outline Dye labeling of retinal axons
- Whole eye injections - Focal labeling to visualize topographic projections
Conditional Gene Regulation - Global Heat Shock to manipulate Hh signaling - Local Heat Shock (24 hpf and 5 dpf)
Tonight and Tomorrow afternoon: Assay gene activation after HS (GFP) Axon Imaging, find a scope and go for it
The fourth annual dye fill/local HS�Zebrafish Course
Photo Contest The Rules
Anything goes
S(he) with the best scope wins? sharp elbows help
S(he) with the best imaging assistant really wins? bribery definitely not disallowed
Upload Images to the iMac by Friday morning?
I. The zebrafish retinotectal system�and RGC labeling methods
Rolf Karlstrom WH ZF Course 2012 Pit Talk
The retinotectal system:�the classic example of sensory mapping
Development of the Zebrafish Retinotectal Projection
30 hours 48 hours
AT/Gfap
Optic Chiasm Tectal
Lobes
A well formed sensory map by 5 dpf
Screened > 3000 genomes
(injected > 120,000 fish)
Identified > 35 essential genes
A large scale screen for genes affecting retinotectal axon guidance
Bonhoeffer Lab Nusslein-Volhard Lab
Tübingen, Germany
Regional DiI/DiO Eye labeling
You won’t do this
• large screen, >35 genes affecting each step of the RT pathway • 15 years later, the majority have now been cloned • Hasn’t been repeated YET. There are more genes out there
Original Papers in Development Zebrafish Screen Issue Dec. 1996, v123 Baier et al., 1996 Karlstrom et al., 1996 Trowe et al., 1996
Your job will be to find one of these mutants
Reviews: Culverwell&Karlsrom2002, seminars in CELL&DEV BIO, v13 Hutson&Chien 2002, Current Opinion in Neurobiology, v12
bel(lhx2) uml(boc)
Finding the genes required for proper wiring
Connectivity defects correlate with OKR defects
Lipophilic dyes (DiI and DiO)
• dissolve into plasma membranes • very bright, bleach slowly, relatively nontoxic • can be used in live or aldehyde-fixed tissues • can be used for antero- or retrograde labelling
DiIC18(3) “DiI” DiOC18(3) “DiO”
Watching it happen #2: Growth cone making a mistake (Chien Lab) Live labeling allows visualization of growth cone behaviors
Laura Hutson, Chien Lab
astray(robo2)
Exquisite labeling of processes in fixed tissue
DiO
DiI
Whole-eye fills and focal application of DiI/DiO�(you will do this)
RGC axons are anterogradely labeled in 6-12 hours Can speed up diffusion by placing at 37°
1) Pressure Injection of 1% DiI or DiO in chloroform
2) Focal application of DiI or DiO crystals
RGCs
lens Optic Nerve
Previous photo contest stand-outs
Confocal image by Phillip Keller, ‘10
$ 8.50 for a 3D movie???? Pixar award
Confocal image by Rita Fior, ‘10
astray(robo2)
Why Conditional gene expression? • Temporal control: to uncover late functions of gene required in early development (or any gene)
• Spatial control: uncover roles in different tissues/cells.
• e.g. manipulate expression of guidance molecules, see how migrating axons or cells respond.
Forebrain patterning mutants Example: late functions of Hh impossible to uncover using the existing mutants
yot(gli2)
Ideally, we want both spatial and temporal control of gene expression
Lots of ways have been developed to do this (not an exhaustive list): Spatial control: cell type-specific enhancers/promoters gal4-UAS transgenes (fly, now fish) lots of gal4 drivers, silencing/variegation has been an issue cre-lox system (mouse, now fish) leaky at permissive temperatures?
Temporal control: Heat shock of transgenes driven by a heat shock sensitive promoter
Time and Space: Local Heat shock Tetracycline (reverse tetracycline-controlled transcriptional transactivator (rtTA) Heat shock or estrogen-receptor-controlled Cre (CreER) Ecdysone receptor-Gal4 chimeric protein to control Gal4 activity
Turns out zebrafish are great for HS control of gene expression -cold-blooded and have a reasonable temperature range (20-33°C as larvae) -30-60 min at 37°C (20 min at 40°C) induces a strong heat- shock response
Shen and Ozacar et al, 2013. Heat-Shock–Mediated Conditional Regulation of Hedgehog/Gli Signaling in Zebrafish DEVELOPMENTAL DYNAMICS 242:539–549
Campbell, Willoughby, and Jensen (2012) Two types of Tet-On transgenic lines for doxycycline-inducible gene expression in zebrafish and photoreceptors and a gateway-based tet-on toolkit. PLoS One v7(12)
Hans S, Freudenreich D, Geffarth M, Kaslin J, Machate A, et al. (2011) Generation of a non-leaky heat shock–inducible Cre line for conditional Cre/lox strategies in zebrafish. Dev Dyn 240: 108–115
Knopf F, Schnabel K, Haase C, Pfeifer K, Anastassiadis K, et al. (2010) Dually inducible TetON systems for tissue-specific conditional gene expression in zebrafish. Proc Natl Acad Sci U S A 107: 19933–19938.
Hesselson D, Anderson RM, Beinat M, Stainier DYR (2009) Distinct populations of quiescent and proliferative pancreatic β-cells identified by HOTcre mediated labeling. Proc Natl Acad Sci U S A 106: 14896–14901
Hans S, Kaslin J, Freudenreich D, Brand M (2009) Temporally-controlled site-specific recombination in zebrafish. PLoS ONE 4: e4640.
Halpern ME, Rhee J, Goll MG, Akitake CM, Parsons M, et al. (2008) Gal4/UAS transgenic tools and their application to zebrafish. Zebrafish 5: 97–110.
Asakawa K, Kawakami K (2008) Targeted gene expression by the Gal4-UAS system in zebrafish. Dev Growth Differ 50: 391–399.
Emelyanov A, Parinov S (2008) Mifepristone-inducible LexPR system to drive and control gene expression in transgenic zebrafish. Dev Biol 320: 113–121
Esengil H, Chang V, Mich JK, Chen JK (2007) Small-molecule regulation of zebrafish gene expression. Nat Chem Biol 3: 154–155.
Huang CJ, Jou TS, Ho YL, Lee WH, Jeng YT, et al. (2005) Conditional expression of a myocardium-specific transgene in zebrafish transgenic lines. Dev Dyn 233: 1294–1303
Nasevicius A, Ekker SC (2000) Effective targeted gene ‘knockdown’ in zebrafish. Nat Genet 26: 216–220.
A few refs on conditional gene regulation in fish:
Tet On System Adapted for ZFish Jensen Lab UMass plus many others Campbell et al, 2012
1) Location: promoter of interest here
2) Gene manipulation constructs:
3) Timing= Add antibiotic
Today’s lab: Conditional manipulation of Hh/Gli signaling.
Gli1, Gli2, Gli3
CyA Tg(hsp70l:shhgfp)
Transcriptional Response Differentiation (homeo/bHLH TFs) Proliferation (cyclins, n-myc) Survival (bcl)
Tg(hsp70l:Gliactgfp) Tg(hsp70l:Gli2DRgfp)
HSP70l Transgenic Lines
Tg(GBS-ptc:gfp) nucleus
Tg(hsp70l:dnPKAgfp)
D
PKA
Shh-N Shh-C
Gli1 activator
hsp70l
tol2
ZnF
Dom Neg PKA
tol2
GFP
Gli2 Dom Rep ZnF
GFP
GFP
GFP 30 min. heat shock 37°
4 hs transgenic lines
30 min. heat shock 37°
Heat shock regulation of Hedgehog Signaling
Meng-Chieh Shen
Heat shock regulated Hh transgenes Gli2 DR
GFP
Shh-N hsp70 GFP
tol2 Shh-C
hsp70
Shh Gli1
nkx6.1 nucleus
cyto
Tg(hsp70;shh-gfp)
-hs +hs
Gli2 DR GFP tol2 hsp70
Tg(hsp70;gli2DR-gfp)
+hs
-hs
Shh Gli1
ptc1 nucleus
cyto
Gli2DR
A. Tuba Ozacar
Blocking Hh cell-autonomously
How long does it take to induce the transgene and make functional protein?
How long does the transgene stay activated?
How long does the trans-protein last/function?
Levels of expression?
Fusion proteins: localization and function?
What controls would you include in each experiment?
You have a transgenic line (hsp70l:gli2DR-PKA) What would you need to know to use this line?
Gli2 DR GFP tol2 hsp70l
slow muscle fibers
wt + hs
Tg + hs
s16 s23
s16
Tg(hsp70;gli2DR-GFP)
slow muscle fiber differentiation requires Hh/Gli signaling
In vivo test for kinetics of transgene activation (Remember this Trick)
Shen and Ozacar et al, 2013 qPCR
Talk review: slit midline repellent and its receptor, Robo
robo mutant - axons cross and recross freely, fewer longitudinals
slit mutant - axons
don’t stay away from
midline
Slit and Robo in flies: midline repellent (slit) and its receptor
+ + + + + + + + + + + + + + + + + + +
Netrin
Bashaw and Goodman 1999
Talk review: slit midline repellent and its receptor, Robo
robo mutant - axons cross and recross freely, fewer longitudinals
slit mutant - axons
don’t stay away from
midline
Slit and Robo in flies: midline repellent (slit) and its receptor
+ + + + + + + + + + + + + + + + + + +
Netrin
Bashaw and Goodman 1999 Prediction for Robo mutant in fish?
Robo/Slit in Fish
Barresi et al, 2005
Complications: even more slits.
Axon Guidance Experiment: Tg(hsp70l:slit1a-GFP)
Timing of HS: Would location matter? Predictions?
Mike Placinta
Spatial control: how to activate gene Expression only in small regions?
Tg(hsp70gfp)
GFP
hsp70l promoter
Pulled optical fibers allow precise heating
The digital thermomenter/thermocouple is key
Unexpected use: Fate mapping
nkx2.2a/pax7
PD PI
Retinotectal lab outline Logistics (1pm-6pm slot): 1-2 PM: Dye Fill Demos in main lab, talk through Local HS device room 158
2-3 PM: Break into 3 groups. Group 1 : Local Heat shock in back room (2 rigs) Demo by Jason or Rolf, then mount and HS 5 dpf and 24 hpf ghoti.
Groups 2,3: Eye Fills
3 PM: Switch, group 2 to local HS,
4 PM : Switch again
~ 5:15: Wrap-up and look at some injected fish!
Imaging, let’s talk about this