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