“The Storyboard Method for Research Strategy and

Management”

Rebecca Sappington, Ph.D. Assistant Professor

Ophthalmology and Visual Sciences, Pharmacology and Neuroscience

Responsibilities of a PI Manage progress within projects as well

as for individuals Make strategic decisions regarding project

direction Recognize benchmarks in project Publications/Abstracts/Presentations

Identify new lines of investigation based on outcomes of current project(s) Grant applications

PIs assemble pieces into a cohesive vision.

Need to see the “big picture”

Not so simple…

Multiple projects with intersecting points of interest Multiple individuals contributing to a single project Multiple individuals contributing to multiple projects with various intersecting points of interest

A successful method of project management is key!

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Needs for Project Management

• Organizing data • Tracking progress • Synthesizing data • Strategizing for future experiments

Goal: Single method of project management that achieves all of the these needs.

The Storyboard

David J. Calkins, Ph.D. Vice Chairman and Director of Research Professor Vanderbilt Eye Institute

Based on storyboards used to produce movies!

“The Waterhorse” Storyboard: Take 1 Craig Woolheater

Advantages: PI • Project Management

– Assess progress of individuals and project with a glance at any time

– Make strategic decisions about project direction – Increase productivity of meetings with

laboratory/project personnel • Publications

– Reduces time to publication – Helps avoid data backlog

• Grants and Presentations – Spend less time looking for data – Data already compiled into an intellectually cohesive

unit – For presentations, simply cut and paste!

Advantages: Lab Personnel

• Increases intellectual investment for personnel at all levels.

• Particularly useful for students, postdocs and collaborators regarding experimental design and preparation for publication.

• Reduces time spent preparing presentations and poster for lab meetings, scientific meetings, graduate programs, etc.

• Teaches and reinforces organizational skills!

Application of The Storyboard

Easy-to-use Interface Microsoft Powerpoint Allows maximum flexibility and easy use

Flexible Organization Components of the storyboard evolve as data set evolves

100% modifiable Fits any discipline or data set

Clinical or Basic Science Application

The Storyboard: A Planning and Documentation Tool for Basic and Clinical Sciences

• Strategic Benefits – Project Initiation and Hypothesis Generation – Information Gathering, Sharing and

Documentation – Benchmark Recognition

• Early Plan versus End Plan – Content of storyboard will be determined by

progress of project – Content should provide information relevant to

current state of project

Project Initiation and Hypothesis Generation

Early Plan – Purpose and Background Information

Often short, bulletpoint statements – Experimental Design

Schematics or brief outline Significant emphasis on method

– Benefits IRB and IACUC applications Biostatistics Consultation

Early Plan Example

Franklin Echevarria Graduate Student

Storyboard Gp130 in the retina

(Expression, Localization, Partners) Franklin Echevarria Started May 2012

Focus of Sappington Lab

• Neuro-inflammation in degenerative diseases – Studied using glaucoma model

• Glaucoma – Second leading cause of irreversible blindness (85M ppl)

• Causes RGC cell death which eventually causes death of optic nerve

• Hallmark symptom is elevated IOP – Two forms

• Open angle- mainly caused by age (begins at old age) • Closed angle- hereditary defect- usually occurs in younger

individuals and progressively gets worse as the individual ages

Neuro-inflammation

• Previous studies implicate cytokines to mediate the inflammatory response. – Focus on the IL-6 cytokine family – All use the signaling receptor gp130

gp130

Complicated signaling pathway 1) IL-6 family member binds to its receptor (yellow). 2) Binding of ligand to receptor causes dimerization of the signaling component gp130 (green) 3) Dimerization of gp130 activates the JAK/STAT pathway which activates a variety of downstream effects. *There is both a soluble IL-6 receptor which can allow activation of gp130 on other cells that isn’t usually activated by IL-6 *There is evidence of a soluble gp130 that can bind the soluble IL-6/IL-6R complex and prevent it from binding to TM gp130 and initiating a gp130 dependent signaling cascade.

Models

• In vitro – Use a pressure chamber to mimic high pressure

system associated with high IOP in the eye • Animal Models (in vivo)

– MOM- acute open angle glaucoma caused by injection of beads into anterior chamber subsequently clogging the trabecular meshwork

– DBA2/J mouse- accepted model of closed angle glaucoma. Mutations causes pigmented cells of the iris to disassociate and the iris itself to collapse increasing IOP and eventually RGC death.

Questions regarding gp130 1. Are there differences in gene expression of gp130 and of

cytokines that use gp130 to signal between 4mo/8mo DBA mice relative to ctrls?

2. Where does gp130 localize to in the retina and is there a change in signal and localization between between 4mo/8mo DBA mice relative to ctrls? – Is there an increase in the expression of gp130 in the glial cells or RGCs

in the RGC area? – Is there an increase in the total number of glial cells expressing basal

gp130? – Is there a change in gp130 localization in any other layer of the retina?

3. What signaling pathway does gp130 utilize in the retina between 4mo/8mo DBA mice relative to ctrls?

4. What co-localizes with gp130 and does this change between 4mo/8mo DBA mice relative to ctrls? – What cytokine receptors of the IL6 family bind to gp130 between

between 4mo/8mo DBA mice relative to ctrls?

Information Sharing, Gathering and Documentation Early Plan

– Purpose More detailed background information Layout of hypothesis

– Experimental Design Geared more towards experiments needed - identifying holes in current data set

Data Slides – Highly variable in content at this stage

Pieces of data that will eventually be organized into a coherent sequence Questions about data Brief description of findings

Early Plan Example

Franklin Echevarria Graduate Student

Hypothesis on gp130 questions 1. I believe there will be an general increase of cytokine

gene expression between young old DBA relative to ctrls. (esp IL6). I may also see increase in gp130 gene expression

2. There may be some increase in gp130. I think more gp130 will be expressed in layers assc with astrocytes and mueller cells and more gp130 associated with RGCs. (NFL & ONL)

3. I think it uses the JAK2/STAT3 pathway. Whether I should look into MAPK or other pathways, I’m not sure.

4. This is will be based on the cytokine gene array. Those with significant increases may show more receptor binding to gp130.

Methods- Model System- Which one? • Utilize MOM injected mice as the glaucoma model?

– Pros • cheaper, faster, more consistent

– Cons • doesn’t take the influence of age into the progression of the disease • No mice readily available. All fixing/tissue prep needs to be done

• DBA2/J mouse model? – Pros

• more realistic progression of disease • Most tissue already available

– Cons • Require C57 mice as ctrls- need to order (NBD really) • Require age matched controls • Fixed tissue held at 4 degrees C. Still ok to parafin embed and section? • Not all mice develop high IOP as they age • EXPENSIVE!

• Concentrate on DBA/2J model as we have the most tissue banked for that already.

Experiments

• Paraffin retina ICC (protein localization, expression)

• Western blot (protein expression) • Co-IP (Binding relationships) • qRT-PCR array (mRNA expression)

mRNA Array • Gene expression of cytokines that use gp130 to

signal – Gp130 – IL6 – IL11 NM_008350 – LIF (Leukemia inhibitory factor) NM_008501 – OSM (Oncostatin M) NM_001013365 – CNTF (ciliary neurotrophic factor) NM_170786 – CLCF1 (cardiotrophin like cytokine factor-1) NM_019952 – CT-1 (cardiotrophin-1) NM_198858 – CT-2 (cardiotrophin-1/neuropoetin) NM_198859

• Was going to use Sigma to do the array, but it was too expensive. DD and HC looking into Vanderbilt’s core to see what they can offer

Paraffin ICC

• ICC Combos – Brn3a/Β-tubulin/gp130 combo – GluSyn/GFAP/gp130 combo

Primary Antibodies Cell species made in anti species Vendor storage Aliquot

ICC dilution

Neuronal Class III β-tubulin monoclonal RGC rabbit Mammal Covance -20°C 5ul 1:1000 gp130 monoclonal NS rat mouse R&D -20°C 1:12.5 GFAP polyclonal Astrocytes rabbit cow DAKO 4°C 1:1000 Glutamine Synthetase Mueller goat human Santa Cruz 4°C 1:250

brn3a RGC mouse monkey/ chicken Millipore -20°C 1:50

Primary Antibodies Secondary ICC dilution

Neuronal Class III β-tubulin monoclonal Rabbit-649 1:200 gp130 monoclonal Dylight Rat-488 1:200 GFAP polyclonal Alexa Rabbit- rhod red 1:200 Glutamine Synthetase Goat- 649 1:200 brn3a Mouse-Rhod red 1:200

ICC Analysis

Animal Name Which eye Slide # Section # Number assc w/ Part of retina and side List of antibodies w/color Optical Zoom C20R-sl23-sc1-Region?-gp130G-brn3aR-BtubB-40x

Section 1 Section 2 C2

0R #

23

gp13

0,DA

PI,

GFAP

6-

12-1

2 FE

Left Right

Regions of Interest

1

2 3

4

1= Outer Retina Left 2= Central Retina Left 3= Central Retina Right 4= Outer Retina Right

Do ICC on 8 slides at a time (2 from each group). When imaging each cohort find the region of the one slide with the highest expression of gp130 and optimize the signal. The settings used for all three channels for that region will be applied to all the pictures taken in that cohort of 8 slides.

Western Blot

• Done to see if there are any changes in protein expression b/w conditions

• Gp130 – Use Heather’s bioluminex data? – Test using western probes to confirm

• Other proteins to test – Housekeeping- Normalization – JAX2, pJAX2, STAT3, pSTAT3- Activation of gp130 via

JAK/STAT pathway – Activation of gp130 via another pathway?

Co-IP

• Done to characterized binding partners of gp130 b/w conditions

• Will be done once array data returns to figure out best possible binding partners (Cytokine Receptors)

Information Sharing, Gathering and Documentation

Late Plan – Focused and cohesive background – Experimental Design

Geared more towards future experiments needed – Identify holes in current data set

– Data slides Brief methods included for each slide (precursor to figure legend) Data presentation is more figure-like More formal description of findings (precursor to figure legend)

– Start putting slides in order that follows a data-driven line of reasoning

Late Plan Example

D’Anne Duncan, Ph.D. Postdoctoral Fellow

Initial Observations Experiments Conducted by:

Simone Abella (Vanderbilt University Undergraduate) Monet Standford (Vanderbilty University Summer Student) Nick Ward (Vanderbilt University Graduate Student)

• Ambient vs. elevated pressure cultures of retinal microglia – Noted a 2-fold increase of CCL5 in microglia-conditioned culture

media at 24h timepoint • IL-6 control to ensure screen worked • CCL5 as a candidate for study within the retina

SearchLight cytokine screen

CCL5

Mouse CCL5 retinal localization Tissue: mouse retina, vertical section; paraffin Magnification: 40X Green: CCL5, 1:6 dilution Blue: DAPI Original Filename: RANTES 40X - 1 to 6 dil - mouse Notes: Interestingly no staining seen in the ONL despite localization in the INL.

At left: Original image used to generate above image.

CCL5 localization in C57 retina

• CCL5 expressed in – Nerve fiber layer (NFL): RGC axons? Astrocytes? – Ganglion cell layer (GCL): RGCs – Inner plexiform layer (IPL): synapses? – Inner nuclear layer (INL): bipolar cells, Müller cells? – Outer plexiform layer (OPL): synapses?

• Expression not observed in ONL.

All layers microglia?

CCL5 localization in C57 NFL and GCL

• Wholemounts for better imaging of glaucoma-related structures • Retinas of C57 mice represent tissue not exposed to elevated IOP.

– Identify the normal cellular distribution for comparison with DBA/2, which develop chronically elevated IOP

• CCL5 localized in RGC somas and axons • Microglia and their processes express CCL5 • Some CCL5 staining appeared to be associated

with the vasculature (at right). – Check for CCL5 localization with astrocytes

Common Elements of the IStoryboard Slides for the “Information” Stage Annotation of data

Short method Statistical tests Representation of data (i.e. median age, sample size) Immunohistochemical labels (antibodies including company)

Interpretation of data Range from one or two sentences to full figure legends

Location of data Electronic location (filename and directory) Physical location

Source of data Personnel Date (for lab notebook references)

Benchmark Recognition Early Plan (Start New Storyboard) – Data slides

Slides in order that reflects a cohesive line of reasoning Most data organized into figures More detailed description of findings Notation of needs for publication, grant, etc.

– e.g. need to re-photograph sample to better highlight conclusion

Late Plan – Data Slides

Publication quality figures with legends Some data not incorporated into figures (will discuss next)

– Complete outline of your publication, presentation or preliminary data

Late Plan Example

D’Anne Duncan, Ph.D. Postdoctoral Fellow

Distribution of CCL5 and its receptors in the retina

Figures for paper submission DD

CCL5 and its receptors

CCR1 CCR5

CCL5

G-protein coupled receptors

CCR3

CCL4 CCL3

Cell motility and migration Cell adhesion Cell survival

Retinal expression---layer specific?

Cell-type specific

expression?

Peripheral Immune System T Cells

Dendritic cells Macrophages Eosinophils

Central Nervous System Microglia

Astrocytes Endothelial cells (BBB)

Neurons

Figure 1: Layer-specific expression of CCL5 and its high affinity receptors in the retina

Westerns qRT_PCR!!!!

N= 24 images (total of 2 mice)

Figure 2: RGC-specific expression of CCL5 and its receptors

Figure 3: Astrocytic-specific expression of CCL5 and its receptors

Figure 4: Muller glia-specific expression of CCL5 and its receptors

Figure 5: Absence of microglia-specific expression of CCL5 and its receptors

Figure 6: Communication of RGCs and Retinal Glia via CCL5 Signaling

Future experiments to complete

• Westerns

• qRT-PCR!!!

Discussion Points

• Constitutive expression of CCL5 and its high affinity receptors in the retina • Also expression in different compartments of the

somas/processes

• Localization of CCL5/CCR3, CCR5 in RGC somas, axons – Most abundant expression of CCL5, CCR3 and CCR5 in

RGL – Other retina retinal layers express these molecules,

but at lower levels.

Project Initiation and Hypothesis Generation

Late Plan – Data slides

Data doesn’t fit into benchmark – Becomes rationale for a new storyboard

Return to the Early Plan – Purpose and Background Information

Data from previous storyboard – Experimental Design

Follow-up to preliminary result – Benefits

Grant proposals

Disadvantages Requires diligent participation by all

members of the laboratory/project team. Lag time between data generation and

incorporation into the storyboard greatly impacts the success of this system!

Lack of participation can also negate many of the advantages!

Thank You!!!

Acknowledgements: Dr. David Calkins