The Storyboard Method for Research Strategy and Management

Transcription

1 The Storyboard Method for Research Strategy and Management Rebecca Sappington, Ph.D. Assistant Professor Ophthalmology and Visual Sciences, Pharmacology and Neuroscience

2 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

3 PIs assemble pieces into a cohesive vision. Need to see the big picture

4 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!

5 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. 4 12

6 The Storyboard Based on storyboards used to produce movies! David J. Calkins, Ph.D. Vice Chairman and Director of Research Professor Vanderbilt Eye Institute The Waterhorse Storyboard: Take 1 Craig Woolheater

7 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!

8 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!

9 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

10 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

11 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

12 Early Plan Example Franklin Echevarria Graduate Student

13 Storyboard Gp130 in the retina (Expression, Localization, Partners) Franklin Echevarria Started May 2012

14 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

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

16 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.

17 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.

18 Questions regarding gp 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?

19 Information Sharing, Gathering and Early Plan Purpose Documentation 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

20 Early Plan Example Franklin Echevarria Graduate Student

21 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.

22 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.

23 Experiments Paraffin retina ICC (protein localization, expression) Western blot (protein expression) Co-IP (Binding relationships) qrt-pcr array (mrna expression)

24 mrna Array Gene expression of cytokines that use gp130 to signal Gp130 IL6 IL11 NM_ LIF (Leukemia inhibitory factor) NM_ OSM (Oncostatin M) NM_ CNTF (ciliary neurotrophic factor) NM_ CLCF1 (cardiotrophin like cytokine factor-1) NM_ CT-1 (cardiotrophin-1) NM_ CT-2 (cardiotrophin-1/neuropoetin) NM_ 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

25 Paraffin ICC species made in anti species Vendor storage Aliquot ICC dilution Primary Antibodies Cell 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 :200 brn3a Mouse-Rhod red 1:200 ICC Combos Brn3a/Β-tubulin/gp130 combo GluSyn/GFAP/gp130 combo

26 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. C20R #23 gp130,dapi, GFAP FE ICC Analysis 1 Regions of Interest Left Right 4 Section 2 Section 1 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 2 3 1= Outer Retina Left 2= Central Retina Left 3= Central Retina Right 4= Outer Retina Right

27 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?

28 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)

29 Information Sharing, Gathering and Late Plan Documentation 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 datadriven line of reasoning

30 Late Plan Example D Anne Duncan, Ph.D. Postdoctoral Fellow

31 Initial Observations Experiments Conducted by: Simone Abella (Vanderbilt University Undergraduate) Monet Standford (Vanderbilty University Summer Student) Nick Ward (Vanderbilt University Graduate Student)

32 SearchLight cytokine screen CCL5 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

33 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.

34 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?

35 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

36 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)

37 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

38 Late Plan Example D Anne Duncan, Ph.D. Postdoctoral Fellow

39 Distribution of CCL5 and its receptors in the retina Figures for paper submission DD

40 CCL5 and its receptors Peripheral Immune System T Cells Dendritic cells Macrophages Eosinophils CCL5 CCL3 CCL4 Central Nervous System Microglia Astrocytes Endothelial cells (BBB) Neurons CCR1 CCR3 CCR5 G-protein coupled receptors Cell motility and migration Cell adhesion Cell survival Retinal expression---layer specific? Cell-type specific expression?

41 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)

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

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

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

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

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

47 Future experiments to complete Westerns qrt-pcr!!!

48 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.

49 Project Initiation and Hypothesis Late Plan Data slides Generation 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

50 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!

51 Thank You!!! Acknowledgements: Dr. David Calkins