Finding a Balance Michelle Juarez, Ph.D. Micro, Immunology, and Pathobiology Wound Healing and Regeneration • Wound Response • Inflammation • Tissue formation • Tissue remodeling
Download ReportTranscript Finding a Balance Michelle Juarez, Ph.D. Micro, Immunology, and Pathobiology Wound Healing and Regeneration • Wound Response • Inflammation • Tissue formation • Tissue remodeling
Finding a Balance
Michelle Juarez, Ph.D.
Micro, Immunology, and Pathobiology
Wound Healing and Regeneration • Wound Response • Inflammation • Tissue formation • Tissue remodeling
Regeneration and Wound Healing Optic Nerve Section Regeneration Chronic non-healing wound
Epidermal barrier formation Mammalian Skin Galko and Krasnow, 2004
Drosophila
Cuticle
Drosophila
: a useful model organism • Fast life cycle • Large samples • Community • Genome tools • Reporters
Drosophila
wound healing studies Razzell et al., 2011
What is the transcriptional response to wound healing?
•
Wound Response Genes
JNK-KKK (msn)
• • • •
Dopa decarboxylase (Ddc) tyrosine hydroxylase (ple) chitin synthase (kkv) transglutaminase1 (CG7356, not shown)
Pearson et al., 2009
PNAS
Schafer and Werner, 2008
Epidermal Wound Assay 50µM wound inducible "enhancer" DNA Fluorescent protein gene
A very large genetic screen has identified many new genes required for a normal transcriptional wound response ple-DsRed 50µM Efren Sandoval-Guillen
Validation of Screen • Conservation and Function • Phenotype in
Drosophila
• Genetic Analyses – Loss-of-function – Gain-of-function analysis
Flotillin: Regeneration & Signaling Schulte, 1998 Stuermer, 2010
Drosophila
Flotillin2 Brain Head Skeleton Lateral View (Saggital Plane)
Purple = Flotillin2 RNA
100µM Dorsal View (Horizontal Plane)
Green = Dopa decarboxylase RNA
Localization of wound reporters Juarez et al., 2011
PLoS Genetics
Gal4 expressing line > UAS-target gene line
armadillo-
GAL4>UAS-
Flo-2 engrailed
-GAL4>UAS-
Flo-2
Flotillin-2 is sufficient to inhibit Flo-2 Localized Wound Response
What mechanisms can reduce a signal?
Source vs. Sink
Mayor & Pagano, 2007
Role of endocytosis during wound response 30 ° C, Non-permissive Temp
wild type, ple WE1 Dynamin, ple WE1
21 ° C, Permissive Temp
wild type, ple WE1 Dynamin, ple WE1
What are the limits of wound response regulation?
Genetic vs. Chemical
Body Cavity Injection Protocol Rachel Patterson
Serine Protease Pathway
Toll Immunity: (SPE) D/V polarity:
(Easter)
Coagulation:
(
“
Thrombin
”
) Melanization:
(ProPO-AE)
2011 Nobel Prize in Medicine and Physiology Hoffmann, Steinman, and Beutler
Flotillin-2 inhibition of Serine Protease
Genome Wide Studies - Micro Array Patterson et al, 2012
PLoS One
How do established wound signals behave in
Drosophila
?
Hydrogen Peroxide Average arrival of first Leukocyte ( ± s.d.) Niethammer et al., 2009 Toledano et al., 2010
H 2 O 2 and Wound Response Signaling H 2 O 2 Flo-2 Localized Wound Response
Drosophila
Dual Oxidase
Duox required to activate WR Duox Trypsin Localized Wound Response
Summary of Results • Flotillin-2 promotes the localization of epidermal wound response reporters • Endocytosis limits wound response to cells surrounding the site of injury • Hydrogen peroxide is a conserved wound signal
Wound Healing and Regeneration Chronic non-healing wound Optic Nerve Section Regeneration
Why is Flotillin conserved?
Parasitic Wasp Infection natural wound assay Journal of Visualized Experiments Small et al, 2012
JoVE
What can we learn from the
Drosophila
wound response?
Chemical Activators of Epidermal Wound Response Genes Methyl -Cyclodextrin
Dopa Decarboxylase(Ddc)
50µM
Epidermal wound response Genetic analyses Cell signaling Biological function Funding: R01 GM077197 to W. McGinnis K12 GM68524 to M. Juarez
Lessons from wound healing •
Drosophila
– Epidermis – Imaginal discs • Vertebrate – Skin – Liver – Heart
Inhibitors of the wound signaling pathway
Lipid Raft
Flotillin-2 Src-family Tyrosine kinases
Microarray experimental set-up “
Early
”
WR genes
30 minutes “
Mid
”
WR genes
60 minutes “
Late
”
WR genes
120 minutes
Unwounded, Puncture-wounded, Trypsin puncture-wounded
2 biological replicates 18 Agilent Drosophila microarrays
Src and Flotillin/Reggie Neumann-Giesen, 2007 Babuke and Tikkanen, 2007 Munderloh et al, 2009
Src42A
wound response
Src42A acts cell autonomously
Do Flo-2 and Src42A interact?
• Genetic double mutants – Src42A , UAS-Flo-2, arm-GAL4 • Chemical double mutants – UAS-Flo-2, arm-GAL4
+ SU6656
– UAS-Src42A.CA; arm-GAL4
+ M
CD
Cell-autonomous inhibition of wound gene transcription
Endocytosis patterns during wound response
Biology of the Cell (2007) 99, 333-342 - M.R. Schmidt and V. Haucke
An epidermal wound transcriptional response/regeneration pathway Duox Flo-2 Src42A Hydrogen peroxide Serine Protease(s) Lipid Raft Trafficking Pathways wound response gene
wild type Grainy head is required for epidermal barrier development
grh -
25µM 25µM
Harden, 2005
Science
Grainy head is required for
Ddc
wound enhancer wild type
Grh
Mace et al, 2005
Science
Genetic Screen
epidermal wound response pathway stitcher ERK Serine protease processed ligand Grh/ AP-1 sites/
Ets21C
!
Ddc
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Wound Duox H 2 O 2 Serine Protease
/
Trypsin
Serine protease processed ligand Spz tube
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