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

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Ddc

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Wound Duox H 2 O 2 Serine Protease

/

Trypsin

Serine protease processed ligand Spz tube

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