Genetic Labeling Techniques

Transcription

1 Genetic Labeling Techniques CRRT 2012 San Diego, California February 14, 2012 Benjamin D. Humphreys, M.D., Ph.D. Principal Faculty and Co-Director, Harvard Stem Cell Institute Kidney Program Assistant Professor, Harvard Medical School Associate Physician, Brigham and Women s

2 Lineage Tracing The marking of a single cell such that the mark is transmitted to the cell s progeny resulting in a labeled clone. Ex: direct observation Ex: application of died agar chips to embryo surface 1905: Ascidian embryo during gastrulation. Edwin Conklin. 1929: Prospective fate maps: Walter Vogt.

3 Fate Mapping (= lineage tracing) Oh God! That one might read the book of fate. William Shakespeare, King Henry IV Historically used by developmental biologists to examine how different cell lineages contribute to formation of 3d organ Genetic fate mapping has revolutionized these studies Non-invasive Molecular precision: Labeled cells defined by gene expression Highly sensitive: unambiguous tracking of cell fate

4 Genetic Fate Mapping No Reporter Expression Promoter loxp X STOP loxp Reporter Kidney-specific promoter Cre Promoter X Reporter Reporter Expression Promoter loxp Reporter Adapted from Igarishi 2004

5 Inducible Fate Mapping Cre-recombinase fused to estrogen receptor (ER) In absence of ligand (Tamoxifen), CreER excluded from nucleus Tam pulse traffics CreER to nucleus, where recombination occurs Fate mark is constitutive and heritable Joyner, Dev. Dyn. 2006

6 Practical Considerations Cre is very efficient Characterization of Cre expression crucial Choice of reporter important LacZ, nlacz, RFP, egfp, mt/mg, MADM All these reporters available from JAX Method of reporter detection important Enzymatic Epifluorescence Immunofluorescence Tamoxifen may be toxic in high doses Muzumdar, 2007

7 Validation of R26-LacZ Lineage Reporter in Kidney

8 Six2 and FoxD1 Expression in Mesenchyme Pax2 merge Mutually exclusive progenitor cell populations Mugford and McMahon, 2008

9 Tissue-specific gene expression in the metanephros Six2-GC; R26R FoxD1-GC; R26R

10 Labeled collecting duct and nephron epithilia Uninjured Uninjured

11 The Origin of Reparative Cells during Kidney Repair Differentiated epithelium derived Ischemic injury stem cell derived Injured tubule Epithelial dedifferentiation and proliferation A. B. C. Extra-tubular stem cells Intra-tubular stem cells Fusion? Regenerated tubule Humphreys, 2006

12 Experimental Strategy What cell types to target?

13 Labeled epithelial cells proliferate Uninjured Injured Humphreys et al, Cell Stem Cell, 2008

14 No dilution of label after repair % Labeled % Labeled con IRI, repair 15 d IRI, repair 30 d 10 Humphreys et al., Cell Stem Cell, con IRI, repair IRI, Repair, IRI, repair

15 The Origin of Reparative Cells during Kidney Repair Differentiated epithelium derived Ischemic injury stem cell derived Injured tubule Epithelial dedifferentiation and proliferation A. B. C. Intra-tubular stem cells Extra-tubular stem cells X Fusion? Regenerated tubule Humphreys, 2006

16 Genetically Marking All Kidney Cell Types Negative control Tubules Pericytes, Mesangium Positive control Collecting Duct Endothelial Cells Humphreys, unpublished

17 Genetically Marking All Kidney Cell Types No Cre Six2-Cre FoxD1-Cre HoxB7-Cre Sox2-Cre Negative control Tubules Pericytes, Mesangium Positive control Collecting Duct Endothelial Cells Humphreys, unpublished

18 Podocyte morphology by scanning electron microscopy (SEM) Andrews P, J Electron Microscopy Technique (1988)

19 Hypothesis: Sporadic podocyte labeling improves contrast between foot processes Slit width: 35-45nm

20 Kozak Podocyte labeling strategy + tamoxifen/4-oht collagen1α1 egfp Cre ER T2 pa R26 or loxp STOP loxp LacZ R26 or mtomato megfp loxp loxp R26/CAG STOP tdtomato loxp loxp

21 Coll1α1GCE;R26Tomato Coll1α1GCE;mTmG Coll1α1GCE;R26LacZ % of all glomeruli % of all glomeruli % of all glomeruli Podocyte labeling results B vehicle tamoxifen/4-oht E one pulse (P0) % labeled per glomerulus C F one pulse (P3) D G % labeled per glomerulus two pulses (P0, P3) % labeled per glomerulus Grgic Humphreys, JASN, in press

22 Podocyte foot processes identified A B C tdtomato laminin merged D * E F G α α Grgic Humphreys, JASN, in press

23 Capillary loop fine structures

24 Capillary loop fine structures tdtomato K laminin L merged M β α * * Grgic Humphreys, JASN, in press

25 *

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27 Current concepts of myofibroblast recruitment in CKD (proposed progenitor pools) tubular epithelial cells bone-marrowderived cell differentiation myofibroblas t activation/ differentiation local interstitial fibroblast endothelial cells pericyte/perivascu lar fibroblast

28 pericyte Experimental Strategy

29 Dramatic expansion of labeled cells during fibrosis

30 Pulse Labeling FoxD1 stromal cells FoxD1-GCE; R26R Tamoxifen pulse At e11.5

31 Expansion of FoxD1-labelled interstitial cells in fibrotic disease Control UUO day 10 Humphreys and Duffield, 2010

32 Newer Applications Kretzschmar and Watt, 2012

33 Newer Applications Kretzschmar and Watt, 2012

34 Funding NIH-NIDDK HSCI Genzyme Tengion Evotec Humphreys Lab Anjali Rao Derek DiRocoo, PhD Steve Fabian, MD Nilka dejesus-gonzalez, MD Ivica Grgic, MD Andreas Hofmeister Rafael Kramann, MD Tetsuro Kusaba, MD Rado Penchev Fengfeng Xu, PhD