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Molecular Final Exam Study Guide

by: Kiara Lynch

Molecular Final Exam Study Guide Bio 413

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Kiara Lynch
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Ch 23: Specialized Tissues, Stem Cells, and Tissue Renewal; Ch 19: Cell Junctions, Cell Adhesion, and the Extracellular Matrix; Heart Regeneration in Adult MRL Mice
Dr. Stefan Samulewicz
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This 18 page Study Guide was uploaded by Kiara Lynch on Tuesday May 10, 2016. The Study Guide belongs to Bio 413 at La Salle University taught by Dr. Stefan Samulewicz in Spring 2016. Since its upload, it has received 16 views. For similar materials see Molecular in Biology at La Salle University.


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Date Created: 05/10/16
CHAPTER 23: Specialized Tissues, Stem Cells, and Tissue Renewal  Permanent cells in the lens of the eye o Unchanged, can’t regenerate o Embryonic lens vesicle- hollow of cells, proliferates and grows, surrounded by CT (collagen type 4)  Capsule- GAGs o Primary lens fiber and anterior lens epithelial cells  Cells closer to retina elongate  Produce crystallines  Water soluble refractory proteins  In middle of lens o Adult lens  Recently formed lens fibers  Core of adult lens consisting of the primary lens fibers formed in the embryo  Nuclei no longer visible  Basal lamina  Photoreceptors o Develop early on and are irreplaceable o Light- 120 million rods, 6-7 million cones (color) o Constantly exposed to light- can be damaging to proteins high turnover  Rhodopsin-transducin o Transmembrane receptor o Cofactor- retinal  Absorbs photon of light  Conformational change cis to trans  conformation change in rhodopsin  Signal transmitted to transducing o Transducin  3 subunits, alpha, beta, gamma  Absorbs light- conformational change- release of alpha subunit  Alpha subunit activated  G protein cascade  hyperpolarization of membrane affects cation channels  Pulse-Chase o H -Leucine  Attach to amino acids and feed to subjects  Large pulse  Taken in and incorporated into proteins  Chase with cold leucine o Cells are permanent but the proteins are replaced  Reach cell- phagocytize, H disappears, proteins recycled, neurons produced  Liver cells- hepatocytes o Arranged in sheets o Exposed to blood vessels- gets toxins out of blood o Simple duplication- replicate mature cell type- different cell duplicates o Stem cell population- contrast with mature cell type where no more differentiation can occur; proliferate and develop o Kupffer cells- phagocytize RBCs o Endothelial cells- line blood vessels o If you remove 2/3 of a rat’s liver it will grow back to normal size in 2 weeks and stop growing once it reaches the right size o What controls the process of liver regeneration?  Kupffer cell- functions in alcoholic liver disease  Hepatocyte- remove part of liver- cells in liver re-enter cell cycle  UPA- protease- urokinase  Changes plasminogen to plasmin; activates other proteases  Wish bone shape receptor  Always present at low level  HGF- hepatocyte growth factor  Moves through bloodstream, signals liver cells to grow  Binds to cMet receptor on surface of liver cells  dimerize to activate  Take out part of liver  upregulation of UPA to 10 fold increase  bind circular UPA  trigger cascade of proteases to break down ECM  inactive pro form of HGF  cleaved to activate  binds cMet receptor  signal to nucleus  re-enter cell cycle  TGF-beta – Trans growth factor  Activation stops proliferation signal – expression increases after wound healing signal  Cells need to re-establish ECM  If not enough time to repair fibroblasts grow and secrete collagen  cirrhosis o Cirrhosis  Remake damaged liver  Resting state  damage  molecular responses  UPA receptor upregulated – binds urokinase  Cuts ties of ECM  liberates pro hepatocyte growth factor- cleaved and becomes activated and binds to CMet receptor  Simultaneous signal to stop growing o Back to resting state; happens as liver reaches appropriate size  Fibroblasts respond to growth signals instead of hepatocytes – secrete collagenous ECM  Endothelial cells in the blood vessels o Loose CT- give structure and shape, few cells, mostly collagen o Smooth muscle- involuntary contracting, moves blood o Elastin- elastic fibers respond to blood pressure o Basal lamina and endothelial lining- filter, barrier for microenvironment  Capillary o Basal lamina o Gaps that control what passes through  Pericyte- support cell o Assisting endothelial cells in process of developing blood vessels o Phagocytize debris associated with damage to blood vessel o Contractile element to move blood o Can differentiate into muscle like cells or adipocytes o Stem cell like characteristics maintain blood brain barrier  Internal o Changes in cytoskeleton- pseudopodial processes o Break through basal lamina and ECM and form a path for blood vessels o Gene expression and behavior changes o Pressure or stress on cell  degradation  need to be replaced  signals o New tissue needs blood supply for nutrients and waste  Tissue grows and blood supply grows with it  Need proper environment  memory  form vacuoles for blood supply o Angiogenesis  Growth of new blood vessels  Happens in absence of blood vessels  Cell growing for a period of time  becomes comfortable with ECM  vacuoles fuse together  blood vessels fuse with other blood vessels o Signaling molecules  If blood supply isn’t close enough, oxygen levels drop  Hypoxia induced factor- low O2 levels o TF in cells with low O2 levels o Heterodimer- alpha and beta subunits  Alpha- rapidly degraded  Beta- constitutively expressed- helix loop helix  Prolile hydroxylase  activates degron on alpha subunit  Ub attached  proteasome  degraded  Vascular endothelial growth factor  Signaling and secreted into EC< to endothelial cells (surface receptors)  Re-enter cell cycle  pseudopodial processes  grow in direction of signal  fuse with blood vessels  relieve hypoxic stress  Epidermis o Stem cells  Maintain telomeres  Replicating indefinitely  Create safe environment o Differentiated cells  Relatively undifferentiated cell that can continue to divide indefinitely, throwing off daughter cells that can undergo terminal differentiation into a cell type  Differentiation levels  Unipotent  Pluripotent  Totipotent  Terminally differentiated- no longer capable of dividing o Protection  Pigment cells (melanocytes)- UV radiation  Immune cells- pathogens  Fibroblasts- collagen o Cytoplasm rich o Layers  Keratin- main structural component; anchored to adherins in membrane that reach between and grab on  form bumps on surface  prickle layer  Basal lamina- anchor and filter, key to maintain stem cell population, where cell division takes place  Differentiation as you move up toward the surface o Hair follicles  Brdu experiment  Bulge region contains cells that take up Brdu (stem cells)  Choice in direction- hair production or keratin production  Skin damage  re-epithelialization o Environmental asymmetry vs divisional asymmetry  Maintain stem cell population or produce terminally differentiated stem cell  Environmental signal- from ECM or other cells influencing the environment  Internal transcription factors  Stay with one cell (stem cell) and absent in other  Gene expression tells other cell to terminally differentiate  Deep wound- affects basal lamina  Environmental asymmetry is the better option o skin heals- environmental factors cause them to both stay stem cells o grows stem cell population o BrdU labeling  Way to identify stem cells  True stem cells don’t divide that often  Short label (pulse) with short chase and still found labeled cells  Transit amplifying cells  Long pulse and chase – stem cells in different places  Rete ridges- epithelial extensions that project into the underlying CT o Two populations of stem cells in basal layer  True stem cells  Divide slowly and not often  2 daughter cells o One stays o One moves and stays in contact with the basal lamina – committed transit amplifying cell  Committed to differentiation  Maintains ability to divide b/c of contact with basal lamina  Divide more rapidly  Only divide at certain times  Original stem cell population communicates with each other  Maintained stem cell population  Growth transit amplifying cells  Still have pockets of stem cells  Genes to maintain stem cells  EGF- epidermal growth factor, PDGF- platelet derived growth factor, Wnt/Hedehog, TGF-Beta, Notch/Notch ligand  Brdu experiment- within stem cell population, when DNA is separated into two daughter cells all Brdu labeled DNA goes in 1 direction and all other DNA goes in another direction  Maintains stem cells without mutations  Lumen of intestine o Secrete digestive enzymes, take in nutrients  Villi increase surface area o Single layer of epithelium o Brdu 2 populations of stem cells in crypts; connection to basal lamina is not relevant here o Pluripotent stem cells  Brush border  Absorbs nutrients  Increases surface area  Most susceptible to damage- protected by goblet cells  Goblet cells  Secrete mucus  Enteroendocrine cells  Signals for the number of cells needed  Paneth cells  Immune cells  Create alpha-difensins- hydrophobic proteins with positive charged domains  bursts in bacterial cells  Secretes proteins that lodge in the membrane of bacterial cells o Normal colon vs cancerous  Normal- certain size of crypts relative to villi  Adenoma- all crypt, little villus, signaling is messed up  Wnt Signaling o Northern and Southern blot, microarray, pulldown experiment  Cell surface receptor- frizzle  binding protein is Wnt  change gene regulation o Wnt signaling molecule binds to frizzle  DSL phosphorylated  dissociates destruction complex  Beta-catenin into nucleus and bind to groucho and TF  transcription  gene expression  proliferation o Destruction complex only works with all substrates o Beta-catenin- makes system responsive o Inappropriate proliferation- expansion of crypts o Notch- receptor protein  signal  differentiation into absorption cell  If no notch and only ligand  secretory cell  EphB/ephrinB o Cell surface receptor in crypt o Stop dividing  move up crypt  signal becomes weaker  converts to ligand EphrinB  leaves cell cycle and moves up villus as non- proliferating cell o Proliferative cells and Paneth cells express EphB proteins- keep them in crypt o Non-dividing differentiating cells migrate up and out of the crypt o Differentiated cells on villus express ephrin proteins keeping them out of crypt o Signals based on location, unequal distribution of factors in daughter cells and signals as they move up epithelium  Crypt cells proliferate  Hedgehog and Wnt signals from the crypt cause BMP4 expression in the villus core  BMP proteins from villus core inhibit expression of Hedgehog and Wnt in the villus epithelium  villus cells do not proliferate  BMP- bone morphagenic protein  Contributes to shutting off proliferation  Blood cells o Bone marrow reproducing blood cells at different concentrations o 2.5 million/second removed from bloodstream o Diversity of WBCs- easy to get blood cell counts o Megakaryocyte processes budding off platelets  Blebbing pieces of membrane to produce platelets for clotting  long processes out of basal lamina o WBCs respond- bloodstream to injured tissue  WBCs in capillary  exposure to mediators of inflammation released from damaged tissue  chemotoxins toward attractants released from damaged tissue WBCs in CT o Selectins  Cell surface transmembrane proteins produced by endothelial cells  Lectin binding domain  Adhesive domain that binds to sugar groups that are post- translationally modified on cells that want to get out- “scotch tape”  P-selectin- lectin domain, EGF-like domain, repeat domains o Integrins  “velcrow”  Transmembrane protein that maintains connection to ECM  Principle receptor cells  2 noncovalently associated subunits alpha and beta  Adhesive competent state- able to bind something outside the cell  Not in endothelial cells  adhesion in competent state  Signaling molecules  surround endothelial cell receptors  receptors activated  adhesive competence  Bone Marrow o Supporting cells  Marrow stromal cells (MSCs)- adipocytes  Nutrition, energy  Fibroblast like cells o Spleen colony assay  Radiating mouse  kill almost all bone marrow cells  get mouse to recover by replacing it with healthy bone marrow  repopulate  healthy mouse  Took cells and introduced a retrovirus  integrates into random spots, unique labels  Some injected cells get stuck in spleen  Secrete ECM, proliferate  Shows what bone marrow cells are colonies in spleen o Stem cells in particular regions  Association with  Osteoblasts  bone, proliferation  MSCs  bind to receptors  Kit receptor- receptor tyrosine Kinase  Bind to ligand  activate  cascade of signaling  change of gene expression  Ras; sarc proteins o Stem Cell Factors  Transmembrane protein- can be alternatively spliced  Diffusible ligand- cleaved off o MSCs produce SCF and bind to KIT or act as a signal to go back to niche o KIT Expression in Hematopoietic Cells- codes for tyrosine protein kinase  Stem cell produces KIT receptor  differentiate  lowering of expression of KIT  Hematopoietic stem cells and progenitors express high levels, mast cells and melanocytes also express it  Proliferation and differentiation signaling o Colony Stimulating factors  Differentiation/cell line specific  Activating the same co-receptor  What [ ] of what CSF at what stage of development will guarantee development of a certain cell type? o Specific Signals  Certain signals get cell up to blast forming stage  Can’t completely regulate cells we create  Controllable parameters o Frequency of cell division o Probability of cell death o Probability that stem-cell daughter will become a committed progenitor cell of the given type o Number of divisions before terminal differentiation o Lifetime of differentiated cells o Marrow Stromal Cells (MSCs)  1867- Cohnheim injected dye  Suspected cells in bone marrow migrate out into tissue  Injected tissue into bone marrow with dye  Cells moved out of bone marrow  stained cells o Blood cells o Stromal cells  Fibroblast like, making CT, secreting ECM in other places  1970s- Freidenstein, non-adherent cells  Developed cultures and got rid of non-adherent cells  Studied adherent cells kept feeding  proliferated, increased ECM, foci developed where cells looked like they were specialized o Cells secreted different ECMs  1980s= tweek culture conditions (CSFs)  Identified colony stimulating factors  Signal molecules and receptors- signal to gene expression, fed with different CSF  Turned into osteoclasts, chondrocytes, adipocytes, and myoblasts o Are bone marrow cells respective to bone or are they like the blood cells?  Mouse collagen type I gene  mutated and replaced with human genome  PCR product  different size bands in + and – cells  Cells moved into other tissues o Applications for MSCs  Extract MSCs, stimulate to become other cells, inject and repair  Degenerative arthritis  Poorly healing bone  Reconstructive surgery  Gene therapy: osteogenesis imperfecta- lack of strength in collagen fibers (brittle bone); replace with cells with the correct gene  Muscle: 4 Types o Heart, smooth, myoepithelial, skeletal o Differentiate into myoblasts – still proliferate, terminally differentiate o TF- HLH, determine stem cells fate, regulate genes o Signals to cells  MyoD  Code for muscle structural genes- myogenin, Myf5, Mrf4  Activate mef2 and keep themselves turned on  Muscle development o DAPI staining  Blue nuclei  Myoblasts- will eventually become muscle cells but proliferate 1 st  Cells fuse into multinucleate fibers  nuclei migrate to periphery o Satellite cells  Muscle stem cells  Still capable of proliferating; association with muscle growth  Limited proliferative capacity  Muscle dystrophy- wear out satellite cells  Dystrophin- muscles contract against an anchored site o no connection to the membrane o Mouse experiment  Alter mouse genetically- inserted a reporter gene under the control of a muscle specific promoter Myosin light chain 3F Beta-galactosidase  Only reports if a cell is a muscle cell  Donor mouse- all of the cells altered  Experimental mouse- slice muscle so it needs repair  Get bone marrow from donor mouse and inject into damaged tissue o Both adherent and non-adherent cells, and some just ad. Cells  Look for expression of donor genes – nuclei of cells  Compare adherent cells vs non-adherent cells and satellite cells in both mice  A,C- satellite cells from donor mouse lot of cells expressing gene  B,D- look similar to A&C  E- adherent cells, expression  F- non-adherent cells, no expression  Recipient mouse- xray and wipe out bone marrow  Replace with donor mouse bone marrow with reporter gene  After time, nuclei begin to express the gene  Cells got to muscle from marrow through bloodstream  Bone marrow is capable of getting out of marrow, traveling through the circulatory system, getting into tissue, and contributing to growth in response to signals o Help satellite cells  Fibroblasts o CT cells- least specialized, produce ECM o Collagen type I- 90% of collagen o Wound healing- locally migrate o Chondrocytes  Fibroblasts can be stimulated to become chondrocytes- secrete collagen type 2  Under poor conditions, revert and secrete collagen type I (in vitro) o Adipocytes  3 main functions  Heat insulation – 2 mm layer  Mechanical cushioning – surrounds internal organs  Energy storage – buffer against energy imbalance, most efficient way to store energy o 1 g fat  9kcal energy o 1 g carb/protein  4 kcal energy o Hydrophobic- less space  more energy  Adipogenesis  Mesenchymal cells (MSC)- spindly  proliferation  determination  preadipocyte  proliferation  differentiation into immature multilocular cells  fuse to 1 fat droplet  mature unilocular adipocytes  MSC  proliferation  differentiation  CCAAT enhancer binding protein  Peroxisome proliferator- activated receptor  P21- cyclin inhibitor  Twist, scleraxis- signaling conversion to adipocyte  Pref-1- pre-adipocyte factor-1  Pref-1  Notch receptor (transmembrane domain, loop, EGF repeats) and ligand o Delta- EGF repeats o Signaling to each other  Ligand to member of the notch family protein receptors o DLK- delta like  Cleavage at site 1 in golgi  transport to plasma membrane  binding to delta and cleavage at site 2 (delta binds to EGF repeats)  cleavage at site 3 (alters gene expression)  notch tail migrates to nucleus  complex containing notch tail and CSL activates gene transcription  transcription of target genes  Soluble diffusible signaling molecule  variety of alternatively spliced versions of Pref-1  EGF diffuse and bind to other receptors  FA-1 – fetal antigen-1: extracellular domain of DLK protein (pref-1)  a differentiated adipocyte could maybe turn back into a pre- adipocyte  Ob gene- obese gene  Codes for hormone leptin that is produced by fat cells that tells your body you have eaten enough – binds hypothalamic region receptor  Fat pad o Inject leptin- lost weight o Lost fatty tissue (triglycerides) not cells (DNA)  Stimulating metabolism – size decrease  Western blot- what proteins produced changes in fat pad o Increase- proteins that metabolize fat o Decrease- proteins that synthesize fat o Pref-1- back to pre-adipocyte like state o Chondrocytes- bones  Osteoblasts, osteoid (uncalcified bone matrix), calcified bone matrix, cell processes in canaliculi, osteocyte  Start to differentiate deeper into bone  Collagen type I  Trapped  no proliferation  long processes – get nutrients, not as close to blood vessel as normal  Calcium phosphate in form of hydroxyappetite  Development of Bone  Cartilage- chondrocytes, collagen type 2  Cells become larger and leave the cell cycle  Fibroblasts in  osteoblasts – collagen type 1 growth  Layer of cartilage- growth plate (disappears)  Thin layer of chondrocytes, more dense bone on outside  Chondrocyte genes- sox9, Runx2 o Signals sent out  Indian hedgehog  Wnt pathway activation  osteoblast  osteocyte o FGFR3 point mutation- cartilage isn’t there, no growth plate bones don’t reach full size  Remodeling  5-10% bone replaced every year  Osteoclasts- digest bone o Blood vessels following o Fibroblasts following secreting CT Chapter 19: Cell Junctions, Cell Adhesion, and the Extracellular Matrix  Process of wound healing o Hemostasis  Epinephrine released to minimize bleeding  From initial injury to 3 hours post injury  Platelet cells  Clot formation, phagocytation, secretion of growth factors  Release of cytokines - platelet derived growth factors that call in other cells to help healing o Inflammation  Leukocytes and macrophages destroy bacteria cleaning the wound of cellular debris  Prevents infection  0-3 days after injury  Swelling, redness, pain, heat o Proliferation  Granulation, contraction, epithelialization – scar formation  Granulation- tissue of fibroblasts, capillaries, and neutrophils  Produce collagen  New capillaries- angiogenesis  Contraction- wound fills with granulation tissue and closes wound  Epithelialization- cells migrate from wound margins, divide, and touch one another to seal wound  Stem cells proliferate in bulge of hair cell or basal layer  Needs to be a moist, heavily vasculated region  Provide covering for fibroblasts to mature o Maturation  Remodeling ECM  Collagen fibers reorganize to improve tensile strength  Scar has less tensile strength than uninjured skin  Collagen synthesis begins with fibroblast  Procollagen fibers  collagen fiber  Weeks to years- more remodeling over time o Way we deal with damaged tissues as opposed to regeneration  Regeneration in hydra, protostomes, deuterostomes o Unidirectional- cut off leg and leg will grow back but the leg will not grow a body o Bidirectional- both halves will regrow the whole o Restoration or new growth by an organism of organs, tissues, or whole body parts that have been lost, removed, or injured o Flat worms- (planarian) can be cut into 279 pieces and all pieces will regrow  Remove some cells, the remaining cells remember which ones are missing  Resulting area devoid of scar tissue, look exactly like original o Urodele o Axololyl  Newts o Can regenerate tails, limbs, jaw, and ocular tissue o Differences from wound healing  Formation of a bulb of relatively undifferentiated cells- blastemal  Consists of rapidly proliferating cells (shown by BrdU experiments)  Most of cells are not specialized  Some of the cells because of the environment are able to dedifferentiate- back to stem cell like state capable of proliferating  Can dedifferentiate or transdifferentiate into other types of cells  Re-epithelialization in amphibians occurs much more rapidly  Proliferate almost instantaneously  Able to migrate across wound site faster and sooner than in mammals  Keratinocytes have integrins in membranes all the time so they don’t need to ramp up expression of genes  Apical ectoderm on blastemal  Where a normal epithelium would cover area and lay down a basal layer, this does not lay down a basal lamina (filter that separates environments)  Signaling molecules can more readily flow back and forth which makes growth easier  Macrophages and leukocytes to the same thing in regeneration  Remodeling process happens more readily and sooner and to a greater degree  Families of proteases  MMP’s matrix metalloprotease o Digest extracellular matrix o Metal ion o Help to remodel ECM  Tissue inhibitor metalloproteases- TMIPs o Secrete protease and activator and soon after secrete its inhibitor so activity is limited o Environment this creates allows dedifferentiation and proliferation o Evidence for dedifferentiation  Newt eye lens regeneration  Cells of the iris (pigmented epithelial cells) will dedifferentiate (stop making pigment) and transdifferentiate into lens cells (produce crystallines)  Can grow cells in culture and they will form foci where crystallines are produced  Human and chicken  In culture, some will differentiate into lens cells  Environment is not conducive to this happening in vivo  Multiple tissue types growing back  Bone, cartilage, muscle, neurons, etc.  Experiments with mytubes  Getting fibroblasts to differentiate into muscle cells  Newt fibers can be turned into myotubes  Grow myotubes in culture, labeled them, and reinserted them into a blastema  Tracked what happened to labeled cells as blastema grew  Labeled cells dedifferentiated as proliferating, non-muscle cells  Found them transdifferentiated into different types of cells  Look for onco gene related proteins to see if it had control over the dedifferentiation process  Western blot of protein retinoblastoma (Rb) o Tumor suppressor gene- keeps cells from proliferating o Binds TFs – active, prevents TF from activating target genes o Inactive- phosphorylated, target genes transcribed  proliferation o Retinoblastoma protein grown in culture at low concentration of serum myotubes – increase concentrations – changes location of retinoblastoma- phosphorylated  Active bottom line, inactive top line o Dedifferentiation linked to the activity of the Rb protein  Signals for location relative to beacon  Distal and proximal cuts on 2 different newts  Cut off one of the arms on each (one D and one P)  Take blastema from distal and attach it to the other newts proximal  Two hands grow- waited til arm grew to turn into hand  Or cut off proximal and attach distal to end o Blastemal knew it was ready to become a hand, but waited to form an elbow  Blastemas get signals for relative locations  People began looking for signaling molecules o Retinoic acid  Coated ceramic bead with retinoic acid (time release capsule)  Cut off arm of newt at different places and implant bead  Retinoic acid sends out signal from its location  Got additional arm parts being grown as retinoic acid signal strength increased  Cells from body under natural conditions are involved in sending out this signal- strength of signal is heard by regenerating cells o Similar to what is seen in fruit flies  Initial stages of healing process o Day 3  B6 mouse – cartilage, scab, clot  MRL mouse – cartilage, loose CT, scab, epidermis growing (doesn’t happen in B6)  re-epithelializes o Day 5  B6- scab/clot, epithelium starting to grow but hasn’t connected and covered scar  MRL- keratinocytes rapidly proliferate, dermal tissue expanding, blastemal begins to form o Day 10  B6- re-epithelializes, scab gone, epidermis is thick, pretty much done growing/healing  MRL- big blastemal, undifferentiated cells, proliferation, epidermis thins, no scab, capillaries forming (blood supply)  Muscle cells begin to form o Day 20  B6- done growing, thick epithelium (separation from cartilage); fibroblast in dense collagen ECM (scar)  MRL- sides of hole grow together and fuse and allow underlying tissue to connect o Day 81- Remodeling- MRL  Muscle forming, cartilage will eventually fuse together  Multiple tissue types  Epidermis fuse  continuous vs hole in B6 mouse  Basal lamina forms (separates epidermis from dermis), persists in B6, digested in MRL  MRL- communication b/w keratinocytes and underlying dermal tissue  can keep growing o Microarray  624 genes identified and spotted onto membrane – done in pairs  Extract RNA, convert to cDNA, wash over microarray and see what hybridizes  Radioactive nucleotide for probe o Measure intensity, quantify cDNA  Pref-1 expression increases after wound o Real Time RT-PCR  Higher level of Pref-1 in B6 but after wound it was lower in B6 than MRL o Laser capture microdissection microscope and IHC  Where was Pref-1 expressed?  Separate dermis from epidermis  Pref-1 expression in dermis, no pref-1 expression in epidermis  Inject Pref-1 into rabbit  makes antibody  IHC  Off end of cartilage was where Pref-1 was located (possible cartilage progenitor cells)  Expressing same way pre-adipocytes do o In site hybridization  What cells are making Pref-1?  Probe for RNA  Both expressing pref-1  Fibroblast like cells in cartilage (possible progenitor cells)  Proliferate and migrate and express protein o Compare protein expression  What made basal lamina go away? Upregulation of proteases  Protease expression  Matrix Metalloproteases (MMP2) o Metal ions at active site o Chew up ECM proteins o B6 higher than MRL o Wound- MRL increases, B6 decreases o MRL upregulates proteases  Native gel- agarose gel with gelatin mixed in – whole gel is protein  Run proteases into gel but don’t denature them  Drop in buffer  activates proteases  Digest gelatin, stain, whole gel turns blue except where proteases are  Compare proteases to active proteases  Stain for cells producing proteases  MRLs more interested in remodeling  Seal hole, remodel ECM  back to original state Heart Regeneration in Adult MRL Mice  Figure 1 o Create heart attack in a mouse o Probe- localization  able to identify where heart attack takes place o Sub lethal- don’t kill mice o Significant enough injury to be visible o Injury needs to be consistent with reproducible degree of damage o Collateral damage- don’t want to injure lungs  6-8 mm incision  Muscle cut exposes diaphragm – limited collateral damage  Figure 2 Days B6 MRL post injury 5 Damaged bottom half, Muscle fibers possibly starting to healthy top half, no form, grow in a line; consistent fingers protruding  with the way muscle fibers line disorganization up; cardiomyocytes; loosely packed  organization 15 Scar tissue Growth 60 A lot of scar tissue Regeneration, little scarring, normal myocardium  Figure 3 o DAPI- stains nuclei blue o BrdU- stains dividing cells o High density of BrdU cells in places of tissue injury o MRL vs B6  B6- BrdU in scar tissue – fibroblasts  MRL- BrdU in heart tissue o Does not tell you what cells they definitely are  Figure 4 o Compare BrdU and DAPI staining o Mitotic index- % of cardiomyocytes labeled with BrdU o B6- low numbers of dividing cells o MRL- high percentage of BrdU relative to total nuclei (10 fold higher than B6)  Range in % of what regenerative animals have o Each bar is an individual mouse- outlier in MRL might be due to difference in injury  Figure 5 o IHC o Stained muscle specific cells  alpha-actinin in cardiomyocytes- Z band on sarcomeres o Stained for dividing cells  BrdU o Colocalization of BrdU and muscle specific proteins  Showed that muscle cells were dividing  Figure 6 o Functional assay o EKG of multiple injured MRL hearts o Measures dimensions of heart chambers o Error bars- show volume of blood during contraction and rest o Damaged chamber- makes up for lack of strength by increasing in size  After 3 months it has regained the ability to pump o Regeneration- whatever grew back gained its original function  Figure 7 o Amino acid analysis, RT-PCR o Quantify hydroxyproline and collagen gene levels o A- levels of hydroxyproline- collagen protein  Shows how much collagen  B6- ECM more dense in collagen after injury  scar  MRL- collagen levels decrease  More proteases being expressed o B- expression of collagen genes  About the same in both mice o Same collagen genes but different amount of protein (expression) due to proteases  P21- peroxisome proliferator (activated receptor) o Regulates cell cycle- Inhibits CDK2 and CDK1  Binds and freezes cell in G1 stage of cell cycle – have to go through synthesis o Fibroblasts from  DNA content  P21 expression o More cells with a lot more DNA and frozen before mitosis  Ready to divide – frozen in G2 o MRL- lacked p21 cells  no expression  can’t control cyclins  divide upon injury o B6 crossed with P21 knockout – healed like MRL o Mate knockout with B6- B6/P21 knockouts  healed o P21 controlled by p53 which is a cancer related gene o Tolerance of DNA damage without going into apoptosis POSSIBLE ESSAYS What happens to Wnt and APC when crelox is knocked out? Is what we saw in the mouse ears regeneration? Is it similar enough to amphibian regeneration?


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