Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Methylation of DNASlide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32Slide 33Slide 34Slide 35Slide 36Slide 37Slide 38Slide 39Slide 40Slide 41Slide 42Slide 43Induction: information from neighbors influences cell fateinducerresponderIf induction tells cells what to do,how do they remember these instructions for a lifetime??DETERMINATIONDETERMINATION: internal and self-perpetuating changes that distinguish a cell & its descendents from other cells and commit them to a specialized course In other words, a cell is set to become a certain kind of cell before it actually becomes that type of cell. And usually there’s no turning back.This concept differs from fate in that for a time a cell’s fate can often be changed by changing environment; it differs from DIFFERENTIATION because this is overt cell specialization. FATE: what a cell will become at a later time--its future!DIFFERENTIATION: the actual elaboration of a cell’s fateDetermination can be assayed by transplantation experimentsLate gastrulaEarly gastrulaTransplant nervous system to epidermis and vice versaCells alter their fate to match that of neighbors i.e. not determinedCells retain their original fates, regardless of neighbors i.e. determined(see also Figure 7.16)Cloning:the ultimatetransplant experimentDolly (and Bonnie)Figure 2.1Cloning: The ultimate transplant experimentFreddy, a cloned leopard frog (Rana pipiens)How does cloning work?See Fig. 2.1 for how cloning works in sheepCloning success drops dramatically asthe age of the nuclear donor increasesDETERMINATION results from heritable changes in gene expressionAll cells have the same genes yet different cells make different mRNAsNon-base change modifications of DNA result in selective gene expressionEvidence for cytoplasmic contributions to selective gene expression3CHDNA Methylation is used in mammals tokeep unneeded genes inactiveSkin cellsBone-specific genes are methylatedMuscle-specific genes are methylatedSkin-specific genes are not methylatedCH3CH3CH33CH3CHCH3CH3CH3Gene expressionMethylation of DNAFigure 2.173CHDNA Methylation is used in mammals tokeep unneeded genes inactiveSkin cellsBone-specific genes are methylatedMuscle-specific genes are methylatedSkin-specific genes are not methylatedCH3CH3CH33CH3CHCH3CH3CH3Gene expressionImprinting results from pre-programmeddifferences in DNA methylation of selected genes in the sperm and eggGenomic imprinting occurs in most mammals* * *“A Silent Struggle”, New York Times, March 14, 2006Igf2r Igf2Imprinted genes affect fetal growthInsulin growth factor 2 Insulin growth factor 2 (Igf2) receptor (Igf2r)growth growthWhat happens if dad’s copy of Igf2 is deleted?Insulin growth factor 2 Insulin growth factor 2 (Igf2) receptor (Igf2r)growth growthWhat happens if mom’s copy of Igf2 isn’t imprinted?Insulin growth factor 2 Insulin growth factor 2 (Igf2) receptor (Igf2r)overgrowth growthBeckwith-Weidemann syndromeWhat if mom’s copy of Igf2r is deleted?Insulin growth factor 2 Insulin growth factor 2 (Igf2) receptor (Igf2r)growth growthWhat happens if mom’s copy of Igf2 is deleted?Insulin growth factor 2 Insulin growth factor 2 (Igf2) receptor (Igf2r)growth growthDNA within cells is not naked!DNA is wrapped around histone proteins as chromatinFigure 2.3Inactive genes are thought to be wrapped up in an inactive "chromatin conformation"Skin cellsBone-specific genes are wrapped up and inaccessibleSkin-specific genes are not wrapped up and so are accessibleGene expressionmRNAMuscle-specific genes are wrapped up and inaccessibleX chromosome inactivation:Silencing one of the two X’s in female cellsXist RNA is associated with the silenced X chromosomePost-translationalmodification ofhistones playsa key rolein regulatinggene expressionX chromosomeinactivationmyoDBy altering methylation, cells can be reprogrammed "Fibroblast-like" mouse cells Treat with 5-azacytidine to reduce DNA methylation Fat cellsMuscle cellsBone cellsExperiment #1Chromatin transferred from myoblasts can transform untreated fat cells. Experiment #2Transformation frequency suggests only one or few of the genes have this abilityUltimately a single gene was identified-myoD-whose expression could convertany other cell type into muscle cellsMyoD is expressed at the right place and time to be a master regulator of muscle developmentSomitesExperiment #3myoD siteDoes the myoD transcription factor bind DNA sites within the promoters of the expected gene types?Bone-specific genes lackmyoD binding sites muscle-specificmRNAmyoDONMuscle-specific genes havemyoD binding sites OFFOFFExperiment #4Fat-specific genes lackmyoD binding sitesScientists "knocked out" the myoD gene to test its predicted role in muscle development Eliminate gene encoding myoD Does mouse develop muscles? Experiment #5!!MyoD and related transcription factors work together to regulate muscle cell fateExperiment #6Cellular memory and the engrailed geneHow do cells remember their decisions?Remember that Wingless isexpressed by specific cellswithin each segmentAnteriorPosteriorWgEnEngrailed is a transcription factor expressed by cells posterior to those that express Wingless.It defines the posterior boundary of each segment.See also Fig. 6.33Here’s the real thing: engrailedMechanism #1: cell-cell signalingEngrailed expression requires Wingless signaling by the neighboring cells.Mechanism #2: autoregulation Later on, engrailed becomes autoregulatory ie. it turns on its own synthesis Segment boundarySegment boundaryONenMore engrailed proteinen geneMechanism #3: specialized chromatin proteins In cells where engrailed is turned off, a set of specific chromatin proteins bind to it and wrap it up in a permanently inactive state. Segment boundarySegment boundaryen geneen geneOFFPermanentlyOFFPolycomb group proteins turn off engrailed in cells it’s not
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