Genetics of Behaviorand Development ILecture 34Adult C .elegansnematode wormWith embryosWith larvaeThe Adult Nematode wormCaenorhabditis elegansMALEHermaphroditeAfter egg fertilization the embryo is laid at ~ 6hand it hatches at ~ 12h to enter the first larvalstage (L1)The C. elegans Life Cycle – only 50 hAdult C .elegansnematode wormWith embryosWith larvaeP0ABP1P2EMSABaABpFertilized egg(zygote)First cell division Subsequent divisionsPattern of early cell divisions from the C. eleganszygote to form six Founder CellsFertilized egg (zygote)PharynxEpidermiszygoteNeuronsVulvaIntestineGermlineSomatic gonadFull lineage of the entire C. elegans bodyL1L2L3L4Adult0h50hhatchPharynxEpidermiszygoteNeuronsVulvaIntestineGermlineSomatic gonadFull lineage of the entire C. elegans bodyL1L2L3L4Adult0h50hhatchJohn SulstonSydney BrennerBob Horvitz (MIT)2002 Nobel Prize inPhysiology or MedicineImportant features of the C. eleganscell lineage• It is the best documented example of a highly reproducible pattern of cell division for any organism• The pattern of cell divisions starting from a wild type C. elegansfertilized egg is nearly always the same• About 10 rounds of cell division create the adult worm that hasmany of the same tissues that we do!• Any one particular cell undergoes relatively few divisions• One hemaphrodite worm can produce 500,000 progeny in a week ona petri dish• Powerful model organism to identify the regulators that controlcell lineage in both time and spaceHow would you identify the regulatory mechanismsthat control the development of cell lineages?Look for mutants that display aberrant lineages and characterize thegenes, of course!Sydney Brenner (and subsequently others) mutagenized C. elegansand screened for “funny looking worms”What does “funny” mean – altered body shapes, e.g., dumpy, small, long;uncoordinated or otherwise behaving strangely.One class of mutants turned out to be lacking a body part…namely thevulva through which the embryos are laid…these animals are unable tolay eggs.These egg-laying mutants were picked for study with the idea thatthere were obvious problems with the execution of at least some celllineages, maybe there would be a problem with other lineages.PharynxEpidermiszygoteNeuronsVulvaIntestineGermlineSomatic gonadFull lineage of the entire C. elegans bodyL1L2L3L4Adult0h50hhatchPharynxEpidermiszygoteNeuronsVulvaIntestineGermlineSomatic gonadFull lineage of the entire C. elegans bodyL1L2L3L4Adult0h50hhatchL1L2Zoom in on particularcell lineages, in thiscase the T lineageAsk whether thedevelopment of thislineage is normal inegg-laying mutantsNormal Precocious RetardedRetardedNormalDistinctivecelldivisionpattern inL1 vs L2PrecociousActuallyskippedover theL1 celldivisionpatternRetardedRepeatsthe L1 celldivisionpatternover andoverRetardedRepeatsthe L1 celldivisionpatternover andoverlin-4 and lin-14 mutants affect postembryonic lineagesH1, H2, M, V1-V6 as well as the T lineageH,G,T,Q,V K,P,W G,T,Q,VM, andGonadal (G)Lineages:These mutants have beencalled HETEROCHRONICmutants – they affect therelative timing of majordevelopmental eventsThe existence of suchmutants indicates thatthere is coordination ofthe temporal sequence ofmany cell fates an animaldevelopsThis temporal coordinationis genetically regulatedHeterochronic MutantsMisregulation of another lineageRetardedlin-29 (lf)Precociouslin-28 (lf)Precociouslin-14 (gf)Retardedlin-14 (lf)Retardedlin-4 (lf)PhenotypeMutantOther Heterochronic mutantsalso identifiedHow would you go about determining theorder in which lin-4, lin-14, lin-28 and lin-29operate to control the timing ofdevelopment?Retardedlin-29(lf)RetardedPrecociouslin-28(lf)RetardedPrecociousPrecociouslin-14(lf)RetardedmixedRetardedlin-14(gf)RetardedPrecociousPrecociousRetardedRetardedlin-4(lf)lin-29(lf)lin-28(lf)lin-14(lf)lin-14(gf)SinglemutantsDouble Mutantslin-4lin-14lin-28lin-29LarvalProgramAdultProgramlin-4lin-14lin-28lin-29LarvalProgramAdultProgramlin-4 encodes small RNAs, 61nt and 21 ntlin-14 encodes a nuclear proteinlin-28 encodes another proteinlin-29 encodes a Transcription factorMicroRNAs control gene expression byhybridizing to specific mRNA speciesTemporal expression of the lin-4 RNA is crucial for theorchestration of lineage specific cell divisionNormal Precocious Retarded Retarded Normal Precocious Retarded Retarded lin-4 RNAlin-14 or lin-28proteinL1 L2hatchRNA orprotein levellin-4lin-14lin-28lin-29LarvalProgramAdultProgramlin-4lin-14lin-28lin-29LarvalProgramAdultProgramThis was the first microRNA regulatory moleculeto be discoveredmicroRNAs are widespread in nature, includinghumans, and are very often involved indevelopment and
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