1Lecture 18: Drosophila melanogasterPolytene chromosomeLife cycleP elements and transformationEmbryogenesisRead 565-578; Fig. 13.17-34“Molecular Biology of the Cell” ed. By Bruce Albertet al. (free online through ncbi books)2Drosophila melanogaster (fruit fly)Small genome 170 Mb (5% of human genome)33% repetitive DNA13,600 genesThree autosome + X and Y (small chr. number)Giant polytene chromosomeFast life cycle (10 days)3Fig. D.34• Giant polytenechromosomes of larvalsalivary gland are keytools– Replicate 10-11 times– 1024-2048 sisterchromatids stay associatedunder perfect lateralregister– Homologous chromosomestay tightly synapsed– Chromocenter – commonregion where centromerescoalesce5Fig. D.46Fig. D.57P-element transposons are critical tools inmolecular genetics• Hybrid dysgenesis– Males from Drosophila strains carrying P elements crossed tofemales that lack P elements– P element becomes highly mobile in germ line of F1 hybrids– Chromosome breakage reduces fertility in hybrids– Progeny of F1 flies carry many new mutations induced by P elementinsertions– Molecular details• P element primary transcript encodes transposase that catalyzestransposition• Cross between P and M strain causes hybrid dysgenesis• Cross between P and P strain does not– Eggs produced by P female have repressor protein that preventstransposition– Repressor coded for by alternatively spliced P element mRNA8Fig. D.7910Transformation: the introduction of cloned DNA into flies• P-elements used asvectors• Insert fly DNA intointact P element andthen into plasmid• Inject into syncytialembryos from M strainmothers• Cross to P males• Mimicking hybriddysgenesisFig. D.8a11Four classes of genes responsible forformation of segments• Maternal genes• Gap genes• Pair-rule genes• Segmentation polarity genes• Function in a hierarchy that progressivelysubdivides the embryo into successively smallerunitsDrosophila embryogenesis12 Figure 21-30. A Drosophila oocyte in its follicle. The oocyte is derived from a germ cellthat divides four times to give a family of 16 cells that remain in communication with oneanother via cytoplasmic bridges (gray). One member of the family group becomes theoocyte, while the others become nurse cells, which make many of the components requiredby the oocyte and pass them into it via the cytoplasmic bridges. The follicle cells thatpartially surround the oocyte have a separate ancestry. As indicated, they are the sources ofterminal and ventral egg-polarizing signals. (From Bruce Albert Book)Drosophila oocyte13 Fig. D.18Drosophila Embryogenesis14Fig. D.1915A parade of 1995 A parade of 1995 NobelsNobels::for their discoveries concerning the genetic control of earlyfor their discoveries concerning the genetic control of earlyembryonic developmentembryonic developmentEric F. Eric F. WieschausWieschausChristianeChristiane N Nüüssleinsslein--VolhardVolhardEdward B. LewisEdward B. Lewis16From BruceAlbert Book17The segmentation pattern of Drosophila larva18Klaus Sander proposed:• Each pole of the egg produces a different substance• These substance form the opposing gradients by diffusion• Concentrations of these substances determine the type ofstructure produced at each position long the body axisMorphogens: Substances that define differentcell fate in a concentration-dependent manner19Fig. D.20Bicoid (Bcd) is a morphogen20How Bcd protein works21Fig. D.21 bottom22Maternal genes interact to producemorphogen gradients• Maternal-effect mutations– Recessive mutations in maternal genes that influence embryonicdevelopment• Maternally supplied components account for formation of bodyplan between fertilization and end of 13 syncytial divisions• Nusslein-Volhard and Wieschaus screened thousands of mutagentreated chromosomes by examining phenotypes of embryos fromhomozygous mutant mothersm/m X+/+Abnormal+/mm/m+/+XNormal+/mF123Zones of expression of four gap genes: hunchback, Kruppel,knirps, and giant in late syncytial blastoderm embryosGap genesThese are zygotic genes24Defects in segmentation frommutations in gap genesFig. D.22bA)B) C) D.)25• Gap genes– Gap mutants show a gap in segmentation pattern atpositions where particular gene is absent– Binding sites in promoter have different affinities formaternal transcription factors– Gap genes encode transcription factors that influenceexpression of other gap genes26Fig. D.23Pair-rule genes27Fig. 19.2Ftz mutant28 Figure 21-38. Modular organization of the regulatory DNA of the eve gene. In the experimentshown, cloned fragments of the regulatory DNA were linked to a LacZ reporter (a bacterial gene).Transgenic embryos containing these constructs were then stained by in situ hybridization to revealthe pattern of expression of LacZ (blue/black), and counterstained with an anti-Eve antibody (orange)to show the positions of the normal eve expression stripes. Different segments of the eve regulatoryDNA (ochre) are thus found to drive gene expression in regions corresponding to different parts ofthe normal eve expression pattern. Two segments in tandem drive expression in a pattern that is thesum of the patterns generated by each of them individually. Separate regulatory modules areresponsible for different times of gene expression, as well as different locations: the leftmost panelshows the action of a module that comes into play later than the others illustrated and drivesexpression in a subset of neurons. (From Bruce Albert Book)29Pair-rule genes• (a) zones of expression at beginningof blastoderm stage– Each gene expressed in sevenstripes• (b) Activation of Eve stripes relieson different cis-regulatory elements30Fig. D.24Segment polarity genes31Segment polarity genes are lowest level ofsegmentation hierarchy• Mutations in segment polarity genes causedeletion of part of each segment and itsreplacement by mirror image of different partof next segment• Regulatory system complex– Transcription factors encoded by pair-rule genesinitiate pattern by regulating segment polarity genes– Interactions between cell polarity genes maintainperiodicity later in development32Segment polarity genes are lowest level ofsegmentation hierarchy• Mutations in segment polarity genes causedeletion of part of each segment and itsreplacement by mirror image of different partof next segment• Regulatory system complex– Transcription factors encoded by pair-rule genesinitiate pattern by regulating