Drosophila development Flashcards
(17 cards)
CNS development: AS-C and asymmetric division
From ventral band/ cells- neuroblasts segregate, delaminate into embryo interior. Leftover cells-> epidermis.
AS-C: Mutation (xs nervous system, no epidermis)+ imaging-> cells that start to exp fam/proneural genes (AS-C)-> neuroblast. Initially all cells cluster, produce AS-C equally. One cells signals more (Delta)-> feedback inhibits AS-C exp in other cells (lateral inhibition)-> less Delta production. Developing neuroblast receives less Notch activation, AS-C exp more.
Asymmetric division/neuroblasts: Neuroblast delaminates (bulbs out of layer of cells like bubble gum) from proneural cluster, cell determinants localise inside. Feedback loops ensure factors @ right pole. Asym div to self-renew+ produce neural progenitor cell (Ganglion mother cell (GMC))- divides only once to make neurons+ glia, diff set by segregation of determinants.
CNS development: prospero and PAR, neuroblast clock
Prospero: ablate cells, image proteins. Prospero segregates only to GMC to limit division to just 1 further cycle, initiate neural programmes of gene exp.
PAR proteins inherited from apical cortex of overlying epithelium. Various adaptor proteins-> cortical MTs oriented properly for asymmetric div. PAR proteins essential to 1st div in worms, too- v conserved. Scaffolding proteins, enzymes, adaptors etc that build+ maintain cortical asymmetries.
‘Neuroblast clock’= series of TFs that reg neuroblast temporal ID. Genetic manipulation-> change exp patterns of NB. WT: exp/ TFs changes each cell cycle. Altering TF exp in NB changes GMC fate. While Prospero in all GMCs ensures they divide only once, TFs ctrl outcome of neurons+ glia.
Early dev, imaginal discs and female reproduction
Drosophila as model: discovery/ many conserved genes+ pathways. Embryo-> larva-> adult.
Larva grows increasing cell size, but not #. DNA replicates but no chromatid sep/ nuclear div-> polytene cells.
Imaginal discs: sac-like epithelial structure in larva. Undergo metamorphosis-> potion of adult during papal transformation, while most larva tissues degenerate during metamorphosis.
Female reproduction: 2 ovaries, ~18 ovarioles each (varies). Oogenesis In germarium- stem cell divides x4, incomplete cytokinesis-> 16 cell germline cyst w/ cells connected by ring canals- 1 cell->oocyte, other 15-> supporting nurse cells. Germline cyst encapsulated by monolayer/ somatic derived follicle cells, making ‘egg chamber’. Ovariole has egg chambers @ diff dev stages- oogenesis has 14 morphologically distinct stages. Germline+ somatic cells interact throughout; mature egg paused. Mature eggs enter oviduct, activated, move to uterus where fertilised by sperm held in female.
mRNA in oogenesis and processes following fertilisation
mRNAs in oogenesis made in nurses, oocyte nucleus transcriptionally off. Early oogenesis- grk+osk mRNAs transported on MT via motors to oocyte. Mid-oogenesis, cytoskeleton remodelled, directing oks, grk, bcd mRNAs to distinct regions of oocyte. Late oogenesis, nurses empty content into oocyte, die; cytoskeleton remodelled again, cytoplasmic streaming (centrifugal mixing) initiate in oocyte. Bcd+ nos anchored @ respective poles. Mature eggs come pre-loaded w/ mRNAs
Following fertilisation+ deposition, embryo dev as syncytial blastoderm (nuclei share cytoplasm). Cellularisation 3hrs post-fertilisation, after 14 rounds/nuclear div. Germband extends+ pole cells invaginate in gastrulation. Germband completes extension, then retracts.
Bcd and nos
Bcd+ nos: in situ antibody stain-> bcd encodes TF w/ homeodomain DNA binding motif. Nos= translational repressor of maternal hunchback mRNA (hb). Before zygotic transcription, maternally exp factors interact to form opposing gradients: bcd vs cad (blocked by Bcd) and hb (blocked by nos) vs nos. Bcd mutant shows double abdomen+ 2 tails.
Genetic screen for maternal patterning genes-> all embryos from homozygous mutant females abnormal (even crossed w/ homo wt males). Reciprocal cross w/ mutant males-> all embryos wt.
Cuticle preps; maternal patterning genes
Cuticle preps: show denticle bands in larva or embryo, showing patterning. Easy, fast, storable for yrs, easy to screen+ generate samples for comparison. Blastoderm stage embryo patterning is reflected in larva cuticle. 4 phenotypic classes (many mutant lines within each) of maternal patterning gene mutants easily seen from cuticle prep- bicoid mutants (anterior), oskar (posterior), torse-like (terminal)+ dorsal/ventral (cactus)
Maternal patterning genes: complementation tests+ gene mapping ID alleles in same/diff genes. ID multiple components of coordinate systems.
Anterior and posterior systems, terminal and dorsoventral; maternal double mutants
Anterior+ posterior systems established by localised mRNA +/protein products, maternally encoded. Terminal (transmembrane receptors Torso)+ dorsoventral (transmem rec ToII) systems depend less on localised products in egg, mediated by signals outside egg (synth by follicle cells) transmitted by receptors in egg mem.
Maternal double mutants- phenotypes additive, indicating genes for each patterning process act independently.
Mutagenic screen to ID zygotic genes
Mutagenic screen to ID all zygotic genes in embryo patterning got Nobel prize (’95). Used balancer chromosome, lethal when homozygous+ DTS (dominant temp sensitive)- in 29C, flies die/infertile. Patterning defects analysed by cuticle preps, heterozygous viable stocks-> mutation could be mapped+ studied. Denticles= specialised cuticle structures secreted by epidermis (cuticle is outside of larva)- diff segments have distinct patterns. In mutants, defects in denticle pattern.
3 major classes of gene affecting segmentation
3 major classes/ genes affecting segmentation:
1) Gap gene- large region depleted. Genes named giant, knirps, kruppel, almost all encode TFs. Antibody stains show exp early, when embryo syncytial. Fade rapidle
2) Pair rule- portion of segment deleted every other segment. Genes odd-skipped, even skipped (eve), fushi-tarazu (ftz). Almost all encode TFs. Antibody stains show exp after gap genes when cellularisation beginning but not complete, exp bands just 1 segment wide. Fade rapidly
3) Segment polarity: each segment missing substantial portion, remainder duplicated. Genes armadillo, hedgehog, gooseberry. Encode array of molecules- TFs, signals, transmem receptors, cell adhesion molecules. Exp after cellularisation when embryo has extended germband. Some remain stable (engrailed marks posterior of every segment throughout life)
Heirarchy of genes and gene copy numbers
Heirarchy: Maternal mutants disrupt gap, pair rule and segment polarity. Gap mutants disrupt pair rule+ segment polarity. Pair rule disrupt segment polarity- i.e., patterning info from each class req for next. E.g. Bcd mRNA+ protein visualised in embryos. Bcd mRNA translated after egg activation. Protein= TF, found in nuclei
Gene copy # impacts protein gradient: e.g. change copy # bcd-> antibody stain/ protein gradient different- morphogen.
Gap genes: Bcd, Hb and Kruppel
Gap genes: Bcd and hb: Maternal genes most likely to act of next group in hierarchy (gap genes). Bicoid mutant mothers’ embryos fail to exp zygotic hb in anterior, suggesting bcd important for Hb proteins. If bcd protein prepped in vitro from exp construct, mixed w/ DNA fragments from hb gene+ use antibody to precipitate DNA bound to Bicoid, find 2 seqs upstream of hb start site bound. Analysis of seqs w/foot printing assay-> 3 sites on fragment A and 2 sites on fragment B protected -> bicoid= seq-specific DNA binding protein, encodes homeodomain TF binding+ targeting gap genes. Construct genes w/ diff promoter fragment lengths coupled to reported seq-> diff amount/Bcd binding-> diff activity, showing bcd regulates hb promoter activity in early embryo. Zygotic hb= direct target/ maternally supplied Bcd. Nanos protein represses translation/maternal hb in posterior (see gradients above), hence 2 mechs ctrl Hb levels. Bcd+Hb are intracellular morphogens acting to reg gene exp of other gap genes.
Hb and Kruppel overlap in a small region ~centre of blastoderm. Overlapping exp domains-> signals for generation of new, more precise patterns.
Pair rule genes: Eve
Pair rule genes: Eve: specific elements in promoters activated only when specific combo/ gap gene TFs bound. Fragments of eve-promoter added upstream of promoter-> discrete module of even promoter activate exp in domains reflecting separate parts of normal pattern. Cross flies carrying reporter construct (eve 2) into gap gene mutants-> in mutant for giant, domain of eve 2 expands into domain normally expressing giant. Normal exp pattern (from proteins visualised together) show gap genes reg eve2+ giant exp in cells adjacent to eve stripe 2 cells. Same concept for other reg genes- bcd+ hb cover the domain (promoters) while giant+ Kr have complementary exp patterns (outside eve stripe 2- inhibitors) for eve 2 exp. Overall, 4 Bcd, 1Hb, 3 Giant+ 3Kr binding sites on eve 2 promoter. Activator+ repressor sites overlap, implying competition/interaction between regulators. Other stripes of eve ctrled similarly- eve regulatory region >20kb, >20 regulators. Promoter acts as microprocessor, integrating positional inputs-> repetitive output.
Segment polarity gene control by pair rule genes, wg and en
Segment Polarity genes ctrled by pair rule: Wg+ En: proteins exp in extended germ band embryo. Segment polarity genes exp in complementary sub-domains in every segment+ only after cellularisation. Visualising pair rule exp w/ antibodies-> initially fuzzy pattern resolves w/ time, suggesting feedback. Analyse seq-> Ftz and Eve have autoregulatory elements in enhancers, downreg each other. Activation/ segment polarity genes follow exp/ pair rule genes. Combinatorial ctrl: En even stripes align w/ front of Ftz stripes, odd w/ Eve stripes, suggesting each segment polarity gene has minimum 2 reg codes. Meanwhile wg where neither eve/ftz exp.
Wg+en cells interact to maintain exp of both. Exp/ segment polarity genes-> denticle secretion (wg/other mutants in pathway have clear cuticle phenotypes).
Initial polarity of egg elaborated through gradually increasing complexity-> div embryo into repeating units.
Homeotic transformation (with 2 e.g.s)
Homeotic transformation= where perfectly formed structure appears in wrong place, often replacing the structure that should be there. Can occur reproducibly. E.g.
posterior features in anterior pb mutant has legs instead of mouthparts, antp mutant has legs instead of antennae- both are dominant gain-of function mutations (1 copy-> phenotype)- dev/ posterior features in anterior usually due to gain of function
Anterior features in posterior usually due to loss of function. Ubx mutant has 4 wings, 2 thorax (there are multiple ‘bithorax’ phenotype mutations)- in wt, T2-> 2nd leg and thorax, T3-> 3rd leg+ haltere- ubx= LoF causing T3 to transform into 2nd T2. In contrast, hm mutant has 2 wt T3, not T2- due to ubx being turned on (gain of function) in wing discs, where it is usually off.
Selector genes, homeotic genes and Hox genes
Selector genes= switches to choose between dev fates e.g. Ubx gene on/off-> T3/2.
Homeotic genes/ drosophila map to 2 clusters chr 3- antp (5 genes) complex+ bx (3 genes) complex.
Hox genes= abbrev/homeobox. Large fam/ related proteins that dictate regional identity in embryo, particularly along A/P axis. Not req for segmentation, but for segment ID. Genetic analysis/mutants affecting ID/ segments in embryo shows when 3 Bx genes deleted, all segments behind T2 develop similarly. Similar analysis deleting diff Bx genes revealed req for thoracic+ abdominal segment IDs. LoF/ Hox genes-> anterior features in posterior segments. Seem to work combinatorially.
Lewis model for Bithorax complex and Ubx
Lewis model for Bithorax complex f(x): moving posterior, segment ID req exp of increasing # Bx genes (posterior genes dominate is exp w/ anterior ones). Antibody stains confirm Hox genes exp in anterior-> posterior seq, w/ collinearity between A/P seq of exp domains and seq of genes along chr 3.
Establishing domains of exp in Hox genes
Ubx: antibody stain shows early exp noticeably stripy in blastoderm stage. Exp req activity of both gap and pair rule genes (hb blocks Ubx exp, ftz promotes it), i.e. Hox exp at blastoderm stages ctrled by gap+ pair-rule genes. Exp of drosophila Hox genes provides cells w/ persistent memory of where they were in blastoderm+ hence what segment they dev into in embryo/larva/adult.
Patterning along the dorsal-ventral axis: Grk, pipe, Dpp and Twist
Patterning dorsal-Ventral axis
Mutants effect inward mvmt of mesoderm. Dorsalising mutants (snake) lack denticle belts; ventralising mutants (cactus) denticle belts run around the whole embryo.
Grk: First events establishing dorsal-ventral axis in oogenesis. Grk mRNA exp localised to future dorsal side. Grk protein signals to overlying follicle cells through EGF receptor Torpedo on ventral side to establish dorsal/ventral follicle cell fates (blocks pipe)
Localised pipe exp in ventral follicle cells during oogenesis provides dorsal protein gradient in early embryo. Ventral cells with Dorsal protein-> mesoderm. Dorsal protein= TF activating/ repressing zygotic genes for differentiation along D/V axis; additional gradients generated by Dorsal apposing gradients. If Dorsal protein not restricted to ventral side (from failure to localise grk mRNA or any part of dorsal axis pathway)-> ventralised phenotype.
Dpp (homolog of vertebrate bone morphogenic proteins (BMPs)) exp dorsally, diffuses ventrally. Sog (homologue of vertebrate chordin)= extracellular matrix protein, Dpp activity antagonist.
Twist exp as result of high conc/ Dorsal in nucleus. Twist expresses TF in mesoderm precursors, causes change in myosin, results in ventral furrow forming.
Gradients of dpp, sog, etc homologues reversed in chordates (‘upside down insects’).
