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Drosophila lecture

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Drosophila early embryogenesis, patterning , fate determination

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Drosophila lecture

  1. 1. EARLY EMBRYOLOGY, FATE DETERMINATION, AND PATTERNING IN DROSOPHILA NEELAM DEVPURA (M.Sc. ,NET) CSIR-UGC JRF Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  2. 2. LIFE CYCLE DROSOPHILA Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  3. 3. DROSOPHILA LIFE CYCLE • Life cycle by days Day 0: Female lays eggs Day 1: Eggs hatch Day 2: First instar (one day in length) Day 3: Second instar (one day in length) Day 5: Third and final instar (two days in length) Day 7: Larvae begin roaming stage. Pupariation (pupa formation) occurs 120 hours after egg laying Day 11-12: Eclosion (adults emerge from the pupa case). Females become sexually mature 8-10 hours after eclosion Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  4. 4. The Drosophila life cycle represents the differentiation of two distinct forms: the larva and the Imago (adult). Embryogenesis: differentiation of the larva Metamorphosis: differentiation of the imago (adult) Imaginal cells are the cells of the adult or imago.
  5. 5. DROSOPHILADEVELOPMENT ¤ Embryonic development in Drosophila is an orderly sequence of change and is controlled by the differential expression of genes. ¤ Drosophila display a holometabolous method of development, meaning that they have three distinct stages of their post – embryonic life cycle, each with radically different body plans: larva, pupa and finally, adult(imago Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  6. 6. Neelam Devpura, GSBTM Presentation, MNVSC DROSOPHILA DEVELOPMENT - OVERVIEW  Cleavage  Fertilization  Gastrulation  Drosophila body plan  Oocyte formation  Genetic control of axis specification  Anterior-posterior  Dorsal-ventral  Segmentation genes  Homeotic genes
  7. 7. Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  8. 8. DROSOPHILAFERTILIZATION Eggs are activated prior to fertilization. - oocyte nucleus has resumed meiotic division - stored mRNAs begin translation Eggs have begun to specify axes by the point of fertilization. Sperm enter at the micropyle. - probably prevents polyspermy. Sperm compete with each other!
  9. 9. EARLY DEVELOPMENT OF DROSOPHILA • Egg is centrolecithal • After fertilization, series of superficial cleavages • Blastoderm is syncytial until 13th cleavage (256 nuclei!) • Nuclei begin dividing centrally, migrate toward the edges • Several nuclei migrate to posterior end, form cell membranes (pole cells) • Give rise to the adult gametes • What cells are like this in mammals?
  10. 10. Superficial Cleavage Syncytial blastoderm stage - zygotic nuclei undergo 8 divisions - nuclei migrate to periphery - karyokinesis continues Cellular blastoderm stage - following division 13, oocyte plasma membrane folds inward - partitions off each nucleus and associated cytoplasm - constricts at basal end Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  11. 11. Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  12. 12. SUPERFICIAL CLEAVAGE Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  13. 13.  Although nuclei share the same cytoplasm, the cytoplasm is not uniform in its makeup • Maternal molecules are distributed differently  Eventually cells will form plasma membranes and the embryo will consist of a cellular blastoderm  Mid-blastula transition occurs slowly, increasing transcription of zygotic genes Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  14. 14. GASTRULATION • At MBT, gastrulation begins, forming mesoderm, endoderm, ectoderm • Cells fold inward to form ventral furrow • Embryo bends to from cephalic furrow • Pole cells are internalized, endoderm invaginates • Ectoderm converges and extends along midline to form Germ Band Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
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  16. 16. GERM BAND • Wraps around the embryo • As it wraps around the dorsal surface, the A-P axis of the embryo is laid down • Body segments begin to form • At the end of germ band extension • Organs are beginning to form • Body segmentation is set-up • Groups of cells called imaginal discs are set aside, these cells will form adult structures Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  17. 17. DROSOPHILA LARVAE • During metamorphosis • 3 “instar” larvae • Pupae • Adult • After gastrulation, 1st instar larvae is formed • Has head and tail end • Repeating segments along axis • Generally the same type of body plan as adult Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  18. 18. DROSOPHILA BODY PLAN • 3 thoracic segments • Each different from each other • 8 abdominal segments • Each different from each other • Able to tell the difference in the larvae based on cuticle • Covering of the embryo • Correspond to the adult segments Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  19. 19. Segments form along the anterior-posterior axis, then become specialized. Specification of tissues depends on their position along the primary axes. A/P and D/V axes established by interactions between the developing oocyte and its surrounding follicle cells T1- legs T2 – legs & wings T3 – legs & halteres Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  20. 20. GENETICS OF AXIS SPECIFICATION IN DROSOPHILA • Controlled by a variety of genes • Maternal effect genes • Gap genes • Pair-rule genes • Segment polarity genes • Homeotic selector genes Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  21. 21. Anterior-Posterior Body Plan Drosophila use a hierarchy of gene expression to establish the anterior-posterior body plan. 2. Gap genes: first zygotic genes expressed Divide embryo into regions - expressed in broad, partially overlapping domains (~ 3 segments wide) - code for transcription factors; transient -activated or repressed by maternal effect genes - Hunchback overlap bicoid gradient Kruppel 1. Maternal effect genes (e.g. bicoid, nanos) Establish polarity: - mRNAs differentially placed in eggs - transcriptional or translational regulatory proteins; transient - diffuse through syncytial cytoplasm - activate or repress zygotic genes Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  22. 22. - even-skipped (red), fuschi tarazu (black) engrailed 3. Pair-rule genes; Establish segmental plan - regulated by combinations of gap genes - code for transcription factors; transient - divide the embryo into periodic units - pattern of seven transverse bands delimit parasegments 4. Segment polarity genes; Set boundaries of segments (i.e. establish A-P for each segment) - activated by pair-rule genes - code for variety of proteins; stable - divide embryo into 14 segmental units Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  23. 23. - 5. Homeotic selector genes; Provide segmental identity - interactions of gap, pair-rule, and segment polarity proteins - determines developmental fat Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  24. 24. Segmentation Genes Cell fate commitment: Phase 1 – specification Phase 2 – determination - early in development cell fate depends on interactions among protein gradients - specification is flexible; it can alter in response to signals from other cells - eventually cells undergo transition from loose commitment to irreversible determination The transition from specification to determination in Drosophila is mediated by the segmentation genes. - these divide the early embryo into a repeating series of segmental primordia along the anterior-posterior axis Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
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  26. 26. Drosophila Body Plan - Egg Stage Translation leads to formations of patterning protein (e.g. morphogen) gradient within the embryo. Body axes are determined in the egg by distribution of maternal mRNAs and proteins. How are asymmetric distributions of messages and proteins established in the egg? Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  27. 27. Oocyte Formation (A-P, D-V Axes) nurse cells contribute mRNA, proteins cytoplasm Drosophila ovariole Oogonium divide into 16 cells 1 oocyte 15 nurse cells all interconnected Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  28. 28. Anterior-Posterior Axis Formation Torpedo (RTK Gurken receptor) present on follicular cells Gurken binding results in “posteriorization” of follicles - posteriorized follicles re-organize egg microtubules; (-) = anterior Gurken protein localized between nucleus and cell membrane - Note – Gurken diffuses only a short distance Nurse cells synthesize gurken (TGF-β family) - gurken mRNA transported toward oocyte nucleus (in posterior region)
  29. 29. A-P Axis: bicoid / Oskar / nanos Nurse cells manufacture bicoid and nanos mRNA - deliver cytoplasm into oocyte bicoid binds to dynein - moves to non-growing (-) end of microtubules oscar mRNA forms complex with kinesin I - moves toward growing (+) end of microtubules Oskar binds nanos mRNA - retains nanos in posterior end “posteriorized” follicles produce organized (+/-) microtubules Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  30. 30. Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  31. 31. Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  32. 32. Posterior Specification - 1 Nanos prevents hunchback translation Oskar binds nanos; remains in posterior At least 9 maternal genes make up the posterior patterning group; including nanos trap: Staufen allows oskar translation staufen, oskar, nanos Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  33. 33. Model of Anterior-Posterior Patterning mRNA in oocytes (maternal messages) Early cleavage embryo proteins hunchback translation repressed by Nanos caudal translation repressed by Bicoid Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  34. 34. Posterior Specification - 2 Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
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  36. 36. Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  37. 37. Bicoid Mutants Martin Klingler Bicoid – homeodomain transcription factor; morphogen Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  38. 38. Manipulating Bicoid Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  39. 39. Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  40. 40. Dorsal - Ventral Axis Formation Further Gurken Effects The oocyte nucleus (with associated gurken) moves anteriorly along the dorsal margin Gurkin/Torpedo interactions “dorsalize” follicle cells Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  41. 41. Dorsal activates genes that create mesodermal phenotype - transcribed only in cells with highest Dorsal concentrations - these genes have low affinity enhancers (lots of Dorsal necessary) Dorsal also inhibits dorsalizing genes Dorsal ventral cells form medoderm DISTRIBUTION OF DORSAL Dorsal: - large amount = mesoderm - lesser amount = glial/ectodermal
  42. 42. Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  43. 43. Zygotic Patterning Genes decapentaplaegic (dpp), zerknüllt (zen), tolloid are dorsal patterning genes - repressed by Dorsal Intermediate dorsal activates rhomboid (no Twist or snail) - determines neural ectoderm - rhomboid + twist = glial cells Intermediate dorsal activates fgf8 - fgf8 repressed by snail - promotes mesodermal ingression dorsal High Dorsal – activates twist and snail (low affinity enhancers) - mesoderm determinants Dorsal (TF) – expressed ventrally; establishes diffusion gradient dorsally ~ 30 genes directly affected by Dorsal Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  44. 44. wingless patched Maternal effect genes bicoid nanos Gap genes huckebein hunchback giant Pair-rule genes even-skipped fushi tarazu Segment polarity genes engrailed hedgehog Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  45. 45. GAP GENE EXPRESSION Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  46. 46. Krüppel Specification by Hunchback Wolpert, 2007 Threshold activation/repression Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  47. 47. Pair-Rule Gene Regulation e.g. – even-skipped - each stripe regulated by a different set of enhancers - expression patterns are stabilized by interactions among other gene products e.g. even-skipped expression limited by Giant Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  48. 48. fushi tarazu – pair-rule gene Segments and Parasegments Segments and parasegments organized from A/P compartments out of phase Cells of adjacent compartments do not mix Expression patterns in early embryos are not delineated by segmental boundaries, but by - parasegments:fundamental units of embryonic gene expression Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  49. 49. HOMEOTIC SELECTOR GENES Homeotic genes specify - head segments - labial palps - antennae - thoracic segments - wings - halteres - legs - abdominal segments Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  50. 50. Eight Genes Regulate the Identity of Within the Adult and Embryo labial (lab) proboscipedia (pb) Deformed (Dfd) Sex combs reduced (Scr) Antennapedia (Antp) Ultrabithorax (Ubx) abdominal A (abd-A) Abdominal B (Abd-B) Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  51. 51.  Homeotic genes encode nuclear proteins containing a DNA- binding motif called a homeodomain.  The products are transcription factors that specify segment identity by activating multiple gene expression events.  The genes are initially activated imprecisely by the concentration gradients of gap gene products. e.g. Ubx is switched on between certain concentrations of hunchback to give a broad band of expression near the middle of the embryo. Later, fushi tarazu and even skipped sharpen the limits of Ubx expression which comes into register with the anterior boundaries of specific parasegments.  The BX-C and ANT-C genes have extensive non-coding sequences (introns) that are critical in regulating their individual expression. Homeotic-Selector Genes Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  52. 52. Homeotic Gene Expression distal-less – jaws, limbs Antennapeida – thoracic Ultrabithorax – abdomen Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  53. 53. Figure . The patterns of expression compared to the chromosomal locations of the genes of the HOM complex. The sequence of genes in each of the two subdivisions of the chromosomal complex corresponds to the spatial sequence in which the genes are expressed. Note that most of the genes are expressed at a high level throughout one parasegment (dark color) and at a lower level in some adjacent parasegments (medium color) where the presence of the transcripts is necessary for a normal phenotype, light color where it is not). In regions where the expression domains overlap, it is usually the most "posterior" of the locally active genes that determines the local phenotype. The drawings in the lower part of the figure represent the gene expression patterns in embryos at the extended germ band stage, about 5 hours after fertilization. Patterns of Expression Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  54. 54. •The direction of homeotic transformations depends on whether the mutation causes loss of homeotic gene function where the gene normally acts or gain of function where the gene normally does not act. •Ultrabithorax (Ubx) acts in the haltere to promote haltere development and repress wing development. Loss of function mutations in Ubx transform the haltere into a wing. •Dominant mutations that cause Ubx to gain function in the wing transform that structure into a haltere. •In antenna-to-leg transformations of Antennapedia the mutants reflect a dominant gain of Antennapedia gene function in the antennae. Homeotic Mutations Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  55. 55. Figure :. Contribution of BX-C genes — Ubx, abdA, and AbdB—to determination of parasegment identity. The numbers above each larva indicate the parasegments; those below, the corresponding segments. The cuticular pattern of larvae is used to assign an identity to each parasegment (PS), which is indicated by color, as depicted in the wild type at the top. Red PS and segment labels indicate abnormal patterns that do not correspond exactly to any found in wild-type larvae. [Adapted from P. A. Lawrence, 1992, The Making of a Fly: Genetics of Animal Design, Blackwell Scientific Publications.] Effects of Mutations in Bithorax Complex Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  56. 56. Expression and lethal embryonic phenotypes of homeotic genes. (Adapted from Akam, 1987.) This phenotypic analysis has been confirmed by the expression patterns of the various genes in various mutant backgrounds. The anterior limits of the domain of expression of a particular homeotic gene are presumably set by a collaboration between gap genes and pair-rule genes (see above). Removal of an anteriorly-acting homeotic has no effect on expression or phenotype in the domain of a more posteriorly acting homeotic gene. •Antp expands posteriorly from PS4 to PS6 in a Ubx mutant •Antp expands posteriorly from PS4 to PS12 in a Ubx AbdA mutant •Antp expands posteriorly from PS4 to PS14 in a Ubx AbdA AbdB mutant •Ubx expands posteriorly from PS6 to PS12 in a AbdA mutant •Ubx expands posteriorly from PS6 to PS14 in a AbdA AbdB mutant etc. Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  57. 57. Homeotic Gene Expression in Mutant Embryos Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  58. 58. Maintaining Hox Gene Expression  The transcription-control regions of some Hox genes contain binding sites for their encoded proteins – autoregulatory loop (e.g. lab and Dfd).  A second mechanism requires proteins that modulate chromatin structure. There are two classes – the trithorax group and the polycomb group.  Early patterning requires repression as well as activation of gene expression. Polycomb proteins have a repressive effect on the expression of Hox genes.  Polycomb proteins bind multiple chromosomal locations to form large macromolecular complexes and this becomes “locked in”.  Trithorax proteins maintain the expression of many Hox genes. These also form large multiprotein complexes at multiple chromosomal sites, but mainain an open chromatin structure and stimulate gene expression. Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  59. 59. Figure 21-52. Fate map of a Drosophila embryo at the cellular blastoderm stage. The embryo is shown in side view and in cross-section, displaying the relationship between the dorsoventral subdivision into future major tissue types and the anteroposterior pattern of future segments. A heavy line encloses the region that will form segmental structures. During gastrulation the cells along the ventral midline invaginate to form mesoderm, while the cells fated to form the gut invaginate near each end of the embryo. Thus, with respect to their role in gut formation, the opposite ends of the embryo, although far apart in space, are close in function and in final fate. (After V. Hartenstein, G.M. Technau, and J.A. Campos-Ortega, Wilhelm Roux' Arch. Dev. Biol.194:213-216, 1985.) Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  60. 60. Terminal Specification - 1 Torso – transmembrane RTK Torso uniformly distributed Torso activated by Torso-like protein - located only at ends of egg Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  61. 61. Terminal Specification - 2 Distinction between anterior and posterior = Bicoid Bicoid = acron formation Torso kinases inactivate an inhibitor of tailless and huckebein Tailless and Huckebein specify termini Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15
  62. 62. Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15 Thank You Discussion Time

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