Drosophila

Drosophila melanogaster (fruit fly)

History:

Thomas Hunt Morgan began using fruit flies in genetic studies at Columbia University in 1910. His laboratory was located on the top floor of Schermerhorn Hall, which became known as “The Fly Room.” They started off experiments using milk bottles to rear the fruit flies and handheld lenses for observing their traits. The Fly Room was the source of some of the most important research in the history of biology. Morgan and his students eventually defined many basic principles of heredity, such as sex-linked inheritance, epistasis, multiple alleles, and gene mapping.

A Model Organism:

The fruit fly is considered an ideal model organism because it is complex enough in that the embryo is similar to some degree to higher eukaryotes, including humans and yet, Drosophila is also easy to study in the laboratory. However, this organism is of particular interest because researchers can simply control gene expression in the embryo by essentially creating gain or loss-of-function conditions. Thus, the fruit fly becomes a living test tube where researchers are able to evaluate the function of genes in a living organism. A number of molecular techniques are also available to help us develop models of the biological function, which are then re-tested in the embryo. In addition, further studies have recently been pioneered using embryonic live imaging to track the various developmental processes that ultimately make Drosophila a unique model organism.

Drosophila melanogaster is one of the most studied organisms in biological research, particularly in genetics and developmental biology. The reasons include:

  • Care and culture require little equipment and occupy little space
  • Low overall cost
  • Small and easy to grow in the laboratory
  • Morphology is easy to identify
  • Short generation time (about 10 days at room temperature)
  • High fecundity
  • Males and females are readily distinguished
  • Virgin females are easily isolated, facilitating genetic crossing
  • The mature larvae show giant chromosomes in the salivary glands called polytene chromosomes-“puffs” indicate regions of transcription and hence gene activity.
  • It has only four pairs of chromosomes: three autosomes, and one sex chromosome.
  • Males do not show meiotic recombination, facilitating genetic studies.
  • Recessive lethal “balancer chromosomes” carrying visible genetic markers can be used to keep stocks of lethal alleles in a heterozygous state without recombination due to multiple inversions in the balancer.
  • Genetic transformation techniques have been available since 1987.
  • Its complete genome was sequences  and first published in 2000.
  • About 75% of known human disease genes have a recognizable match in the genome of fruit flies, and 50% of fly protein sequences have mammalian homologs.

Genome: 1.5 X 108 bp per haploid genome (30X E. coli; 2X C. elegans; .04X human)
4 autosomes and X or Y chromosome.  12,000 genes

Figure: Drosophila melanogaster chromosomes (wikipedia)

Embryology:

Egg is elongate, 0.4 mm long, moderately yolky.
Cytoplasmic at periphery & around pronucleus.
Chorion forms tough protective coating arond egg.
Has small opening, called micropyle, for sperm entry.
Micropyle end forms anterior ventral structures in larva – egg is already asymmetric.

After fertilization, nuclei divide every 8-10 min., without cell division.
9 nuclear divisions create 512 nuclei, which then migrate to periphery to form “syncytial blastoderm.”
Some nuclei move early to posterior, become pole cells and establish germline.

4 more rapid, synchronous nuclear divisions.

Plasma membrane then invaginates to enclose nuclei and form cellular blastoderm.

Morphogenesis begins with gastrulation – furrowing and invagination.

Imaging the Entire Embryonic Development of a Fruit Fly

End result is 1st instar larva with head, 3 thoraxic segments, 8 abdominal segments.

During embryogenesis, PIWI proteins serve a necessary function during mitosis. These proteins are responsible for chromatin structure and spindle formation, and function through an epigenetic pathway. To learn more, click here.

Post-Embryonic Development:

After hatching, larva undergoes 3 molts:  2nd instar, 3rd instar, pupa
During pupation, larval tissues resorbed, imaginal discs expand.
Adult emerges, with external organs (eyes, antennae, legs, wings, genitalia, etc.) formed from imaginal discs.

To learn more about the xenobitic response regulation of metamorphosis in Drosophila, click here.

Drosophila imaginal discs http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Student/fly_webo4/web%20presentation/fly_diagram.jpg

19 imaginal discs (9 pairs plus 1 fused pair)
Each disc predetermined as to developmental fate.
Contains hundreds to thousands of cells.
Distinguishable by location, size and shape.
First seen as thickening of epidermis that later invaginates.
Single-layered epithelium, partly folded, attached by stalks to larval epidermis.
Can be dissected & transplanted to abdomen of adult flies, where they remain in larval state, slowly grow.
Can be serially transferred every 2 weeks from adult to adult.
Once put back into larva, and larva induced to pupate, transplanted disc will evert and form original predetermined adult structure.
Isolated discs will also evert & form correct adult structures in Petri dishes upon exposure to ecdysone, a steroid hormone that regulates molting.

Maternal Effect Genes and Establishment of the Body Plan:

4 sets of maternal effect genes establish the embryonic axes.

anterior system:  Exuperantia, Swallow, Staufen -> Bicoid

maternal Bicoid mRNA is localized to anterior end of oocyte by other maternal germline genes – Exuperantia, Swallow and Staufen.  Fertilization causes translation of Bicoid mRNA to create a concentration gradient of Bicoid protein by diffusion.  Concentration gradient of Bicoid protein regulates expression of zygotic gap genes such as Giant and Hunchback, and Bicoid and gap gene proteins regulate pair-rule gene expression and expression of homeotic genes (Orthodenticle, Empty spiracles and Buttonhead) that define head and anterior segments.

posterior system:  various -> Nanos, Pumilio -> maternal Hunchback, Caudal

majority of posterior system genes required for localization of Nanos and Pumilio to posterior pole.  Nanos is the posterior determinant, but is not a transcription factor.  Instead, Nanos represses translation of maternal mRNAs for Bicoid and Hunchback.  Pumilio appears to regulate the diffusion or activity of the Nanos protein.  Oskar is another posterior system gene; posterior localization of maternal Oskar mRNA specifies pole cells.  Caudal gene expression is zygotic, and encodes a homeodomain protein, with vertebrate homologues (Cdx).  Caudal is thought to be the posterior transcription factor.  Mutations in mouse Cdx-2 cause death early in development 3-5 days p.c. for homozygous mutants, or varying degrees of homeotic transformation of vertebrae and skeletal structures for heterozygotes, with high incidence of multiple intestinal adenomatous polyps (Chawengsaksophak et al., 1997).

terminal system: Torsolike; Trunk, etc. ->  Torso, L(1)pole hole ->  gene Y?

Torso encodes a transmembrane receptor tyrosine kinase.  Maternal Torso mRNA is not translated until after fertilization.  Neither Torso mRNA nor protein show terminal localization; they are distributed uniformly throughout the egg and embryo.  Trunk, Fs(1)Nasrat and Fs(1)pole hole presumably generate a localized signal that activates Torso at the termini.  L(1)pole hole (aka D- raf) is a Drosophila homolog of mammalian c-Raf, and transduces the Torso signal.  One final gene in this pathway, Torso-like, acts in the maternal somatic follicle cells, near the posterior pole of the oocyte.

dorso-ventral system:  Nudel, Pipe, Windbeutel -> Snake, Easter, Spatzle etc. -> Toll, Tube, Pelle, Cactus , Dorsal

Toll encodes a transmembrane receptor with an extracellular domain similar to human thrombin receptor and intracellular domain similar to the mammalian IL-1 receptor.  Like Torso, Toll is expressed uniformly throughout the embryo at the syncytial blastoderm stage.  Upstream genes, including Snake and Easter (both encode serine proteases) and Spatzle, generate the ventral signal or ligand in the perivitelline fluid for Toll.  Downstream genes encode a signal transduction pathway for Toll; Dorsal is homologous to mammalian transcription factor NFkB, Cactus is homologous to IkB, and Pelle encodes a serine/threonine protein kinase.  Cactus and Dorsal form an inactive complex localized in the cytoplasm.  Activation of Toll results in phosphorylation of Cactus by Pelle, and release of Dorsal which transits to the nucleus and acts as a transcription factor.  The function of Tube is still unknown.  Dorsal protein in ventral nuclei represses expression of Zerknullt (Zen) and Decapentaplegic (Dpp) and induces expression of Twist and Snail.  Finally, at least 3 genes are required in the maternal somatic follicle cells.  Pipe, Nudel and Windbeutel are expressed in ventral follicle cells to generate a ventral signal in the eggshell or the perivitelline fluid to activate proteases encoded by Snake and Easter.

How is the initial polarity of the egg determined by interactions with the follicle cells?

Torpedo (aka DER) is an epidermal growth factor (EGF) receptor homolog whose expression in dorsal follicle cells is required for dorso-ventral patterning of the embryo.  Torpedo mutations cause ventralized embryos.  Gurken is a transforming growth factor (TGF) alpha homolog with an EGF repeat, whose expression in the germline (oocyte or nurse cells) is also required for follicle cells to adopt a dorsal identity.  Thus Gurken may act as a signal from the oocyte nucleus to dorsalize adjacent follicle cells.

A-P patterning, or localization of Bicoid mRNA at the anterior pole and Oskar mRNA at the posterior pole, depends on microtubules.  Initially, the location of the oocyte at one end of the nurse cells signals the adjacent follicle cells to adopt a posterior fate.  Microtubules are directed with their minus ends toward the posterior pole, directing the flow of nutrients from the nurse cells towards the posterior.  Then a signal from the follicle cells redirects the microtubules in an opposite direction, and the oocyte nucleus moves anteriorly and to one corner.  Mutations affecting Notch and Delta in the follicle cells all prevent this reversal of microtubules, and cause Oskar mRNA to localize to the middle and Bicoid mRNA to localize at both ends (A-P duplication).

Mutants in Gurken, Torpedo and Cornichon show anterior follicle cell types at both poles and A-P duplication.  Mutants also fail to reverse microtubules, and the nucleus remains centrally localized.  Gurken mRNA localizes at posterior pole in wild type oocytes, and later at anterior dorsal margin after microtubule repolarization and movement of the oocyte nucleus.

Zygotic Genes and Establishment of the Antero-Posterior Segmentation Pattern:

Gap genes:

  • Huckebein (anterior)
  • Tailless
  • Giant
  • Hunchback (zygotic expression)
  • Kruppel
  • Knirps
  • Giant
  • Tailless
  • Huckebein (posterior)

All gap genes encode transcription factors.

Pair-rule genes:

  • Fushi tarazu (ftz)
  • Hairy
  • Even-skipped

Segment polarity genes:

  • Wingless -> Frizzled (Bhanot et al., 1996) -> Zw3:Armadillo
  • Engrailed

Homeotic selector genes:

  • Antennapedia complex (ANT-C)
  • Bithorax complex (BX-C)
  • head genes:  Orthodenticles (Otd), Empty spiracles (Ems), Buttonhead (Bhd)

Induction of Mesoderm and Mesodermal Structures

Twist is a basic helix-loop-helix (bHLH) transcription factor required for early mesoderm, initially expressed in entire mesoderm, but later restricted to somatic mesoderm and reduced in visceral mesoderm (Michelson, 1996).
Twist appears to induce skeletal muscle; ectopic expression in ectodermal cells suppresses epidermal and nerve differentiation and activates myogenesis (Baylies and Bate, 1996).
However, in mouse, Mtwist may repress myogenesis (Spicer et al., 1996).

Induction of Neural Structures and Tissues Along Dorso-Ventral Axis:

Decapentaplegic (Dpp), a TGF  homologue, is a dorsalizing morphogen.
Screw is another TGF  homologue responsible for dorsalmost structures.
Tolloid encodes a metalloprotease that activates Dpp protein.
Thickveins and Saxophone encode type I receptors for Dpp.
Punt encodes a type II receptor, a serine/threonine receptor kinase, for Dpp.

Imaginal disc identity

Imaginal disc identities are specified by the homeotic selector genes as well as the dorso-ventral patterning genes. Wing discs express the Wingless gene; loss-of-function mutants lack wings. Leg discs express the Distal-less gene; loss-of-function mutants lack legs. Eye discs express the Eyeless gene; ectopic expression of Eyeless can induce formation of eyes on antennae, legs, and even wings!

Limb Patterning:

Segment-polarity genes function in larvae to determine pattern in imaginal discs.
Hedgehog (Hh) determines the posterior compartments of wings and legs.
Hedgehog induces expression of Dpp at A/P boundary.
Wingless (Wg) is required for formation of  polar coordinate system.

Posterior cells express and require Engrailed (En) and secrete Hedgehog (Hh) protein.
Anterior cells express Cubitus interruptus (Ci), a zinc-finger protein (Gli is human homolog) (Pennisi, 1996).
Ci required in anterior cells to suppress Hh; anterior cells without Ci express Hh and adopt posterior properties without expressing En (Dominguez et al., 1996).
Ci also transduces Hh signal in anterior cells; cells with increased levels of Ci induce expression of Dpp in a Hh-independent manner.
Patched (Ptc), a transmembrane protein, induced by Hh; it represses expression of other genes (TGF beta and Wnt genes) induced by Hh.  The human Patched homolog is a tumor suppressor; mutations in this gene are linked to basal cell nevus syndrome (Johnson et al., 1996).
Smoothened (Smo), a seven-transmembrane domain protein related to Frizzled, may be receptor for Hedgehog (Perrimon, 1996).

Armadillo involved in Wg signal transduction; Wg signal causes rise in intracellular level of free Armadillo.  Armadillo a component of cell-cell adhesive junctions.
Pangolin, or dTcf, loss of function mutants disrupt A-P patterning, block expression of Wg-induced genes (Brunner et al., 1997).  Armadillo:dTcf heterodimer binds to promoters of Wg-responsive genes; binding required for transcriptional activation.  dTcf has DNA-binding activity, Armadillo may interact with basal transcription machinery.

Videos:

Drosophila Embryo Development

Drosophila Gastrulation

WWW Resources:

FlyBase

Biology Times: articles on HOX genes

www.wikipedia.org/wiki/Drosophila

References:

Baylies, M.K. and M. Bate, 1996.  twist:  A myogenic switch in Drosophila, Science 272:1481- 1484.

Bhanot, P., M. Brink, C.H. Samos, J.-C. Hsieh, Y. Wang,  J.P., Macke, D. Andrew, J. Nathans and R. Nusse, 1996.  A new member of the frizzled family from Drosophila functions as a Wingless receptor, Nature 382:225-230.

Brunner, E., O. Peter, L. Schweizer and K. Basler, 1997.  Pangolin encodes a Lef-1 homologue that acts downstream of Armadillo to transduce the Wingless signal in Drosophila, Nature 385:829-833.

Chawengsaksophak, K., R. James, V.E. Hammond, F. Kontgen and F. Beck, 1997.  Homeosis and intestinal tumours in Cdx2 mutant mice, Nature 386:84-87.

Dominguez, M., M. Brunner, E. Hafen and K. Basler, 1996.  Sending and receiving the hedgehog signal:  control by the Drosophila Gli protein Cubitus interruptus, Science 272:1621- 1625.

Gonzalez-Reyes, A., H. Elliott and D. St. Johnston, 1995.  Polarization of both major body axes in Drosophila by gurken-torpedo signalling, Nature 375:654-658.

Johnson, R.L., A.L. Rothman, J. Xie, L.V. Goodrich, J.W. Bare, J.M. Bonifas, A.G. Quinn, R.M. Myers, D.R. Cox, E.H. Epstein Jr. and M.P. Scott, 1996.  Human homolog of patched, a candidate gene for the basal cell nevus syndrome, Science 272:1668-1671.

Lecuit, T., W.J. Brook, M. Ng, M. Calleja, H. Sun and S.M. Cohen, 1996.  Two distinct mechanisms for long-range patterning by Decapentaplegic in the Drosophila wing, Nature 381:387-393.

Lemons D, McGinnis W (2006) Genomic evolution of Hox gene clusters. Science 313:1918-1922.

Marques G., M. Musacchio, M.J. Shimell, K. Wunnenberg-Stapleton, K.W.Y. Cho and M.B. O’Connor, 1997. Production of a DPP Activity Gradient in the Early Drosophila Embryo through the Opposing Actions of the SOG and TLD Proteins  Cell 91:417-426.

Michelson, 1996.  A new turn (or two) for twist, Science 272:1448-1450.

Ng, M., F.J. Diaz-Benjumea, J.-P. Vincent, J. Wu and S.M. Cohen, 1996.  Specification of the wing by localized expression of wingless protein, Nature 381:316-318.

Pennisi, E., 1996.  Gene linked to commonest cancer, Science 272:1583-1584.

Perrimon, N., 1996.  Serpentine proteins slither into the wingless and hedgehog fields, Cell 86:513-516.

Ruiz i Altaba, 1997.  Catching a gli-mpse of hedgehog (minireview), Cell 90:193-196.

Smith, J., 1996.  How to tell a cell where it is (News & Views), Nature 381:367-368.

Spicer, D.B., J. Rhee, W.L. Cheung and A.B. Lassar, 1996.  Inhibition of myogenic bHLH and MEF2 transcription factors by the bHLH protein twist, Science 272:1476-1480.

St. Johnston and Nusslein-Volhard, 1992.  The origin of pattern and polarity in the Drosophila embryo, Cell 68:201-219.

One Response to Drosophila

  1. Connie Williams says:

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