A quick and simple introduction to Drosophila melanogaster

What is it and why bother about it?

Drosophila melanogaster is a fruit fly, a little insect about 3mm long, of the kind that accumulates around spoiled fruit. It is also one of the most valuable of organisms in biological research, particularly in genetics and developmental biology. Drosophila has been used as a model organism for research for almost a century, and today, several thousand scientists are working on many different aspects of the fruit fly. Its importance for human health was recognised by the award of the Nobel prize in medicine/physiology to Ed Lewis, Christiane Nusslein-Volhard and Eric Wieschaus in 1995.

Why work with Drosophila?

Part of the reason people work on it is historical - so much is already known about it that it is easy to handle and well-understood - and part of it is practical: it's a small animal, with a short life cycle of just two weeks, and is cheap and easy to keep large numbers. Mutant flies, with defects in any of several thousand genes are available, and the entire genome has recently been sequenced.

Life cycle of Drosophila

The drosophila egg is about half a millimeter long. It takes about one day after fertilisation for the embryo to develop and hatch into a worm-like larva. The larva eats and grows continuously, moulting one day, two days, and four days after hatching (first, second and third instars). After two days as a third instar larva, it moults one more time to form an immobile pupa. Over the next four days, the body is completely remodelled to give the adult winged form, which then hatches from the pupal case and is fertile within about 12 hours. (timing is for 25°C; at 18°, development takes twice as long.)

Research on Drosophila

Drosophila is so popular, it would be almost impossible to list the number of things that are being done with it. Originally, it was mostly used in genetics, for instance to discover that genes were related to proteins and to study the rules of genetic inheritance. More recently, it is used mostly in developmental biology, looking to see how a complex organism arises from a relatively simple fertilised egg. Embryonic development is where most of the attention is concentrated, but there is also a great deal of interest in how various adult structures develop in the pupa, mostly focused on the development of the compound eye, but also on the wings, legs and other organs.

The Drosophila genome

Drosophila has four pairs of chromosomes: the X/Y sex chromosomes and the autosomes 2,3, and 4. The fourth chromosome is quite tiny and rarely heard from. The size of the genome is about 165 million bases and contains and estimated 14,000 genes (by comparison, the human genome has 3,400 million bases and may have about 22,500 genes; yeast has about 5800 genes in 13.5 million base bases). The genome was (almost) completely sequenced in 2000, and analysis of the data is now mostly complete. Several other insect genomes have now been sequenced, including many Drosophila species, and the genomes of mosquito and honey bee, and these are starting to show what is common among all insects, and what distinbuishes them from each other.

Polytene Chromosomes

These are the magic markers that first put Drosophila in the spotlight. As the fly larva grows, it keeps the same number of cells, but needs to make much more gene product. The result is that the cells get much bigger and each chromosome divides hundreds of times, but all the strands stay attached to each other. The result is a massively thick polytene chromosome, which can easily be seen under the microscope.

Even better, these chromosomes have a pattern of dark and light bands, like a bar code, which is unique for each section of the chromosome. As a result, by reading the polytene bands, you can see what part of the chromosome you are looking at. Any large deletions, or other rearrangements of part of a chromosome can be identified, and using modern nucleic acid probes, individual cloned genes can be placed on the polytene map. The standard map of the polytene chromosome divides the genome into 102 numbered bands (1-20 is the X, 21-60 is the second, 61-100 the third and 101-102 the fourth); each of those is divided into six letter bands (A-F) and those are subdivided into up to 13 numbered divisions (the picture above shows band 57). The location of many genes is known to the resolution of a letter band, usually with a guess to the number location (e.g. 42C7-9, 60A1-2). The polytene divisions don't have exactly the same length of sequence in them, but on average, a letter band contains about 300kb of DNA and 15-25 genes.

Drosophila melanogasterWithin a few years of the rediscovery of Mendel's rules in 1900, Drosophila melanogaster (the so-called fruit fly) became a favorite "model" organism for genetics research.

Some of the reasons for its popularity:

The flies are small and easily reared in the laboratory.

They have a short life cycle The figure shows the various stages of the life cycle (not all drawn to the same scale). A new generation of adult flies can be produced every two weeks.

They are fecund; a female may lay hundreds of fertilized eggs during her brief life span. The resulting large populations make statistical analysis easy and reliable.

The giant ("polytene") chromosomes in the salivary (and other) glands of the mature larvae.

o These chromosomes show far more structural detail than do normal chromosomes, and

o they are present during interphase when chromosomes are normally invisible.

More recently, Drosophila has proven in other ways to have been a happy choice. Its embryo grows outside the body and can easily be studied

at every stage of development. The blastoderm stage of the embryo is a syncytium

(thousands of nuclei unconfined by cells) so that, for example, macromolecules like DNA injected into the embryo have easy access to all the nuclei.

The genome is relatively small for an animal (less than a tenth that of humans and mice). [View]

Mutations can targeted to specific genes. [Link to discussion of transposons.]

Second Example: Drosophila

In Drosophila and other insects, cleavage involves repeated mitosis but without cytokinesis. So the daughter nuclei remain suspended within the single egg compartment. After several thousand nuclei have been formed, they migrate to the margins of the egg. Only then

do plasma membranes form around each nucleus forming true cells. But, as for Xenopus, the genes that will be expressed by those cells are regulated by the cytoplasmic constituents they found themselves surrounded by. And that, again like Xenopus, is determined by where those molecules end up in the egg.

Bicoid

For example, Drosophila eggs have a gradient of mRNA transcribed from a gene designated bicoid (bcd). The transcripts are deposited in the egg by "nurse" cells surrounding it. Once within the egg, they are transported (along microtubules) toward the anterior. The result is a concentration gradient of bicoid mRNA extending from a high level at the anterior of the egg to a low level at the posterior.

After fertilization, the mRNAs are translated into bicoid protein. High levels of the protein lead to the formation of the head of the larva.

Nanos

Conversely, the posterior of the egg has a high concentration of mRNA encoding the nanos protein, which is needed to form the structures of the tail of the larva.

Three demonstrations:

1.

Remove some of the bicoid-rich cytoplasm from the anterior of the fertilized egg and replace it with nanos-rich cytoplasm from another egg. The result: a larva (nonviable) with a tail at each end.

2.

Inject the anterior of the fertilized egg with nanos mRNA. The result: another double-posterior larva.

3.

Make female fruit flies that are transgenic for a recombinant gene containing:

the structural gene for nanos coupled to the 3´ anterior-directing signal of the bicoid gene.

This causes nanos mRNA, instead of bicoid mRNA, to be deposited in the anterior of her eggs. The result: more double-posterior larvae (on the left).

A normal larva is shown on the right. The bright object at the right end of the normal

larva and at both ends of the double posterior larva is the tip of the tail. These micrographs are courtesy of Elizabeth Gavis and Ruth Lehmann, in whose lab the third demonstration was performed.

Third Example: The Mud Snail

The mud snail, Ilyanassa obsoleta, is a small gastropod that lives in mud flats along the Atlantic coast.

Like other protostomes, cleavage of the zygote produces daughter cells that are already committed to their fate. In other words, even as early as the two-cell stage, the cells are no longer totipotent. Unlike humans and other deuterostomes, then, identical twins cannot form.

In the 12 December 2002 issue of Nature, J. David Lambert and Lisa Nagy reported another mechanism by which two daughter cells become committed to different fates even though they have inherited the same genome.

They traced the distribution in the cells of early embryos of the messenger RNAs (mRNAs) encoding 3 proteins that are known to be important in the development of other animals such as Xenopus and Drosophila.

IoEve, which is Ilyanassa obsoleta's version of even-skipped (eve) in Drosophila; IoDpp, which is the snail's version of

o decapentaplegic (dpp) in Drosophila and the genes encoding

o b one m orphogenic p roteins (BMP2 and BMP4) in vertebrates

IoTld, which encodes the snail's version of a protein called tolloid in Drosophila.

One of the first events when an animal cell prepares to divide by mitosis is the duplication of its centrosome and separation of the duplicates. [Link to discussion.]

Lambert and Nagy found that

in

interphase the messenger RNAs were distributed diffusely throughout the cytosol, but as the cell got ready for cleavage, the mRNAs collected at only one of the now pair of

centrosomes. They were collected there by traveling along the microtubules that radiate out from the centrosome.

As cleavage continued, the mRNAs moved from the centrosome to a spot on the inner surface of the plasma membrane. They got there by traveling along actin filaments.

At cytokinesis, this patch of accumulated mRNAs was incorporated exclusively into the smaller daughter cell.

Centrosome sorting (of proteins in this case) also plays a role in determining whether embryonic cells of Caenorhabditis elegans remain in the germline or become the somatic cells of the worm. [Link to discussion.]

What comes next?

Development in Xenopus and Drosophila (and probably that of animals in general) passes through three rather different (although often overlapping) phases:

establishing the main axes (dorsal-ventral; anterior-posterior; left-right). This is done by gradients of mRNAs and proteins encoded by the mother's genes and placed in the egg by her. It has been discussed here.

establishing the main body parts such as the notochord and central nervous system in vertebrates

see Organizing the Embryo: The Central Nervous System

and the segments in Drosophila

see Organizing the Embryo: Segmentation

These are run by genes of the zygote itself.

filling in the details; that is, building the various organs of the animal. (Our example will include the wings, legs, and eyes of Drosophila.)

see Embryonic Development: Putting on the finishing touches

(http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/D/Drosophila.html)

The fruit flies in this exhibit show just a few of the mutations that occur in natural fruit fly populations.

The genetic instructions to build a fruit fly-or any other organism-are imprinted in its DNA, a long, threadlike molecule packaged in bundles called chromosomes. Like a phone book made up of different names and addresses, each chromosome consists of many individual sections called genes. Each gene carries some of the instructions for building one particular characteristic of an organism.

To build a complete organism, many genes must work precisely together. A defect in a gene can cause a change in the building plan for one particular body part-or for the entire organism.

Mutations are neither good nor bad: some may be beneficial for an organism; others may be lethal. By creating new gene versions, mutations are a driving force for changes in evolution, sometimes leading to new species.

Biologists learn about the proper function of any gene by studying mutations. If a defective gene causes short wings, for instance, scientists know that the healthy version of the gene is responsible for correct wing formation.

 

Normal Fruit Flies

 

These are normal fruit flies, or "wildtypes." Notice the shape and length of their wings. Now compare them with the other fruit flies here.

Short-Winged Flies

 

Notice the shortened wings of these flies. Flies with vestigial wings cannot fly: they have a defect in their "vestigial gene," on the second chromosome. These flies have a recessive mutation. Of the pair of vestigial genes carried by each fly (one from each parent), both have to be altered to produce the abnormal wing shape. If only one is mutated, the healthy version can override the defect.

Curly-Winged Flies

 

Notice the curled wings of these flies. They have a defect in their "curly gene," which is on the second chromosome. Having curled wings is a dominant mutation, which means that only one copy of the gene has to be altered to produce the defect. In fact, if both copies are mutated, the flies do not survive.

Normal Fruit Flies

 

These normal fruit flies, or "wildtypes," have black-and-tan striped bodies. Compare them with the other fruit flies here.

Yellow Flies

Notice that these flies are yellower than normal flies. They have a defect in their "yellow gene," which is on the X chromosome. Since the yellow gene is needed for producing a fly's normal black pigment, yellow mutant flies cannot produce this pigment.

Ebony Flies

Notice that these flies have a dark, almost black, body. They carry a defect in their "ebony gene," on the third chromosome. Normally, the ebony gene is responsible for building up the tan-colored pigments in the normal fruit fly. If the ebony gene is defective, the black pigments accumulate all over the body.

Normal

These are normal fruit flies, or "wildtypes." Notice that their eye color is bright red. Compare them with the other fruit flies here.

Fruit Flies

Orange-Eyed Flies

Notice that these flies have orange eyes. They have a defect in their "white" gene, which normally produces the red pigments in the eye. In these flies, the white gene only works partially, producing fewer red pigments than it should.

White-Eyed Flies

 

These flies have white eyes. Like the orange-eyed flies, they also have a defect in their "white" gene. But in these flies, the white gene is totally defective: it produces no red pigment at all.

Normal Fruit Flies

These are normal fruit flies, or "wildtypes." Notice the antennas sticking out in front of their red eyes. Compare these flies to the other fruit flies here.

Eyeless Flies

Notice that these flies have no eyes. They have a defect in their "eyes absent" gene, which normally instructs cells in the larvae to form an eye.

Leg-Headed Flies

Notice that these flies have abnormal, leg-like antennas on their foreheads. They have a defect in their "antennapedia" gene (Latin for "antenna-leg"), which normally instructs some body cells to become legs. In these flies, the antennapedia gene falsely instructs cells that would normally form antenna to become legs instead.

Mutant Fruit Flies (and other aspects of "Diving Into the Gene Pool") were made possible through support by the United States Department

of Energy, Office of Energy Research.

(http://www.exploratorium.edu/exhibits/mutant_flies/mutant_flies.html)

Drosophila melanogaster is a small, common fly found near unripe and rotted fruit. It has been in use for over a century to study genetics and lends itself well to behavioral studies. Thomas Hunt Morgan was the preeminent biologist studying Drosophila early in the 1900's. Morgan was the first to discover sex-linkage and genetic recombination, which placed the small fly in the forefront of genetic research. Due to it's small size, ease of culture and short generation time, geneticists have been using Drosophila ever since. It is one of the few organisms whose entire genome is known and many genes have been identified.

Fruit flies are easily obtained from the wild and most biological science companies carry a variety of different mutations. In addition these companies sell any equipment needed to culture the flies. Costs are relatively low and most equipment can be used year after year. There are a variety of laboratory exercises one could purchase, although the necessity to do so is questionable.

Why use Drosophila?Teachers should use fruit flies for high school genetic studies for several reasons.

1. They are small and easily handled 2. You can anesthetize them easily and manipulated individuals with very unsophisticated equipment. 3. Drosophila are sexually dimorphic (males and females are different), making it is quite easy to differentiate the sexes.4. It is easy to obtain virgin males and females, as virgins are physically distinctive from mature adults. 5. Flies have a short generation time (10-12 days) and do well at room temperature. 6. The care and culture requires little equipment, is low in cost and uses little space even for large cultures.

By using Drosophila, students will:

1. Understand Mendelian genetics and inheritance of traits 2. Draw conclusions of heredity patterns from data obtained 3. Construct traps to catch wild populations of D. melanogaster 4. Gain an understanding of the life cycle of D. melanogaster, an insect which exhibits complete metamorphosis 5. Construct crosses of caught and known wild- type and mutated flies 6. Learn techniques to manipulate flies, sex them, and keep concise journal notes 7. Learn culturing techniques to keep the flies healthy 8. Realize many science experiments cannot be conducted and concluded within one or two lab session

National standards covered in these lessons:

Content: 1. Organisms require a set of instructions for specifying traits (heredity) 2. Hereditary information is located in genes. 3. Combinations of traits can describe the characteristics of an organism.

Inquiry: Students will 1. Identify questions and concepts that guide scientific investigations 2. Design and conduct scientific investigations 3. Formulate and revise scientific explanations and models using logic and evidence 4. Communicate and defend a scientific argument

The genetics of Drosophila are well known and several web sites feature the complete genome. In additions, many gene loci are known and these, too, are in the public- domain web sites. Therefore, those teachers or students wishing to see where their mutations occur have a ready reference available.

Since Drosophila has been so widely used in genetics, there are many different types of mutations available for purchase. In addition, the attentive student may find mutations within their own cultures since, due to a short generation time, mutations are relatively common compared to other animal species.

Classification:

Domain: Eukarya Kingdom: Animalia Phylum: Arthropoda Class: Insecta Order: Diptera Family: Drosophilidae

Genus: Drosophila ("dew lover") Species: melanogaster ("dark gut")

 

Life cycle of D. melanogaster

D. melanogaster exhibits complete metamorphism, meaning the life cycle includes an egg, larval (worm-like) form, pupa and finally emergence (eclosure) as a flying adult. This is the same as the well-known metamorphosis of butterflies and many other insects. The larval stage has three instars, or molts.

Life cycle by day 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 (pupal 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

·The time from egg to adult is temperature- dependent. The above cycle is for a temperature range of 21-23 degrees C. The higher the temperature, the faster the generation time, whereas a lower (to 18 degrees C) temperature causes a longer generation time. ·Females can lay up to 100 eggs/day.·Virgin females are able to lay eggs; however they will be sterile and few in number.

After the eggs hatch, small larvae should be visible in the growing medium. If your media is white, look for the black area at the head of the larvae. Some dried premixed media is blue to help identify larvae however this is not a necessity and with a little patience and practice, larvae are easily seen. In addition, as the larvae feed they disrupt the smooth surface of the media and so by looking only at the surface one can tell if larvae are present. However, it is always a good idea to double check using a stereomicroscope. After the third instar, larvae will begin to migrate up the culture vial in order to pupate.

The Drosophila life cycle consists of a number of stages: embryogenesis, three larval stages, a pupal stage, and (finally) the adult stage!

from the Carolina Drosophila manual

Embryogenesis in Drosophila Following fertilization, mitosis (nuclear division) begins. HOWEVER, cytokinesis (division of the cytoplasm) does not occur in the early Drosophila embryo, resulting in a multinucleate cell called a syncytium, or syncytial blastoderm. The common cytoplasm allows morphogen gradients to play a key role in pattern formation. At the tenth nuclear division, the nuclei migrate to the periphery of the embryo. At the thirteenth division, the 6000 or so nuclei are partitioned into separate cells. This stage is the cellular blastoderm. Although not yet evident, the major body axes and segment boundaries are determined.  Subsequent development results in an embryo with morphologically distinct segments.    

from LIFE: The Science of Biology, Purves et al, 1998

Genetic Analysis of Drosophila development Much of what we understand about Drosophila development is based on the isolation and characterization of developmental mutants by three scientists, Ed Lewis, Christiane Nusslein-Volhard, and Eric Wieschaus, who were awarded the Nobel prize for their work in 1995.  Lewis did pioneering research on late embryogenesis, while Nusslein-Volhard and Wieschaus concentrated their efforts on understanding early embryogenesis.

Nusslein-Volhard and Wieschaus set out to identify EVERY GENE required for early pattern formation in the Drosophila embryo. They looked for recessive embryonic lethal mutations, and classified them according to their phenotype before death. That is, they looked for and analyzed dead embryos. Images of some of the mutants they identified are shown below. Notice the differences in segmentation patterns between the wildtype, shown on the left, and the mutant embryos. An online historical essay about their work is here.    

images from the  October 30, 1980 issue of Nature

A cascade of gene activation sets up the Drosophila body plan

The maternal-effect genes, including bicoid and nanos, are required during oogenesis. The transcripts or protein products of these genes are found in the egg at fertilization, and form morphogen gradients. The maternal-effect genes encode transcription factors that regulate the expression of the gap genes. The gap genes roughly subdivide the embryo along the anterior/posterior axis. The gap genes encode transcription factors that regulate the expression of the pair-rule genes. The pair-rule genes divide the embryo into pairs of segments. The pair-rule genes encode transcription factors that regulate the expression of the segment polarity genes.  The segment polarity genes set

the anterior/posterior axis of each segment.  The gap genes, pair-rule genes, and segment polarity genes are together called the segmentation genes, because they are involved in segment patterning.

from LIFE: The Science of Biology, Purves et al, 1998

But how do these segments take of individual identities?

In normal flies, structures like legs, wings, and antennae develop on particular segments, and this process requires the action of homeotic genes. Enter Ed Lewis, who discovered homeotic mutants - mutant flies in which structures characteristic of one part of the embryo are found at some other location.  

Homeotic mutations, such  as Antennapedia, cause a misplacement of structures. These two scanning electron micrographs show fly heads. On the left is a wildtype fly. On the right is a fly with the dominant Antennapedia mutation - and legs where the

Photographs by  F. R. Turner,  Indiana University

from ZYGOTE

antennae should be! 

The homeotic genes encode transcription factors that control the expression of genes responsible for particular anatomical structures, such as wings, legs, and antennae. The homeotic genes include a 180 nucleotide sequence called the homeobox, which is translated into a 60 amino acid domain, called the homeodomain. The homeodomain is involved in DNA binding, as shown in the images below.    

from HOX Pro db

Homeobox-containing (or HOX) genes are found in many organisms, including worms, fish, frogs, birds, mammals, and plants. Interestingly, HOX genes are found in clusters, and the relative gene order within these clusters in conserved between organisms.  That is, the order of related HOX genes in Drosophila and in mice is the same! In addition, the order of HOX-genes on the chromosome is related to where they are expressed along the anterior/posterior axis.

(http://biology.kenyon.edu/courses/biol114/Chap13/Chapter_13A.html)

Drosophila melanogaster

Drosophila melanogaster, or fruit-fly is widely used in scientific and medical research. This 3mm-long insect usually accumulates around spoiled fruit. It has been used in genetics and developmental biology for almost a century, and today several thousand scientists are working on many different aspects of its biology.

The importance of Drosophila as an animal model was realised by Thomas Hunt Morgan, who was awarded the 1933 Nobel Prize for physiology or medicine after demonstating that genetic information is carried on chromosomes using drosophila. Since then this tiny insect, which breeds rapidly and is easily kept in a labaoratory, has performed a crucial role in genetics research.

Its importance for human health was recognised more recently by the award of the Nobel prize for medicine in 1995, for work on the genetic control of early embryonic development. Mutant flies with defects in any of several thousand genes are available, and the entire genome has recently been sequenced.

Drosophila helped in the development of drugs to combat pathogens responsible for a range of diseases from skin infections to pneumonia and meningitis. Recent research with fruitflies has focused on the pathology of Alzheimer’s disease, for although the flies have a very simple brain they have highly developed muscles and nerves.

(http://www.animalresearch.info/en/science/animalsused/drosophila)

 Drosophila melanogaster

© istockphoto/janeff