Principles of Neurobiological research

• Drosophila as a model genetic organism


  • Four chromosomes, 180Mb
  • ~13,600 genes
  • Easy to keep large numbers
  • Two nervous systems constructed, one in the larva and a second for the adult
  • Neurogenesis studied in the embryo, larva, adult
  • Synaptic plasticity studied in the larva
  • Behaviour and functional activity generally studied in the adult
• Drosophila life cycle


  1. ~10 days at 25°C

  2. ~14 days at 22°C

  3. ~21 days at 18°C

  4. 5-6 days to 3rd instar larva

  5. 4 days at 25 degrees -> adult
• Drosophila genes


  14,000 genes -> useful properties; they small so its easy to keep a large number of flies relatively easily
• Drosophila nervous systems


  • Have 2 nervous systems developed at different stages; embryo + larva stagte -> theyre not completely distinct but develop at different times during development
• Drosophila in research has been very successful & has attracted many nobel prizes in physiology and medicine
• Morgan (at Columbia) -> worked with them before we know DNA was inheritable molecule in cells -> he used the model to see how genes are inherited and how chromosomes are stored / hereditary
• 6 nobel prizes awarded across 10 scientists for their work / contribution to the scientific community using drosophila e.g. molecular mechanisms in circadian rhythm
• Drosophila as a model genetic organism

  • Four chromosomes, 180Mb
  • ~13,600 genes
  • Easy to keep large numbers
  • Two nervous systems constructed, one in the larva and a second for the adult
  • Neurogenesis studied in the embryo, larva, adult
  • Synaptic plasticity studied in the larva
  • Behaviour and functional activity generally studied in the adult
• Practical advantages of Drosophila as a model system


  • Short generation span (10-12 days)
  • Ability to generate lots of progeny
  • Very adaptable to food sources (easy to culture in the laboratory)
  • Adaptable to changes to temperature (for genetic manipulations and culturing)
  • Easy to store due to small size
  • Generate lots of progeny; single female -> 100 eggs in 4/5 days -> quick amplification
  • Easy to culture in lab; e.g. in fruit bowls, wines etc -> grow on simple yeast food source in lab (cheap)
  • Can adapt to many temperatures / areas -> very wide-spread -> can survive in all geographic locations across different species; don't need to be overly cautious when keeping them in lab
  • Easy to store; small size
  • Drosophila are easy to culture – store them in vials & let them grow in incubators where temperature can be altered for optimum growth at 25 degrees
• Drosophila life cycle


  1. ~10 days at 25°C

  2. ~14 days at 22°C

  3. ~21 days at 18°C

  4. 5-6 days to 3rd instar larva

  5. 4 days at 25 degrees -> adult
• Genetic advantages of Drosophila as a model system

  • Entire genome sequenced – one of the earliest sequenced genomes allows a full characterisation of the genetic elements that contribute to the biology of the model organism (development, behaviour, disease, aging) – making it a sufficient model for a wide range of studies
  • Rapid identification of genes/mutations - was possible using traditional methods before the genome was sequenced; tech now allows for quicker / easier gene coding
  • Functional genomic approaches can be applied in drosophila (mutagenesis, protein function, genetic interactions) - made easier to research
  • Large-scale annotation of the genomic elements (gene expression, gene function and genomic modifications during development) – lots of molecular information / interactions can be understood from drosophila
• Drosophila reduced complexity from a genetic & cellular point of view -> drosophila has around 13,000 genes & 100,000 neurons (more complicated than worms) -> this is much simpler than the human brain considering humans have around 86 billion neurons and roughly the same amount of non-neuronal cells
• Drosophila genes


  14,000 genes -> useful properties; they small so its easy to keep a large number of flies relatively easily
• Drosophila conserved neurobiology


  • Flies and mammals have similar complexity of neural cell types - more so than worms
  • Flies and mammals use the same neurotransmitters – similar use of neurotransmitters
  • Flies have similar electrophysiology to vertebrates, utilise similar Na+, K+ and Ca2+ channels
  • Flies utilise many of the same synaptic proteins – e.g. SNARE for neurotransmission vesicular docking
  • 75% of human disease genes have close homologs in Drosophila – although this is not a complete 100% overlap, there's a good chance if you see a gene in human causing disease there will likely be a drosophila homologue of it which can be studied e.g. ANK1 gene for hereditary spherocytosis; inherited blood disorder – problem with RBCs -> spherical shape RBCs instead
• Neuroanatomical tools used in drosophila

  • Specific brain structures, e.g. mushroom body (MB), or sets of neurons can be labelled using antibodies, 'enhancer traps', or binary expression systems, e.g. the GAL4/UAS system; can get high resolution images of networks in neuronal activity / population of neurons and their activity
  • Cell specific transactivator (GAL4) can drive expression of genes cloned downstream of a response element (UAS) – upstream activator system
  • Can be used to drive expression of markers (membrane labelled GFP), toxic proteins, activity modulators, other genes etc
• Drosophila nervous systems


  • Have 2 nervous systems developed at different stages; embryo + larva stagte -> theyre not completely distinct but develop at different times during development
• The GAL4-UAS binary expression system is not native to drosophila; was taken from yeast (bakers) system adapted and put into drosophila (also gets used in other systems)
• Drosophila in research has been very successful & has attracted many nobel prizes in physiology and medicine
• The GAL4-UAS binary expression system


  1. The GAL4 transactivator system is used to express a gene of interest, e.g. gene X, in a particular tissue

  2. Transgenic flies expressing the yeast transcription factor GAL4 expressed in cell or tissue specific pattern drive expression of genes downstream of a GAL4 binding site (UAS)

  3. When flies carrying UAS-geneX are mated to flies carrying a GAL4 line, then geneX will be expressed in the same place as GAL4

  4. Collections of GAL4 flies exist that drive expression in a wide variety of neuronal cells, e.g. eye, CNS, PNS etc

  5. GAL4 activity can be modulated by use of GAL80 repressor or use of a hormone inducible system
• Morgan's work with drosophila


  Worked with them before we know DNA was inheritable molecule in cells -> he used the model to see how genes are inherited and how chromosomes are stored / hereditary
• How the Gal4/UAS system works – to study gene expression

  1. You have 2 lines of drosophila – a driver line (Gal4) and a reporter line (UAS)

  2. Each driver line contains Gal4 protein and a tissue specific promotor gene which only allows gal4 to be produced in certain tissues – e.g. gut only or brain only etc depending on where you want it

  3. Where the tissue specific promotor is active, Gal4 will be active and when the promotor is not expressed, Gal4 is not expressed and thus the Gal4 protein will not be produced

  4. UAS reporter line – reporter gene is the gene the researchers want to study e.g. GFP, upstream of it is the UAS. UAS can only be activated when bound to Gal4 protein

  5. Thus, researchers cross breed the 2 lines of drosophila (Gal4 & UAS reporter line) -> new fly expresses Gal4 & UAS

  6. In the specific cells where the tissue specific promoter is active, Gal4 gene can produce Gal4 protein -> Gal4 protein binds UAS -> triggers the reporter gene of interest associated with the UAS -> this tells researchers exactly where their target gene is being expressed within the fly
• Nobel prizes awarded for work with drosophila

  • 6 nobel prizes awarded across 10 scientists for their work / contribution to the scientific community using drosophila e.g. molecular mechanisms in circadian rhythm
• Specific antibodies allow drosophila neurons to be individually identified


  • Antibodies are used in immunostaining techniques and visualised by microscopes – this can be used to identify specific neurons e.g. neuroblasts, ganglion mother cells (GMCs) are different and will thus showcase differential staining patterns under microscopes – in these cases their heterogeneity means specific antibodies will work on one or the other
• Practical advantages of drosophila as a model system

  • Short generation span (10-12 days)
  • Ability to generate lots of progeny
  • Very adaptable to food sources (easy to culture in the laboratory)
  • Adaptable to changes to temperature (for genetic manipulations and culturing)
  • Easy to store due to small size
  • Generate lots of progeny; single female -> 100 eggs in 4/5 days -> quick amplification
  • Easy to culture in lab; e.g. in fruit bowls, wines etc -> grow on simple yeast food source in lab (cheap)
  • Can adapt to many temperatures / areas -> very wide-spread -> can survive in all geographic locations across different species; don't need to be overly cautious when keeping them in lab
  • Easy to store; small size
  • Drosophila are easy to culture – store them in vials & let them grow in incubators where temperature can be altered for optimum growth at 25 degrees
• Mushroom body neurons


  Bilateral structure containing densely packed neurones in the anterior regions of protostome brains – they are associated with processing olfactory sensory inputs, olfactory discrimination & olfactory learning – essential in drosophila for learning & memory
• Genetic advantages of drosophila as a model system

  • Entire genome sequenced – one of the earliest sequenced genomes allows a full characterisation of the genetic elements that contribute to the biology of the model organism (development, behaviour, disease, aging) – making it a sufficient model for a wide range of studies
  • Rapid identification of genes/mutations - was possible using traditional methods before the genome was sequenced; tech now allows for quicker / easier gene coding
  • Functional genomic approaches can be applied in drosophila (mutagenesis, protein function, genetic interactions) - made easier to research
  • Large-scale annotation of the genomic elements (gene expression, gene function and genomic modifications during development) – lots of molecular information / interactions can be understood from drosophila
• Forward genetics in Drosophila

  • Forward genetics is the standard genetic approach whereby genes involved in a particular process are identified by generating genetic mutations that disrupt the process under study
  • Mutant phenotypes are identified, often using non-biased screens, and the gene that has been disrupted is characterised
  • Many neurobiological processes can be studied in this way from development to behaviour
  • Can be used in the study of human disease by screening for mutations with a phenotype comparable to the disease pathology
• Drosophila has reduced complexity from a genetic & cellular point of view -> drosophila has around 13,000 genes & 100,000 neurons (more complicated than worms) -> this is much simpler than the human brain considering humans have around 86 billion neurons and roughly the same amount of non-neuronal cells
• Pick a phenotype and find the gene for it by inducing genetic mutations and seeing how the phenotype changes – important in developmental & behavioural processes, and human disease
• Conserved neurobiology of drosophila


  • Flies and mammals have similar complexity of neural cell types - more so than worms
  • Flies and mammals use the same neurotransmitters – similar use of neurotransmitters
  • Flies have similar electrophysiology to vertebrates, utilise similar Na+, K+ and Ca2+ channels
  • Flies utilise many of the same synaptic proteins – e.g. SNARE for neurotransmission vesicular docking
  • 75% of human disease genes have close homologs in Drosophila – although this is not a complete 100% overlap, there's a good chance if you see a gene in human causing disease there will likely be a drosophila homologue of it which can be studied e.g. ANK1 gene for hereditary spherocytosis; inherited blood disorder – problem with RBCs -> spherical shape RBCs instead
• Drosophila genetics in neurobiological research


  • Forward genetic approaches (random mutagenesis using chemical, radiation and molecular approaches – see what phenotypes change accordingly)
  • Reverse genetic approaches (analysing genes of interest through gain- and loss-of function approaches)
  • A range of molecular genetic tools to label and mis-express genes in specific cell populations (e.g. antibodies, GAL4/UAS, FLP/FRT, MARCM)
• Neuroanatomical tools used in drosophila

  • Specific brain structures, e.g. mushroom body (MB), or sets of neurons can be labelled using antibodies, 'enhancer traps', or binary expression systems, e.g. the GAL4/UAS system; can get high resolution images of networks in neuronal activity / population of neurons and their activity
• Genetic screens in drosophila have identified mutations that affect axon outgrowth
• GAL4/UAS binary expression system

  • The GAL4 transactivator system is used to express a gene of interest, e.g. gene X, in a particular tissue
  • Transgenic flies expressing the yeast transcription factor GAL4 expressed in cell or tissue specific pattern drive expression of genes downstream of a GAL4 binding site (UAS)
  • When flies carrying UAS-geneX are mated to flies carrying a GAL4 line, then geneX will be expressed in the same place as GAL4
  • Collections of GAL4 flies exist that drive expression in a wide variety of neuronal cells, e.g. eye, CNS, PNS etc
  • GAL4 activity can be modulated by use of GAL80 repressor or use of a hormone inducible system
• PNS of drosophila embryo -> neuronal markers of neurons
• Repeated patter
• How the Gal4/UAS system works – to study gene expression

  1. You have 2 lines of drosophila – a driver line (Gal4) and a reporter line (UAS)

  2. Each driver line contains Gal4 protein and a tissue specific promotor gene which only allows gal4 to be produced in certain tissues – e.g. gut only or brain only etc depending on where you want it

  3. Where the tissue specific promotor is active, Gal4 will be active and when the promotor is not expressed, Gal4 is not expressed and thus the Gal4 protein will not be produced

  4. UAS reporter line – reporter gene is the gene the researchers want to study e.g. GFP, upstream of it is the UAS. UAS can only be activated when bound to Gal4 protein

  5. Thus, researchers cross breed the 2 lines of drosophila (Gal4 & UAS reporter line) -> new fly expresses Gal4 & UAS

  6. In the specific cells where the tissue specific promoter is active, Gal4 gene can produce Gal4 protein -> Gal4 protein binds UAS -> triggers the reporter gene of interest associated with the UAS -> this tells researchers exactly where their target gene is being expressed within the fly
• Forward genetic approaches in Drosophila genetics


  • Random mutagenesis using chemical, radiation and molecular approaches - see what phenotypes change accordingly
• Reverse genetic approaches in Drosophila genetics


  • Analysing genes of interest through gain- and loss-of-function approaches
• Molecular genetic tools used in Drosophila


  • Antibodies
  • GAL4/UAS
  • FLP/FRT
  • MARCM