Drosophila Axis Determination Flashcards
Why was Drosophila chosen to study early embryonic development?
- Well-characterized genetically + established techniques for mutating and mapping genome positions.
- Easy to culture + Short lifecycle
- Development mutants are easy to identify because the exoskeleton of Drosophila larvae has several landmark features that can be used to identify mutants defective in aspects of anterior/posterior and dorsal/ventral polarity
- Once a mutation has been mapped in Drosophila, it is relatively easy to clone the gene.
What genetic analysis and standard Recombinant DNA techniques can be used to analyse gene function in Drosophila?
- Characterization of mutant phenotypes:
i. A detailed characterization of the phenotypes of mutants is important for determining which structures are affected by a particular mutation. - Nucleotide sequence analysis of clone, and subsequent determination of the amino acid sequence:
i. May reveal function of the protein product, by comparison with other well characterized protein sequences in public databases. - The spatial and temporal patterns of expression can be examined at the RNA level (by in situ hybridization) and at the protein level (by using immunocytochemistry)
- Genetic experiments:
i. Can be carried out to determine whether different genes interact.
What is a synctium?
Embryo is which the nuclei divide and migrate in a common cytoplasm
What are the 4 regions defined along the dorsal/ventral axis?
- Mesoderm -> internal soft tissue like muscle & connective tissue
- Ventral ectoderm -> neural tissue and ventral epidermis
- Dorsal ectoderm -> only to epidermis
- Amnioserosa -> extra-embryonic membrane that is sloughed off during embryonic development
What are segmentation genes?
Divide the body into the correct number of parts
What are homeotic genes?
Establish the identity of parts
What are maternal-effect genes?
= These genes are transcribed in the nurse cells in the ovary of the mother, and mRNA transcripts are passed into the developing oocyte.
- The phenotype of embryo is dependent on maternal genotype.
- The phenotype of the offspring is independent of the genotype of the father.
o i.e., A recessive mutation will only produce a mutant animal when the mother is a mutant homozygote.
Zygotically-active genes
- Expressed in the zygote during early development. Recessive mutations in zygotically-active genes elicit mutant phenotypes in homozygous mutant animals.
- Grouped into three classes on the basis of their phenotype:
o Gap mutants
o Pair-rule mutants
o Segment polarity mutants
Method of in situ hybridization
Reveals stage of development at which a gene is turned on and the tissues in which it is expressed
o RNA transcript marks cells in which gene is expressed
o If a DNA probe is made that hybridizes to the RNA from a particular forming homologous base pairs
o After incubation, wash embryos so probes that have bound non-specifically will leave
o Treat embryo’s with an antibody that will stick to the probe.
o Antibody is coupled to enzyme which reacts with 2 chemicals in developing solution -> blue colour will form wherever the probe is stuck.
o Allows us to determine presence of RNA which means the gene was expressed
Immunocytochemistry
- Antigen = protein you want to visualise (target of antibody)
- Primary antibody = recognizes protein of interest
- Epitope = protein sequence that antibody binds to
- Secondary antibody = recognizes and binds to primary antibody and is modified so you can visualise it (conjugated to a fluorescent dye molecule)
- Colormetric indirect detection: Secondary antibody is enzyme which leads to tissue colouring
- Direct detection: Primary antibody has fluorescent molecule. This is worse.
- Inject peptide (10-20 aa long) into rabit, rabbit immune system will generate antibody against this peptide which can be isolated from blot serum of rabbit.
- Secondary antibody formation: take FC region of primary antibody and inject into second animal – immune system produces antibodies against FC region of antibody. Then conjugated to fluorescent molecule.
- Can reuse secondary antibody in different experiments due to high level of conservation in FC region of antibodies
- Cell fixation: crosslink proteins to terminate all biological processes in cells.
- Permeabilization: Add detergent which makes membranes more permeable so antibodies can get in
- Add primary antibody
- Add secondary antibody
- Add antifade mounting media & mount to slide
- Microscopy
What was the experimental approach to work out the model of how drosophila embryo is patterned along the A/P axis?
- Isolation of recessive mutants (grouping into segmentation or maternal effect genes)
- Cloned genes and determined their nucleotide sequence.
- Mapped spatial and temporal patterns of expression of genes and proteins using in situ hybridization and DNA probes. Also raised antibodies to use immunocytochemistry to find the proteins.
- Did genetic experiments to determine the epistatic relationship between mutants (To work out the order of expression).
a. Co-ordinate genes (bicoid, nanos, caudal, hunchback, torso) provide initial positional information required for activation of Zygotic-effect genes. - Manipulated expression of genes and assayed their effect on the expression of other genes, and embryonic development.
What are the 3 classes of maternal-effect genes?
- Anterior mutants
o E.g. Bicoid – missing acron segment - Posterior abdomen mutants
o E.g. Nanos – ‘dwarf’ in Spanish missing part of posterior - Terminal mutants
o E.g. Torso – missing both ends
What is the phenotype of a Bicoid mutant?
- Loss of anterior structures (Acron) and duplication of posterior structures.
- Has duplication of posterior segments in place of acron – forms mirror image
What does Bicoid code?
o Encodes a transcription factor that is expressed in a [conc] gradient in the anterior of any develop
What is the localisation of bicoid mRNA and protein?
mRNA localised to the anterior, the protein expressed on a gradient from the anterior.
How was it tested whether Bicoid was required to form the identity of anterior structures?
o Cytoplasm from anterior of fertilised WT egg transplanted to anterior of bicoid mutant egg -> Seen that some anterior structures develop (i.e. rescues mutant).
o Cytoplasm from anterior of fertilised WT egg transplanted to middle of bicoid mutant egg -> Seen that some anterior structures develop in the centre of the embryo.
o Proves that Bicoid rescues the mutant phenotype and sufficiently pattern the head region.
Expression patterns of hunchback, nanos and caudal
Nanos:
- mRNA expressed posteriorly
- After fertilisation, protein (still posterior) but diffuses slightly out.
- Wherever there is nanos protein there is no hunchback protein until it is below a threshold level.
Maternal hunchback:
- mRNA expressed at low levels throughout the embryo
- Protein not expressed in posterior due to nanos
- Activated in anterior by Bicoid
Caudal:
- Caudal mRNA evenly distributed throughout oocyte
- Protein has gradient. High in posterior, low in anterior (opposite of Bicoid)
What is the bicoid-caudal interaction?
- Bicoid suppresses translation of caudal in anterior of embryo
- Bicoid binds to 3’UTR of caudal mRNA transcripts and represses translation
- Unusual for a transcription factor!
What is the Nanos-hunchback interaction?
- Nanos represses translation of hunchback in posterior of the embryo
- Pumilio encodes RNA binding protein – found in even concentration throughout developing embryo
- In Anterior:
o Pumilio binds to 3’ end of hunchback mRNA – adds polyA tail
o Poly-A tail is important for efficient translation of hunchback mRNA
o Increases translation
o Promotes anterior structures - In Posterior:
o Nanos binds to Pumilio -> Pumilio can’t add extra Adenines = shortening of polyA tail
o Inhibits translation
o No hunchback results in posterior abdominal formation
How is the localisation of bicoid and nanos mRNA transcripts accomplished?
- Result of polarised microtubule network in the oocyte [+ end in posterior; - end in anterior]
- Nurse cells in anterior of egg chamber, secrete all the maternal effect genes into developing oocyte in the posterior of the egg chamber.
- Gurken mRNA synthesised in nurse cells, translated in oocyte. Gurken protein associates with oocyte nucleus.
- Oocyte nucleus (with associated Gurken) moves towards terminal follicle cells (posteriorly)
- Terminal follicle cells differentiate into posterior follicle cells in response to Gurken signalling
- Posterior follicle cells signals to oocyte and activates protein kinase A
- Protein kinase A orientates microtubules so that growing (+) end is at posterior of egg.
- Bicoid mRNA binds to dynein, a motor protein that associates with non-growing (-) end of microtubules
- Oskar (another maternal effect gene) secreted into oocyte. Oskar mRNA binds to kinesin1, a motor protein that associates with the growing(+) end of microtubules -> oskar mRNA moves to posterior.
- Nanos mRNA binds to Oskar protein and is localised to posterior
What is the genetic hierarchy leading to segmentation?
- Maternal-effect genes establish morphogenic gradients in the egg.
- Gradients ensure that gap genes are expressed in certain broad domains of the embryo - Gap genes activate pair-rule genes in a series of 7 stripes
- Pair-rule gene products restrict expression of segment polarity genes to series of 14 stripes, one per segment
- Embryo is divided into 14 segment-sized units
Patterns of expression of Gap genes in WT embryos
- As hunchback tails off, we get Kruppel
- As Kruppel tail off we get knirps
- As knirps tails off we get giant
What regulates expression of Gap Genes
- Expression of gap genes is initially dependent upon maternal-effect genes
o (eg. zygotic hunchback is dependent upon bicoid) - Kruppel expression is positively and negatively regulated by hunchback
- Gap genes reinforce the expression patterns of each other
o Zygotic hunchback (hb) gene expression is dependent on bicoid
o In the posterior of the embryo, Knirps gene expression repressed by bicoid, activated by high [caudal]
o Kruppel gene expression is repressed by high [hb], but is activated by low threshold [hb]
How did scientists show that zygotic hunchback was dependant on bicoid?
- Created a construct with the zygotic phase hunchback (z-hb) promoter fused to a reporter gene (lacZ – easy assay for B-galactosidase activity) and cloned the TATA box from a heat-shock promoter upstream of transcriptional start site of lacZ (because well characterized and easily available)
o TATA box important for recruiting RNA Pol to start of transcription - Analysed z-hb promoter, and found 2 Bicoid response elements in the promoter
- Analysed distribution of B-galactosidase in 3 embryo’s using immunocytochemistry
- Control:
o Just the construct above, to show that the gene was transcribed as it should be. - Partial Promoter:
o 1 of the 2 bicoid response elements was removed
o Saw that transcription still occurred, but some expression was lost - Promoter lacking bicoid response element:
o All expression is lost - Also changed the dosage of bicoid and tested to see if it had an effect on lacZ expression:
o Higher doses resulted in higher expression.
