Drosophila Axis Determination

Question 1

Why was Drosophila chosen to study early embryonic development?

Answer 1

• 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.

Question 2

What genetic analysis and standard Recombinant DNA techniques can be used to analyse gene function in Drosophila?

Answer 2

1. 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.
2. 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.
3. 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)
4. Genetic experiments:
   i. Can be carried out to determine whether different genes interact.

Question 3

What is a synctium?

Answer 3

Embryo is which the nuclei divide and migrate in a common cytoplasm

Question 4

What are the 4 regions defined along the dorsal/ventral axis?

Answer 4

1. Mesoderm -> internal soft tissue like muscle & connective tissue
2. Ventral ectoderm -> neural tissue and ventral epidermis
3. Dorsal ectoderm -> only to epidermis
4. Amnioserosa -> extra-embryonic membrane that is sloughed off during embryonic development

Question 5

What are segmentation genes?

Answer 5

Divide the body into the correct number of parts

Question 6

What are homeotic genes?

Answer 6

Establish the identity of parts

Question 7

What are maternal-effect genes?

Answer 7

= 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.

Question 8

Zygotically-active genes

Answer 8

• 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

Question 9

Method of in situ hybridization

Answer 9

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

Question 10

Immunocytochemistry

Answer 10

• 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

1. Cell fixation: crosslink proteins to terminate all biological processes in cells.
2. Permeabilization: Add detergent which makes membranes more permeable so antibodies can get in
3. Add primary antibody
4. Add secondary antibody
5. Add antifade mounting media & mount to slide
6. Microscopy

Question 11

What was the experimental approach to work out the model of how drosophila embryo is patterned along the A/P axis?

Answer 11

1. Isolation of recessive mutants (grouping into segmentation or maternal effect genes)
2. Cloned genes and determined their nucleotide sequence.
3. 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.
4. 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.
5. Manipulated expression of genes and assayed their effect on the expression of other genes, and embryonic development.

Question 12

What are the 3 classes of maternal-effect genes?

Answer 12

• 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

Question 13

What is the phenotype of a Bicoid mutant?

Answer 13

• Loss of anterior structures (Acron) and duplication of posterior structures.
• Has duplication of posterior segments in place of acron – forms mirror image

Question 14

What does Bicoid code?

Answer 14

o Encodes a transcription factor that is expressed in a [conc] gradient in the anterior of any develop

Question 15

What is the localisation of bicoid mRNA and protein?

Answer 15

mRNA localised to the anterior, the protein expressed on a gradient from the anterior.

Question 16

How was it tested whether Bicoid was required to form the identity of anterior structures?

Answer 16

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.

Question 17

Expression patterns of hunchback, nanos and caudal

Answer 17

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)

Question 18

What is the bicoid-caudal interaction?

Answer 18

• 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!

Question 19

What is the Nanos-hunchback interaction?

Answer 19

• 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

Question 20

How is the localisation of bicoid and nanos mRNA transcripts accomplished?

Answer 20

• 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

Question 21

What is the genetic hierarchy leading to segmentation?

Answer 21

1. Maternal-effect genes establish morphogenic gradients in the egg.
   - Gradients ensure that gap genes are expressed in certain broad domains of the embryo
2. Gap genes activate pair-rule genes in a series of 7 stripes
3. Pair-rule gene products restrict expression of segment polarity genes to series of 14 stripes, one per segment
4. Embryo is divided into 14 segment-sized units

Question 22

Patterns of expression of Gap genes in WT embryos

Answer 22

• As hunchback tails off, we get Kruppel
• As Kruppel tail off we get knirps
• As knirps tails off we get giant

Question 23

What regulates expression of Gap Genes

Answer 23

• 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]

Question 24

How did scientists show that zygotic hunchback was dependant on bicoid?

Answer 24

• 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.

Question 25

Pattern of expression of pair-rule genes

Answer 25

• Expression of each pair-rule gene in 7 stripes divides the embryo into 14 parasegments.
• Each pair-rule gene expressed in alternating parasegment
• Each row of nuclei in each parasegment express a unique combination of pair-rule genes

Question 26

Regulation of eve (even-skipped) in stripe 2

Answer 26

• Low levels of bicoid, kruppel and giant
• High levels of hunchback
• Activators:
  o Dependant on Bicoid & Hunchback
• Repressors:
  o Kruppel represses posterior expression of stripe 2 enhancer which restrics the posterior boundary of stripe 2.
  o Giant represses anterior expression of stripe 2 enhancer which restricts the anterior boundary of stripe 2.

Question 27

Regulation of fushi-tarazu expression

Answer 27

• Ftz first expressed at low levels in broad band in segmented region of embryo a t division cycle 14 (when cellular blastoderm starts to form)
• In next 30mins Ftz expression is enhanced or repressed (by eve) by primary pair-rule proteins.
• Ftz also activates its own expression.
• Linked heat shock promoter to eve (silent in absence of heat shock; activated after pulse of heat -> eve expressed throughout embryo)
  o No heat shock = alternating expression of ftz and eve.
  o Heat shock = eve expressed broadly everywhere, and ftz expressed nowhere -> direct evidence that eve represses expression of ftz

Question 28

Patterns of expression of segment-polarity genes

Answer 28

• Turned on during germband extension
• Segment polarity genes are expressed at the cellular blastoderm stage in one row of cells in each parasegment
• Engrailed is expressed in the anterior of each parasegment
• Engrailed expressed in posterior of each segment following segmentation

Question 29

What are the 3 phases of regulation of expression of segment-polarity genes?

Answer 29

1. Expression of engrailed and wingless is initiated by pair-rule genes
2. Cell signalling between engrailed expressing cells and wingless expressing cells regulates the continued expression of en and wg.
3. En and Wg expression is independently maintained

Question 30

Cell signalling between engrailed expressing cells and wingless expressing cells

Answer 30

1. Receiving wingless signal :
   a. Absence of wingless:
   i. Zw3 protein phosphorylates Armadillo
   ii. P-Armadillo is sent for proteolysis
   b. Presence of Wingless:
   i. Binds to Frizzled receptor
   ii. Activates Dsh
   iii. Dsh inhibits Zw3
   iv. Armadillo translocated into nucleus, acts as transcription factor of engrailed, hedgehog
2. Receiving hedgehog signal:
   a. Absence of Hedgehog:
   i. Patched receptors binds to smoothened receptor
   ii. Restrains smoothened protein
   iii. Cubitus interruptus (Ci) tethered on microtubules
   iv. Ci cleaved on microtubules, translocates to nucleus where it acts as repressor
   b. Presence of Hedgehog:
   i. Hedgehog binds to patched receptor
   ii. Patched releases smoothed protein
   iii. Smoothened releases Ci from microtubules
   iv. Ci translocates into nucleus -> activates transcription of wingless, patched.

Question 31

What is the conserved collinear regulation of Hox genes?

Answer 31

• Hox genes are organized into 2 separate clusters of genes, the Antennapedia and bithorax complex.
• The pattern of expression of the genes in these 2 complexes shows a correlation between the physical order they appear in on the chromosome (in the 3’-5’ direction), and the order of their expression in both space and time.
• This means that a gene which is first on the chromosome (3’ side) will be expressed first in development, and it will be expressed in the most anterior portion of the developing embryo.
• These 2 complexes of gene clusters are conserved in most animals.
• However, in many of these cases the complexes have been duplicated.
• Even though the number of sets of these Hox genes is different (mice have 4 sets of them), the order of the actual genes themselves within the clusters has been fully conserved.
• This means that in each complex, homologous genes (in Fig. 1 an example would be b-1 in mice and lab in Drosophila) will have the same position along the chromosome, and be expressed in the same special and temporal order during development.
• This conservation of chromosome positioning and its correlated spatial and temporal order of expression between species is what is referred to as “conserved collinear regulation of Hox genes”.