Drosophila

Question 1

What four classes of genes are responsible for segmentation of AP axis

Answer 1

• Maternal genes (loaded in the egg)
• Gap genes
• Pair-rule genes
• Segmentation polarity genes
• They function in a heirachy that progressively subdivides the embryo into successively smaller units

Question 2

What are the heirachy of events during embryonic development in terms of the segmentation classes of genes? And given example genes.

Answer 2

1. Maternal
   
   1. bicoid - establishes polarity
2. Gap
   
   1. hunchback - divide embryos into regions
3. Pair-rule
   
   1. even-skipped - stablish segmental body plan
4. Segment polarity
   
   1. engrailed - set boundaries of segment

Question 3

How were the genes involved in segmentation first identified?

Answer 3

Through mutants with defects in segmentation. Such as hunchback mutant

Question 4

How to study an embryonic lethal mutation?

Answer 4

• Mosaics produced by FLP, a site-specific recombination enzyme from yeast.
• Create genes that are homozygous in adult that were hetrozygous earlier in development
• Cross a heterozygous mutant male with a homozygous WT female
• FRT sites are placed on trans chromosomes to induces translocation.
• FLP is under control of a heat shock promoter
• During mitosis (recombination events) two different daughter cells are formed
• Tag the the WT gene with GFP so mutant cells can be distinguished from WT.
  
  • WT cells will glow GFP

Question 5

How to follow mutations in flies?

Answer 5

• Drosoohila only have three chromosome. Each chomosome can have a visible marker. Mutations can be linked to visible marker. However, genetic recombination can disrupt this can be solved by:
  
  • In males there is not genetic recombination
  • Use of balancer chromosomes

Question 6

What are balancer chomosomes?

Answer 6

• Chromosomes with multiple inversions that supress recombination
• A dominant marker is included in balancer chromsome
• A recessive lethal mutation is also incorporated into chromsome
  *

Question 7

Give examples of how FLP/FRT can be used in drosophila

Answer 7

• Allows analysis of cell-type specific gene function (especially critical for lethal genes)
• Defines which cells require gene activity: distinguished between cell autonomous and non-cell autonomous
• Helps distinguish between correlation and causation
• Construct can be used to switch genes on
  
  • A stop cassete flanked by FRT sites in front of GOI
• Or can be used to switch genes off
  
  • A stop cassete flanked by FRT sites in front of gene encoding for RNAi of your GOI

Question 8

What is positive and negative clone marking?

Answer 8

• Negative: the WT gene is fused with a GFP reporter
• Positive: Gal80 on WT gene represses GAL4 production on same WT chromosome which inturn represses UAS-driven GFP on mutant chromsome. After translocation by FLP, Gal80 is no longer present in mutant cells therefore, GFP is expressed in mutant cells
  
  • This can reveal a small subset of neurons
  • Depending on FLP driver, they might be related clonally or via gene expression

Question 9

How can FLP/FRT be used to study maternal effect genes?

Answer 9

• Somatic recombination in germline
• Heterozygous germlime mozaic will make homozygous mutant eggs
  
  • only 10% though so it is difficult to screen
  • By combining Dominant female sterile gene (DFS) to the WT chromsome, cells that do not recombine to make homozygous mutant cells will not be able to make eggs. Now 100% of eggs are mutant!
• This method has superceeded the transplantation method
  
  • This involved transplanting mutant pole cells to a WT mother and vice versa

Question 10

How it is the involvement of Gurkin in patterning the axis

Answer 10

• Gurken patterns both AP and DV
• grk mRNAs are expressed in nurse cells and transported long microtubules (MTS) where they localise in the oocyte at the MT minus end via the dynenin motor.
• Grk encodes TGF-alpha ligand that signals to overlying follicle cells
  
  • AP: Defines posterior follicle cells (PFCs). PFCS signal back to oocyte and repolarise MT cytoskeleton (unknown mechanism). Gurken then relocalises to ventral anterior corner for DV patterning
  • DV: Grk drives overlying FCs to be dorsal. Ventral FCs synsthesis a specialised vitelline membrane that sequesters signalling component. After fertilisation ventral proeteolytic cascade ctivates signalling that generates DV transcription gradient

Question 11

What is pole plasm?

Answer 11

• The pole plasm is formed during oogenesis and cantains a determinant for germ cells, importantly are the products of oskar
• Osk nucleates formation of pole plasm
• osk activity is necessary:
  
  • osk mutant embryos lack pole cells. The number of pole cells depend on osk levels
• osk activitiy is sufficient to nucleate pole plasm:
  
  • anterior osk mRNA causes anterior-ole plasm.

Question 12

How is the DV axis formed?

Answer 12

• The protein encoded for by the maternal dorsal is localised in the egg chamber in a ventral to dorsal gradient.
• The Dorsal protein is a transcription factor
• The high point of the Dorsal protein gradient is dependent on signalling from the ventral follicle cells. This signal is only acitve ventrally because it is repressed on the dorsal side by a previous inhibitory signal in the oocyte (gurkin).
• The Dorsal protein gradient itself works by egulating various zygotic genes such that each ventral to dorsal stip of cells has a different a combination of transcription factors active.

Question 13

How is Osk localised?

Answer 13

• mRNA is made in nurse cells and transported to oocyte by dynein and to the posterior end of the oocyte by kinesin
• Transport specificty is due to osk mRNA 3’UTR and internal RNA signal
• mRNA is anchored at oocyte posterior
• non-localisd mRNA is translationally repressed
• Anchored mRNA is derepressed
• osk protein promotes mRNA anchorage, i.e positive feedback

Question 14

How is the AP axis formed after gurkin and oscar?

Answer 14

• In the anterior mRNA for bicoid is deposited in the egg and a Bicoid protein gradient upregulates various genes to generate regional subdivisions
• bicoid encodes the anterior morphogen
• Bicoid protein gradient regulates the expression of many zygotic gap genes, such as hunchback and kruppel. Each of these genes have a promter of different sensitivity to Bicoid, so they become upregulated at different AP levels.
• The anterior system depends on the deposition of nanos
• Gap genes are transcription factors that influence expression of other gap genes. Gap mutants show a gap in segmentation patterns where a particular gap gene is absent.
• The upregulation of pair-rule genes represents the first formation of a reiterated pattern in the embryo. E.g. of pair rule genes are: hairy and ftz.
  
  • To form a stripe it is necessary that the gene been turned on at one AP level and then off again at another level.
• The semgment polarity genes functions to create the parasegment boundaries of the early embryo. Once upregulated they maintain the expression through a positive feedback loop.