Discussion Paper 4 Flashcards
How many segments is a drosophila melanogaster embryo divided into?
14
What are the advantages of using drosophila in genomic studies? (4)
- well-sequenced genome
- simple genome with only 4 chromosomes
- fast life cycle/generation time
- genes show a lot of conservation and have functional overlap with mammal genes
The discussion paper examines flies at this stage in their life
Larval stages
What key event occurs during the fly larval stage?
A 2nd wave of neurogenesis
What are the 3 regions of the fly CNS present in the larval stage?
Optic lobe
Mushroom body
Ventral nerve cord
The fly ventral nerve cord innervates…
The thoracic and abdominal segments
What is the specific motor neuron name which is examined in this paper?
RP2
What muscle fiber does RP2 innervate?
Da2
What is the broad overarching question of the paper?
What are the mechanisms that underlie asymmetric cell divisions and cell fate decisions?
The specific scientific objective of this paper is to determine…
The temporal requirement of Notch signaling during RP2/sib cell development
What is the assay in Figure 1?
Immunolabelling for Eve (Even-skipped)
Why did the authors choose to examine Eve for figure 1?
Eve is a marker in GMC cells, which give rise to RP2 and sib cells. RP2 cells stay Eve+, but sib cells become Eve- when they mature (but initially, they are Eve+)
What are the advantages of the Eve assay in figure 1?
- Robust
- Illuminates a pretty simple pathway which mechanism can be addressed
What are some weaknesses of the Eve assay from Figure 1?
Sib cell loses it and you lose track of it, don’t even know if the cell lives or dies
What cell gives rise to the GMC-1 cells?
Type I neuroblast: NB4-2
Previous studies determined that genetic inhibition of (1) signaling causes GMC-1 to divide into two RP2 cells
Notch
Prior to the findings of this paper, it was thought that signaling from this molecule was involved in determining sib cell fate after GMC-1 division
Notch
Describe the previous findings of other studies related to Numb and RP2 specification
Previous studies found that Numb localizes asymmetrically to the basal end of GMCs and is inherited only by the basal daughter cell, and that this promotes RP2 specification because Numb inhibits Notch signaling
How does Numb inhibit Notch signaling?
Promotes endocytosis of Notch, and promotes Notch ubiquitination and degradation
Where is Numb located in the cell?
Along the basal membrane (it is a membrane-associated protein)
What is the underlying question of Figure 1?
Is notch signaling required for GMC development prior to cell division?
What is the experimental approach used to address the question of Figure 1?
Use a temperature-sensitive loss-of-function mutation for Notch - they switched the temperature to “non-permissive” at different times prior to cell division (in this figure, E and M refer to Early and Middle)
What is the main takeaway from Figure 1?
When Notch is mutated early on, this results in the most symmetric divisions (get 2 Rp2 cells)
When Notch is mutated in the middle stage, this results in 2 RP2 cells of unequal size - intermediate phenotype
What is the underlying question for figure 2B and 2C?
Is Notch required prior to cell division for asymmetric NUMB expression and asymmetric cell division
What is the most important finding demonstrated by this figure?
To get proper basal localization of Numb, you must have Notch signaling prior to GMC-1 division
What is the experimental approach used by this figure?
Used temperature-sensitive Notch mutations to knock out notch at early, middle, and late time periods, and then did immunolabelling for Numb and Spectrin
The findings of figure 2 demonstrate that Notch is required for (2)
- basal localization of numb
- positioning of the cleavage furrow
In Figure 3, what is the value of examining Mastermind (Mam) mutants?
Wanted to confirm that the canonical Notch signaling pathway underlies the GMC-1 phenotype
What are the most important findings demonstrated by this figure?
In the stronger loss of function, you get 2 RP2 cell duplicates of equal size, in the weaker loss of function, you get some equal and some asymmetric cell division
What are the important findings of this figure?
When you use the strong mam mutation (mamIL42), this results in symmetrical localization of Numb
What is the underlying question of Figure 4?
Does Notch regulate asymmetry through Insc?
What conclusion can be drawn from this figure?
Notch signaling is required prior to cell division for insc apical localization
What is the experimental approach used in this figure?
Did temperature-sensitive Notch knockouts, and mutated mam (using the strong mutation) and then did immunolabelling for insc
What is the main question being addressed in figure 4b and 4c?
Is Insc causal to numb asymmetry?
What are the key takeaways from figures 4b and 4c?
4B: loss of insc results in symmetric, duplicated RP2 cells
4C: Insc is required for basal localization of numb
What question is being addressed by figure 4d?
Is numb required for apical localization of insc?
What is the underlying question addressed by figure 4f?
Does notch gain of function override numb and lead to sib cell fate and asymmetric cleavage?
What is the experimental approach being used by Figure 4f?
Give the cells a shit ton of Nintra using the Hsp70 promoter (Hsp70 = heat shock protein, only active at high temps - inducible model!)
Highlight the most important findings from figure 4f
Both cells become sib cells, and the cells divide asymmetrically - therefore, ectopic expression of Nintra results in double sib cell fate - overrides Numb
