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Biology 467 > Discussion Paper 4 > Flashcards

Discussion Paper 4 Flashcards

1
Q

How many segments is a drosophila melanogaster embryo divided into?

A

14

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2
Q

What are the advantages of using drosophila in genomic studies? (4)

A
  1. well-sequenced genome
  2. simple genome with only 4 chromosomes
  3. fast life cycle/generation time
  4. genes show a lot of conservation and have functional overlap with mammal genes
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3
Q

The discussion paper examines flies at this stage in their life

A

Larval stages

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4
Q

What key event occurs during the fly larval stage?

A

A 2nd wave of neurogenesis

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5
Q

What are the 3 regions of the fly CNS present in the larval stage?

A

Optic lobe
Mushroom body
Ventral nerve cord

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6
Q

The fly ventral nerve cord innervates…

A

The thoracic and abdominal segments

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7
Q

What is the specific motor neuron name which is examined in this paper?

A

RP2

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8
Q

What muscle fiber does RP2 innervate?

A

Da2

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9
Q

What is the broad overarching question of the paper?

A

What are the mechanisms that underlie asymmetric cell divisions and cell fate decisions?

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10
Q

The specific scientific objective of this paper is to determine…

A

The temporal requirement of Notch signaling during RP2/sib cell development

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11
Q

What is the assay in Figure 1?

A

Immunolabelling for Eve (Even-skipped)

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12
Q

Why did the authors choose to examine Eve for figure 1?

A

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+)

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13
Q

What are the advantages of the Eve assay in figure 1?

A
  1. Robust
  2. Illuminates a pretty simple pathway which mechanism can be addressed
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14
Q

What are some weaknesses of the Eve assay from Figure 1?

A

Sib cell loses it and you lose track of it, don’t even know if the cell lives or dies

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15
Q

What cell gives rise to the GMC-1 cells?

A

Type I neuroblast: NB4-2

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16
Q

Previous studies determined that genetic inhibition of (1) signaling causes GMC-1 to divide into two RP2 cells

A

Notch

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17
Q

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

A

Notch

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18
Q

Describe the previous findings of other studies related to Numb and RP2 specification

A

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

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19
Q

How does Numb inhibit Notch signaling?

A

Promotes endocytosis of Notch, and promotes Notch ubiquitination and degradation

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20
Q

Where is Numb located in the cell?

A

Along the basal membrane (it is a membrane-associated protein)

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21
Q

What is the overall hypothesis of this paper?

A

Asymmetric cell division of GMC is mediated by Notch signaling prior to cell division

22
Q

What is the underlying question of Figure 1?

A

Is notch signaling required for GMC development prior to cell division?

23
Q

What is the experimental approach used to address the question of Figure 1?

A

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)

24
Q

What is the main takeaway from Figure 1?

A

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

25
The conclusions drawn from figure 1 demonstrate that Notch is required prior to cell division for (2)
1. sib cell specification 2. asymmetric daughter cell size
26
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
27
In this study, 7.5 hours marks the time at which...
GMC cells have not yet divided
28
For the purposes of this study, GMC division is complete by...
8 hours
29
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
30
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
31
The findings of figure 2 demonstrate that Notch is required for (2)
1. basal localization of numb 2. positioning of the cleavage furrow
32
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
33
What is the experimental approach used to address the question proposed by figure 3?
Loss of function analyses using Mam mutants - used 2 mutants, one "strong" mamIL42, and one "weak" mamHD10/6
34
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
35
What are the important findings of this figure?
When you use the strong mam mutation (mamIL42), this results in symmetrical localization of Numb
36
What is the underlying question of Figure 4?
Does Notch regulate asymmetry through Insc?
37
Previous studies have noted these two key observations about Insc
1. insc is apically localized in dividing GMCs 2. apical insc localization restricts numb to the basal end of the GMC
38
Describe 3 functional characteristics of insc
1. has Sh3 homolog domains (for prot-prot interactions) 2. ankyrin repeats (for prot-prot interactions) 3. has cytoskeletal attachment sites
39
What conclusion can be drawn from this figure?
Notch signaling is required prior to cell division for insc apical localization
40
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
41
What is the main question being addressed in figure 4b and 4c?
Is Insc causal to numb asymmetry?
42
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
43
What question is being addressed by figure 4d?
Is numb required for apical localization of insc?
44
What are the key findings from figure 4d?
Numb is not required for apical localization of insc
45
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?
46
Previous studies have demonstrated when you knock out numb, what fate of the 2 GMC daughter cells do you get?
get 2 sib cells
47
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!)
48
Why did the authors decide to use Nintra instead of Notch for figure 4f?
Because they wanted Notch to be active regardless of the presence of Delta
49
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
50
What was the key contribution of this paper to the field?
Previously, it was thought that Notch functioned after GMC division to influence cell fate, but this paper determined that Notch is required before GMC division

Biology 467 (20 decks)

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