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BMS 109 > Dev 3- drosophila > Flashcards

Dev 3- drosophila Flashcards

1
Q

Drosophila relationship to us in evolution?

A

500million evolutionary years away, but homologues of genes e.g. 530/950 human disease genes have homologues in the fly.

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2
Q

How can the life span of the drosophila be altered?

A

By temperature- life cycle to adulthood 10days at 25degrees, but 21 days at 18degrees.
Full lifespan 40-60

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3
Q

Life cycle stages of the drosophila?

A

Embryogenesis- cleavage- gatrulation- hatching

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4
Q

Dates for the drosophila?

A

1910- Morgan discovers a white fly- look at mutation.
1913- Genetic map of the drosophila
1914/16- bridge showed chromosomes contain DNA.
1927- Muller found x-ray to cause mutations and chromosome rearranging.

1980- Drosophila used for mutagenesis experiments

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5
Q

What are enhancer traps?

A

An enhancer trap is a method in molecular biology that allows hijacking of an enhancer from a gene. The enhancer trap will contain a transposible element for random insertion into the genome and a reporter gene for identification of the spatial regulation of the enhancer.
e.g. GAL4 yeast, or LacZ for ecoli

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6
Q

Which experimental methods were developed in the drosophila during the 80’s,90’s and 2000’s?

A
  • Enhancer trapping
  • Transgenics
  • Gene misexpression
  • Clonal mutant analysis
  • RNAi
  • ‘omic’ techniques- proteome, transcriptome
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7
Q

When was the Drosophila genome published?

A

2000

1.5million bp’s

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8
Q

Genome of the Drosophila- bps? Genes? Protein coding?

A

1.5million bp’s
16,700 genes
14,000 protein coding genes.

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9
Q

Coutship in the drosophila?

A

Highly reproducible, so is innate behaviour that is coded in the genome. 15mins courtship to mating.

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10
Q

How does spermatogenesis happen in the Drosophila?

A

-Hub cells produce Unpaired ligand (part of JAK/STAT pathway) which maintains stem cell fate, whereas cells further away differentiate.
so the sperm cells gradually differentiate from germline stem cells as they move away.

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11
Q

How does oogenesis happen in the drosophila?

A

Stem cells divide 4 times (16 total) without segregating (endoreduplication) into a cyst, and one of the two centre cells with 4 connections become the oocyte, while the other 15 become nurse cells.
Divides by meiosis to activate so can be fertilised by sperm to form a pronucleus/

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12
Q

After fertilisation structure of the oocyte?

A

The oocyte grows in the posterior half of the egg, with the nurse cells at the anterior. There are rotective follicle cells that develop around the outside, and secrete egg cell (chorion)

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13
Q

What are polytene chromosomes?

A

Giant chromosomes common to drosophila, large due to many rounds of endoreplication.

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14
Q

Function of the Nurse cells?

A

Make the materal contributions such as proteins and RNA which are transferred into the developing oocyte via ring canals (cytoplasmic dumping via holes in the cytoplasm) moving using Microtubules. Follicle cells squeeze pressure on the nurse cells so they give off these contents.
Oocyte grows at expense of the nurse cells,

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15
Q

How is the fertilised egg drosophila structure formed?

A

Chorion egg secreted by the follicle cells.

Vitelline membrane- hydrophobic so protects egg from drying out.

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16
Q

After fertilisation what cell rearrangments are there?

A

After 14 nuclear divisions, nuclei migrate to the edge of the embryo and form a synctial blastoderm. Membrane ingrows up between these nuclei to form the individual cells creating a cellular blastoderm.

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17
Q

Mesoderm movements in the drosophila embryo after during and after gastrulation?

A

Mesoderm ingresses into the blastoderm during gastrulation from the ventral location. This elongates but it caannot puncture the egg so the embryos tail forms anterior and folds up on top of the embryo. (saved good image)

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18
Q

Experiment done in 1980 to look for mutations in drosophila segmentation?

A

Christiane Nüsslein-Volhard and Eric Wischaus undertake a saturation mutagenesis to identify genes involved in the development and patterning of the larval cuticle.
(saturation experiment= random codon(s) mutated to give all possible AA’s in a position)

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19
Q

1980 segmentation mutation experiment findings?

A

27,000 mutated genes, Over half lethal, but 580 found to cause phenotypes that could be seen in the embryo.
These were then divided into 139 complementation groups. (only 139 genes but hit these around 5 times)

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20
Q

Complimenting mutations?

A

If -/+ x +/- and complement then these are two different mutations and no -/- is created.
FAIL TO COMPLIMENT- mutation is in the same gene, so create the -/- and the phenotype is shown.

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21
Q

What is Knirps? mutation?

A

It is a gap gene. Mutation causes a shorter phenotype with missing segments (GAP in body giving ‘little boy’ german translation)

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22
Q

Gap gene exmples?

A

Hunchback, Knirps, Giant, Tailess, Kruppel

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23
Q

Sequence of genes in the drosophila?

A

Maternal genes-> Gap genes-> Pair rule-> Segment polarity

e.g. bicoid and nanos- hunchback and knirps etc- Eve and Ftz- Hh, Engrailed, Wg

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24
Q

Example of maternal gene?

A

Morphogen with mRNA located anteriorly but also codes for a diffusible protein transcription factor which goes into the nuclei of the cells on the outside of the syncytium

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25
Q

Bicoid mutant? (-/-)

A

loss of anterior structures. Only forms 5 segments instead of the 7 in WT. The front two segments only form at high levels of bicoid which is absent. These other 5 segments are pushed forward as the concentration they form at is now more anterior.

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26
Q

If take cells from the most anterior tip containing bicoid to a bicoid mutant what happens? Control for this?

A

This partially rescues the mutant.
Controls: if etopically transplant from the posterior not rescued, or if transplant the anterior to the middle section ectopic head structures form in the thorax.

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27
Q

If have 4 gene copies of bicoid?

A

Still gives the 7 bands of patterning, but these are pushed posteriorly. If higher concentration created anteriorly then the designated conc for the segment will appear further posteriorly.

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28
Q

How does the bicoid transcription factor work anterior to posterior?

A

Anteriorly, the binding sites are weak , therefore lots of bicoid is needed in order to turn on expression of the target genes, whereas posteriorly they are stronger so low bicoid levels are needed.

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29
Q

How do the Gap genes work? (briefly)

A

The combinations of these present and absent, leads to te activation of the pair rule genes. They diffuse from a source through the embryo, but the boundaries are created as they have a short half life, so cant diffuse that far. Gives 7 stripes.

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30
Q

Example of the gap genes action?

A

e.g. the overlap of hunchback and bicoid give the 2nd Eve (pair rule) Gene expression. The A. giant band and Kruppel expression gives the boundaries to this stripe.

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31
Q

What genes cause the jump from 7 to 14 stripes?

A

pair rule genes cause the 14 stripes of the segment polarity genes.

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32
Q

What do the segment polarity genes do?

A

These pattern the segments, deciding whether they are to become naked cuticle or hary denticle stripes.

33
Q

How do the 7 stripes go to 14?

A

High levels of Eve or Ftz which are in the alternating 7 stripes (COME BACK TO)

34
Q

How do the 7 stripes go to 14?

A

High levels of either Eve or Ftz upregulates the expression of hedgehog. This is a secreted factor which causes the secretion of the Hedgehog protein into the most posterior cells of a segment. The diffusing hedgehog upreulates the expression of wingless in neighbouring cells. Where posteiorly the high levels of Eve and Ftz repress Wg expression, this is uregulated in the cells anterior to Hh.
Wingless codes for a secreted factor which upregulates Engrailed expression in the cell just posterior, which in turn upregulates the Hedgehog here further.
The result is that the cells most anterior of this segmention with intermediate levels of Eve or Ftz are free from singalling, but posteriorly wingless then hedgehog and engrailed are expressed.
The anterior, where there is no expression = denticles
Posterior, where there is= naked cuticle.
Each of the 7 stripes is then divided into Anterior and posterior, making 14 stripes.

35
Q

Time scale for the 7 to 14 stripes of the drosophila?

A

5-9hrs.

36
Q

Eve specifies the …. stripes, whereas ftz specifies the ….?

A

Even skipped: Odd (K/O skips the even stripes)

Ftz- even.

37
Q

relevence of para-segments?

A

Anterior of the parasegment=P of the segment e.g.where there is signalling of hedgehog.

Parasegments= the eve and Ftz stripes
Segments=naked cuticle to denticles.

38
Q

K/O of hedgehog?

A

Loss of naked cuticle covered in denticles- and shorter embryo.

39
Q

K/O of wingless?

A

Loss of naked cuticle covered in denticles.

40
Q

How does Hh and Wg lead to naked cuticle, and absent anterior denticles etc?

A

Selector genes: Turning on certain HOX genes, along the A/P axis. Occurs in the same order as in the genome e.g. the most anterior gene within the genome is the 1st.
e.g. Ubx, AbdA etc

41
Q

long bandvs short band insects?

A

Drosophila: long band insect, as all 14 segments are defined v quickly and complicated- all segments at the same time. embryogenesis within 24hours
Whereas short band: Can see segmentation pattern happening say starting with the head and thorax then abdominal segments bud off one by one from the Posterior disc.

42
Q

Short band insect mechanism?

A

e.g. Strigamia maritima
Segmentation controlled by Delta and Notch in adjacent stripes.
Activation of Notch by Her/Hes4 on one cells sets up feedback loop of oscillation, as this inhibits Delta in this cell, resuting in less activation of Notch in the next cell.
High Notch in cell 1 -> activates Her/Hes4 which represses delta ligand in cell 1 -I Notch in next cell 2 -I Her/Hes4 so more Delta to cell 3 ->causing high Notch in cell 3.

Causes bands of high, low, high, low etc

43
Q

Intermediate band insect example?

A

Beetles.

44
Q

What are imaginal discs?

A

Sheets of epidermal cells that are set aside during embryogenesis and grow thoughout larval life to form legs, wings, haltere, eyes, genitals, antennae etc during metamorphis.
These grow and evert into the adult strucutres during the pupal stage

45
Q

What are larval instars?

A

Stages between shedding (ecdysis), where the larva gets too big for the outer casing so sheds the casing and makes a new one.

46
Q

Imaginal disc growth example wing? Evidence?

A

Grows from 30-50,000 columnar epithelium by 3rd larval instar.

47
Q

Pupal, larval definition?

A

Larva- immature stage e.g. maggot
Pupa- undergoing metamorphis
Adult- after metamorphis- mature stage e.g. fly

48
Q

Eye imaginal disc growth?

A

Evert (turn inside out) and bulge from the diencephalon to increase size by 8 times.

49
Q

Pax 6 drosophila homologue? Mutant?

A

Eyeless

-/- = eyeless

50
Q

What is Pax6?

A

A homeobox transcription factor that acts back to the nucleus to activate expression of hox genes. Drosophila homologue is Wingless.

51
Q

Human Pax6 inserted into the Drosophila?

A

At 16 cell stage

Extra eyes over organism

52
Q

What experiment shows how conserved PAX6 is?

A

Insert mouse Pax6 ectopic expression- form ommatidia eyes.

53
Q

Pax6/eyeless necessary or sufficient?

A

Both

54
Q

Drosophila other genes required for eye development?

A 
Eyeless
Eyesgone
Optix
Teashirt
Dachshund
Sine oculis
55
Q

O word denoting the fucntional uni structure of the Drosophila eye? How many do they have?

A

Ommatidia- have 800 per eye. Are the hexagonal shaped structures that tesselate together to form the compound eyes.

56
Q

How many photoreceptors are in each o-structure of the eye?

A

Ommatidia- 8 photoreceptors (R1-8)

57
Q

Cells in an Ommatidia?

A 
8 photoreceptors (R1-8)
4 cone cells
2 primary pigment cells
6 secondary pigment cells
3 tertiary pigment cells
(most also a bristle, neuron, sheath cells and a socket)
58
Q

Cone cells in drosophila eye do what developmentally?

A

Secretes the overlaying lens

59
Q

Location of the cells in the ommatidia in the most anterior cross-section?

A

4 cone cells in centre, with pigment around. 3 bristles on the corners, 3 tertiary pigment cells on other 3, with secondary making the hexagonal edges.

60
Q

Function of the red pigment granules around the cone cells?

A

Insulates each eye unit so that is only recieves light from straight ahead and not light that is refracted from other angles.

61
Q

Pigment cells mutant in eye?

A

White eye, very poor vision as see refracted light

62
Q

R1-8 stands for?

A

rhabdomere 1-8 thin photoreceptor cells.

63
Q

Struture of the overal drosophila eye?

A

Compound eye of a Neurocrystalline lattice, with a mirror image of ommitidia either side of the equator dividing it into dorsal and ventral.

64
Q

Advantage of the equator reflection of the drosophila eye?

A

May allow a higher density of photoreceptors in the centre of the eye between the ground and sky boundary

65
Q

What is the structure of a rhabdomere?

A

Microvilli packed with rhodopsin

66
Q

How important is vision for the fly?

A

Very 2/3 of fly brain is visual processing

67
Q

How does the drosophila eye connect to their brain?

A

Connect to the brain via the optic stalk
R1-6 project into the lamina
R7.8- project into the medulla

68
Q

Arrangement of rhabdomeres in the ommatidium?

A

R7 is in the centre until half way down the ommatidium, where R8 is then found. The other 6 Rhabdomeres are arranged around these.

69
Q

How is the centre R8 cell singled out?

A

Lateral inhibition- one outcompetes the others and inhibits those surrounding.

70
Q

How is the ommatidium formed?

A
  1. Eye Imaginal disc growth x8 during larval life
  2. Photoreceptors connect to actin rich morphogenic furrow which forms.
  3. The first mitotic wave is just ahead of the furrow.
  4. Furrow moves posterior to anterior across the disc (2hrs per ommatidia row). G1- cell cycle arrested in the furrow.leaving behind photoreceptors in its wake.
  5. Stepwise addition of the photorectors by lateral inhibition (R8 first).
  6. R8 cells recruit additional (20) cluster cells (2 hrs per cluster row), forming the ommatidium.
71
Q

How are more cells recruited to the preclusters of ommatidium?

A

Preclusters-> R8 selected, then R5 and R2, then R4, R3, then R1 and R6 last.
R8, R5 and R2, secrete the EGF (epithelial growth factor) ligand Spitz, to EGF receptors on neighbouring cells, leading to the recuitment of R4 and R3 then R1 and R6.
R7 is last.

72
Q

Mutants lacking R7?

A

Cannot see UV light

73
Q

Flies and Uv light?

A

Normally flies are Phototactic so move towards/away from light.
Without the R7 the flies go to the white light over UV.

74
Q

How do you experimentally get a R7 mutant? Genes?

A

Mutant for Boss or Sev

75
Q

Experiment to show is function of Boss/sev is necessary?

A

Make mutant cells for Boss/Sev and allow these to proliferate
WIl have a gradient of some areas recruited cells purely from these mutated, whereas others partially and not at all.
Can work out the boundary of where can see etc to work out which are required.

76
Q

Sev/Boss necessary/sufficient?

A

Both can be removed from the outer photoreceptors without losign R7.
Sev is required for R7
Boss required for R8

77
Q

Structure of Sev?

A

Sevenless- RTK to Boss, activates the Ras pathway, expressed in all cone cells and R photoreceptors except R2

78
Q

Structure of Boss?

A

Bride of Sevenless- A membrane tethered ligand that is specific for Sev, expressed only in R8.

79
Q

Signalling cascade of Boss/sev?

A

R8 Boss -> binds to Sev RTK -> Ras-GEF activates Ras-GDP making it Ras-GTP -> downstream signalling in R7.

BMS 109 (20 decks)

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