lecture 26

Question 1

What are the aims of these lectures?

Answer 1

• focus on drosophila melanogaster as a model organism for the study of mechanisms that regulate organ and/or body size
• extrinsic hormone/peptide circuits within the whole animal that regulate organ size
• intrinsic systems (salvador/warts/Hpo signal transduction pathway) that regulate organ growth (imaginal discs)

Question 2

What experiments revealed that there are both extrinsic and intrinsic factor that regulate organ size?

Answer 2

• 1930s that peoples started to think about the regulation of organ size
• two types of experiments done
  1. if you starve an animal during development, it will be smaller than its nutritionally supplied/well-fed counterparts. Same for mammals: if you have mouse that have been nutritionally deprived, their progeny will be smaller. Nutrition has a profound influence on body size. This is an example of an extrinsic factor.

2. intrinsic factors: simple experiment by Twitty and Schwind, took the limb from a larger embryo and grafted it onto a smaller embryo (axolotl). As they developed, the grafted limbs maintained the body size of the original animal from that it came. Therefore there must be systems within organs/body tissues/organisms that regulate organ size.

Question 3

What are examples of extrinsic factors that regulate organ growth?

Answer 3

• include nutrition and hormones
  A. environmental regulation of body size: increasing temperature, smaller, increasing protein, bigger
  B. physiological regulation of body size: hormones,
  C. genetic regulation of body size: male or female (female much larger)
  D. coordination of organ growth: organ systems crosstalk so that the brain doesn’t outgrow/pace heart, limbs etc

Question 4

What is the difference between extrinsic and intrinsic factors that control organ size?

Answer 4

Extrinsic

• hormones and nutrient status (glucose, fats, amino acids, ecdysone, insulin)
• cell-cell and organ-organ communication

Intrinsic
- genetic programmes within the cell that are unaffected by neighbouring cells e.g. salvador/warts/Hpo pathway

Question 5

What is the drosophila life cycle?

Answer 5

• egg - 24 hours
• 1st instar larva
• 2nd instar larva
• 3rd instar larva
• pupa
• adult
• life cycle takes 10 days –> fantastic for studying in a laboratory
• cheap compared to mouse studies
• at end of each larval stage cuticle is malted
• all the energy and nutrients that are going to make the adult are present in the pupal stage - no more feeding, climbs up wall

Question 6

What is nutrient dependent growth control in Drosophila?

Answer 6

• Minimum viable weight: threshold minimum weight to survive metamorphosis or eclose to adult when starved
• critical weight: minimal size at which starvation no longer delays metamorphosis
• larvae feed during larval stage 2, then they undergo a transition from larval stage 2 to larval stage 3, larvae senses that it is fed enough and that there is enough energy stored to allow the larvae to pupate, and undergo metamorphosis
• unique biological system
• these terms allude to the fact that in the drosophila there is this critical period during which the animal knows through sensing systems that it is time to undergo metamorphosis
• if you starve the animal you get smaller adults because you haven’t reached the minimal viable weight

Question 7

What is hormone dependent growth control in Drosophila?

Answer 7

• Critical weight - insulin like peptides are released from the Fat Body and Imaginal Discs
• Ecdysone released from the Ring Gland
• larvae feed on sugars
• this signals in the fat body (liver equivalent) and the brain (ring gland/neurosecretory cells)
• as larvae feeds, fat body has the ability to detect amount of amino acids, sugars, fat in the body
• fat body releases insulin like peptides that enter the circulatory system and are eventually detected by both the neurosecretory cells (also secreting insulin like peptides) and the ring gland
• when critical balance of secreted insulin like peptides is reached, the ring gland releases a hormone called ecdysone
• ecdysone travels throughout the animal via the circulatory system
• tells the animal to stop feeding and begin the process of morphogenesis
• crosstalk between organs can thereby regulate developmental timing etc, when it should stop feeding
• if imaginal discs are undeveloped (release insulin like proteins), then development will be delayed

Question 8

What is the ring gland?

Answer 8

• sits between the lobes of the adult brain
• releases ecdysone
• prothoracic gland and corpus allutum

Question 9

How is the ring gland important for organ size?

Answer 9

A) wildtype ring gland
B) small ring gland
C) pupae from larvae with reduced ring gland size
- starved larvae
- non-starved but reduced larval ring gland size = bigger pupae

• decreasing ring gland size, increase time to pupation, increase organ and body size

Question 10

What do insulin like peptides in the wing disc regulate?

Answer 10

• growth and developmental timing
• dILP8 secreted by wing discs and regulates organ size
• eight insulin like peptides in drosophila
• imaginal discs express ILP - unexpected, also wing disc, fat body (salivary gland doesn’t express ILP)
• eyeful - eye disc keeps growing, never mutate
• expression of ILP8 is dramatically higher in eyeful mutants
• this suggests that secretion of ILPs from the imaginal discs is also feeding back onto the ring gland and affecting the secretion of ecdysone
• what happens then is that in the developing larvae, there are a number of organs, fat body, neurosecretory cells in the brain, imaginal discs and fat body that are all secreting ILPs, (and the ring gland)
• all those ILPs are ultimately sensed by the ring gland
• searching for equivalent system in mammalian embryo
• coordinated growth of insulin like peptides

Question 11

What is the insulin signalling pathway?

Answer 11

• small secreted proteins (30kDa)
• enter the circulatory system
• bind the insulin receptor
• through a series of kinases and phosphatases they ultimately regulate a transcription factor called FOXO
• FOXO goes into the nucleus and binds a number of gene promoters where it will regulate the transcription of target genes
• genes involved in the regulation of protein synthesis, degradation and cell death
• FOXOs part of sensing amino acids and glucose
• regulate organ size

Question 12

What two hormone systems within Drosophila larvae signal between organs to regulate growth?

Answer 12

• insulin-like-peptides (ILPs) in wing discs and fat body

- ILPs signal to Ring Gland to secrete Ecdysone to initiate morphogenesis

Question 13

What are the molecular mechanisms that control organ size?

Answer 13

Extrinsic

• energy production/nutrient status
• • insulin/PI3 kinase pathway (Akt, Dp110)
• hormones (Ecdysone)

Intrinsic

• cell proliferation
• • cycle regulators e.g. cyclin E (cyc E), Retinoblastoma (Rb)
• Apoptosis/Cell death (reaper (rpr), sickle (skl), Drosophila inhibitor of apoptosis (diap1)
• Cell size
• • myc
• protein synthesis
• • minutes (M)
• • myc

Question 14

What are drosophila imaginal discs?

Answer 14

• precursors of adult organs
• imaginal discs are comprised of epithelial cells
• imaginal discs are the cornerstone of genetic analysis of organ size

Question 15

How do drosophila wing discs develop?

Answer 15

• drosophila wing disc has an excellent cell fate map
• in the ~30,000 cell wing disc, fates of almost all are known
• cells ‘in here’ will form the notum = sort of shoulder of drosophila
• pouch cells will form adult wing blade
• certain cells will go on to form vein tissue
• drosophila wing disc has known rates of cell proliferation
• some cells are actively dividing and some are dividing less e.g. wing pouch vs notum
• cells in hinge region don’t divide a great deal
• cells in pouch region undergo a great deal of cell division
• developing imaginal discs align within the larvae, within pupal case
• to form adult structures have to undergo a huge amount of cell migration

Question 16

How do drosophila eye-antennal discs develop?

Answer 16

• eye made up of ommatidia and bristle cells –> precise organ organisation
• stems back to organisation in the larvae
• drosophila eye disc has an excellent cell fate map
• posterior to the morphogenetic furrow all cells are fully differentiated –> these are the cells that will contribute to the adult eye and the ommatidia
• photoreceptors etc are present here
• drosophila eye disc cell fate is coupled to cell proliferation
• • very very coordinated process
• when undifferentiated cells divide at random
• apical constriction –> arising the morphogenetic furrow
• when cells enter the morphogenetic furrow they cease to divide - G1 morphogenetic arrest
• begin to differentiate
• proliferation tightly coupled to division
• after differentiated will only undergo one more division
• drosophila has a precise pattern of cell division

Question 17

What is the patterning of the eye/ommatidia?

Answer 17

• cone cells in the centre
• underneath which sit photoreceptors
• surrounded by pigment cells
• hexagaonally shaped
• surrounded by insulating cells that allow for discrete neural circuits
• bristle cells in corners
• precise pattern

Question 18

How is drosophila eye disc cell fate coupled to cell death?

Answer 18

• drosophila pupal eye disc has a precise pattern of cell death
• e.g. hpo mutant
• these cells don’t undergo apoptosis in the development of the eye
• precise pattern for cell death
• doesn’t happen in the wing disc
• normally eye disc cells die so there aren’t any extra cells to disrupt the pattern

Question 19

What are key learning points?

Answer 19

• development is an integrated process that incorporates pattern formation, morphogens and organ size control
• body/organ size control is regulated by extrinsic (nutrients and hormones) mechanisms and intrinsic mechanisms (SWH pathway)