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What are the aims of these lectures?

- 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)


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

- 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.


What are examples of extrinsic factors that regulate organ growth?

- 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


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

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

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


What is the drosophila life cycle?

- 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


What is nutrient dependent growth control in Drosophila?

- 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


What is hormone dependent growth control in Drosophila?

- 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


What is the ring gland?

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


How is the ring gland important for organ size?

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


What do insulin like peptides in the wing disc regulate?

- 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


What is the insulin signalling pathway?

- 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


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

- insulin-like-peptides (ILPs) in wing discs and fat body
- ILPs signal to Ring Gland to secrete Ecdysone to initiate morphogenesis


What are the molecular mechanisms that control organ size?

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

- 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


What are drosophila imaginal discs?

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


How do drosophila wing discs develop?

- 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


How do drosophila eye-antennal discs develop?

- 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


What is the patterning of the eye/ommatidia?

- 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


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

- 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


What are key learning points?

- 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)