What does the development of an organism require?
- regulation of organ size/growth
By what are developmental programmes dictated?
- a cell’s history (intrinsic mechanisms)
- (extrinsic mechanisms) interactions with the cellular environment including cell-cell interactions and hormones
How have drosophila been a useful tool?
Useful to find genes involved in development, but in particular genes involved in cell growth or cell death
How did advanced Drosophila genetics create a novel screen design for growth control genes?
- drosophila FRT-mediated mitotic recombination
- 3 types of tissue: wildtype, heterozygous, mutant
- heterozygotic mutant/+ parent cell
- low level heat shock with hs-FLPase
- exchange in small % of cells
- mitosis
- homozygotic daughter cells
- site specific recombination events
- basically a way of generating embryos that have all three types of tissue
- mosaics
- mutant tissue in the context of wildtype tissue
What are ‘loser’ cells?
- genetic mutations cause cells to grow slowly i.e. ‘loser’ cells
- in adult tissues you never see mutant clones –> losers
- higher fitness, less fitness
What will mutations in genes that restrict cell growth allow?
- over-representation of mutant tissue
- induce clones during mitosis using FLP/FRT system in the eye-antennal disc that result in mutant cells that outcompete loser cells
- insightful screen
What did the FLP/FRT ‘loser’ genetic screen identify?
- identified genes that regulate organ size
- Hpo pathway
- genes that were required to repress or negatively regulate cell growth
What is the SWH growth pathway?
- identified by the screen
- Salvador/Warts/Hpo
- salvador: mutants had larger eyes, also other tissues e.g. notum, wing, leg
- hpo: clones overgrow, head capsule, eyes, nodum
- these genes are conserved in mammals: Hpo = Mst1/Mst2 (2 hpo genes in mice/mammals)
- discovery of pathway that regulates organ growth in drosophila but also mammals, zebra fish, mice etc (and humans)
How does the SWH pathway regulate organ size?
- via inhibiting normal patterns of cell death
- increased number of inter-ommitidia cells in mutants, disrupting patterns of the eye
- loss-of-function clones of SWH pathway genes inhibit cell death by increasing dIAP1 expression
- loss of function clones of SWH pathway genes increase cell proliferation by increasing cycE expression
- two things happening in mutant cells:
- inhibition of cell death
- upregulation of cell proliferation
What do the core components of the SWH pathway form?
- a kinase cascade
- Hpo and Salvador (Sav) physically interact
- Warts (Wts) and Mob as tumour suppressor (Mats) physically interact
What kind of protein is Hpo?
- serine threonine kinase
- binds salvador
What kind of protein is Wts?
- kinase
What are key regulatory mechanisms of controlling this pathway?
- phosphorylated Wts
- phosphorylated Hpo
What is Yki?
- Wts phosphorylates co transcription activator Yki and excludes Yki from the nucleus
- phosphorylation of Yki creates a binding site niche for 14-3-3 proteins to bind
- these proteins exclude Yki from the nucleus
- unphosphorylated Yki enters the nucleus and activates target gene expression e.g. cycE, dIAP1
- Yki is a co-transcriptional activate
- binds Sd which is the DNA binding protein
- so the whole idea of the pathway is to maintain Wts in a phosphorylated state so that it can donate a phosphate group to Yki
- this phosphorylated Yki is excluded from nucleus from binding 14-3-3 protein, remains in the cytoplasm
- when the pathway is inactivated –> Yki enters nucleus –> activates genes involved in cell growth (e.g. cycE) or downregulates apoptosis (dIAP1)
What is apico-basal cell polarity?
gives cells a top and bottom
cells are highly structured
joined together through adherens junctions (in drosophila through septate junctions)
- all proteins involved in regulating wts are in the apical part of the cell
What are upstream regulators of the Hpo pathway?
- SWH pathway is regulated by apically localised cadherin (Fat) and members of the FERM family of proteins Kibra, Ex and Merlin (KEM)
- Fat stabilises Wts protein levels therefore pYki levels are maintained
- KEM complex - unknown, involved in negatively regulating the pathway
- Fat is a cadherin protein - transmembrane
- involved in the regulation of Dachs –> stabilises wts
- in the absence of Fat you get tissue overgrowth
What are Fat and Dachsous?
- atypical cadherins
- upstream regulators of the Hpo pathway
- Fat has a number of cadherin repeats on the extracellular domain, EGF repeats, Laminin repeats, intracellular region involved in the regulation of Dachs
- Fat is also involved in cell-cell interactions
- Fat binds a second cadherin protein called Dachsous (Ds)
- Ds is also a cadherin protein that has a number of repeats
- doesn’t have the EGF/Laminin repeats
- also has an intracellular domain
What is the Dachsous pathway upstream of Hpo?
- Fat and Ds bind together in the extracellular portion of the cellular space
- Fat binds Dachs and affects the stability of Wts
- Ds binds a kinase complex Riquiqui and Minibrain that phosphorylates Wts
- subsequently leading to the phosphorylation of Yki and downregulation of genes required for growth e.g. cycE and diap1
- adherens system
- Wts and its phosphorylation status is actually regulated by a number of cellular inputs
What are the inputs on Wts?
- FERM domain proteins
- KEM complex
- Fat and Dachsous
- senses whether cells are tightly packed or loosely adhering to each other
- regulates activity of pathway
- when fat and ds aren’t engaged proliferation is encouraged, but once there’s enough cells within the tissue space, Fat and Ds find and interact with each other to inhibit further growth
speculative
Is the SWH pathway conserved in mammals?
- yes
- advent in technology has allowed a short turnover in discovering homologs of drosophila proteins in mammals, and how these pathways work in mammals
- all proteins have conserved homologs in mammals –> alludes to the fact that this is a core, conserved pathway
YAP/TAS = Yki
TEAD = Sd
What did the genetic screen in Drosophila identify?
- genes that inhibit growth
- some of these genes are components of the SWH pathway
What does the SWH pathway control?
- organ growth by coordinately regulating cell proliferation and cell death
- genetics and biochemistry have established the core SWH pathway
- core components of the SWH pathway are conserved between flies and mammals
What controls organ size?
Extrinsic mechanisms
- nutrient status
- hormones
Intrinsic mechanisms
- cell proliferation
- cell death
- cell size
- protein synthesis
how are these processes integrated? What is the role of the SWH pathway in this?
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