lecture 24

Question 1

What are cell adhesion molecules?

Answer 1

• cell adhesion is critical in morphogenesis
• for the formation and maintenance of cellular structures (e.g. epithelia)
• for the ability of cells to move with respect to their environment (e.g. cell migration and axon guidance)

Cell-cell adhesion: e.g. E-cadherin—E-cadherin in adherens junctions
Cell-ECM adhesion: e.g. integrin to laminin

Question 2

What is the role of the cytoskeleton in morphogenesis?

Answer 2

• regulation of the actin and microtubule cytoskeleton is crucial for many morphogenetic cell behaviours
• important for changing shape, movement etc

Question 3

What sort of control is required for cell migration?

Answer 3

• molecular control of cell migration is complex
• cell migration and other motile behaviours (e.g. axon guidance, branching, morphogenesis) involve highly complex regulation of many cellular processes such as cytoskeletal remodelling, receptor-ligand interactions, and vesicle trafficking

Question 4

What is Cell Migration at a Glance?

Answer 4

• cell has polarity: front and back/trailing and leading edge, definitely heading in one direction
• one thing migrating cells do is extend protrusions
• thin protrusions = filopodia
• broad, sheet-like protrusions = lamellipodia
• actin filaments sliding against each other under the influence of myosins
• focal adhesions controlled by integrins
• adhesion dyanmics
• integrin to actin cytoskeleton
• polarity regulated by complexes like CDC42, apical complex
• adhesions at the back need to be released, microtubules bring something that dissolves the adhesions

Question 5

What is regulation of actin polymerisation and contractility?

Answer 5

• the assembly and contraction of actin filaments
• crucial for many cell behaviours such as cytokinesis, cell shape changes, and cellular protrusions
• myosin II is found in muscle and these migrating cells/normal cells (e.g. cytokinesis)
• heads pull on actin filament to make them slide together
• Myosin light chain regulates whether the myosin is contractile or not
• myosin regulatory molecule is active when phosphorylated
• MLC kinase and MLC phosphatase regulate phosphorylation of light chain
• two main types of polymerisation
• molecules help a polymer add monomers to the growing end (even though they will also spontaneously grow)
• diaphenous
• Arp2/3 complex (actin related protein)
• binds to an existing filament and then nucleates a new filament that comes off at 70º exactly

1. contraction of networks of actin filaments is controlled by myosin II . myosin is comprised of a heavy chain (MHC) and a regulatory light chain (MLC)
2. myosin contractility depends on MLC phosphorylation, which is regulated by MLC kinase and MLC phosphatase
3. elongation of actin filaments is promoted by formins such as Diaphanous (mDia1) which bind to the end of actin filaments
4. formation of new branches is promoted by the Arp2/3 complex which binds to the side of actin filaments

Question 6

What are Rho small GTPases?

Answer 6

• regulate actin structures
• Rho GTPases are molecular switches that cycle between an inactive, GDP-bound form and an active, GTP-bound form
• when active they can bind, and activate downstream effector proteins
• they are activated by Rho GTP exchange factors (RhoGEFs)
• they are inactivated by Rho GTP Activating Proteins (RhoGAPs)
• different Rho GTPases control formation of different actin structures
  e. g. Cdc42-GTP = filopodia, Rac1-GTP = lamellipodia, RhoA-GTP = stress-fibres

Question 7

How do Rho small GTPases regulate the actin cytoskeleton?

Answer 7

three key GTPases are:

• RhoA: activates myosin contractility, via Rho kinase, which inhibits MLC phosphatase, and polymerisation through mDia
• Cdc42: stimulates polymerisation through mDia and formation of new branches via WASP, which activates the Arp2/3 complex
• Rac: stimulates new branches, via WAVE, which activates the Arp2/3 complex, and inhibits contraction through the kinase PAK

Question 8

What is genetic regulation of cell behaviour?

Answer 8

• cells have a large range of behaviours, but how are these behaviours controlled during development?
• answer: differential gene expression

Question 9

What is an example of genetic regulation of cell behaviour?

Answer 9

• Twist regulates mesodermal cell behaviour
• the Drosophila presumptive mesodermal cells express the transcription factor Twist
• during gastrulation these cells fold inwards (i.e. invaginate), undergo an EMT, and then migrate out over the ectoderm
• all of these behaviours are controlled by twist

Question 10

What does Twist do?

Answer 10

• transcription factor in presumptive mesodermal cells
• turns on 3 key genes (fog, snail and heartless)
• fog: initial furrowing, bending
• • myosin apical constriction
• snail: EMT
• • inhibits E-cadherin, epithelial cell-cell adhesion
• heartless: migration
• • FGF-receptor, motility

Question 11

What is Fog?

Answer 11

• target of Twist
• controls epithelial sheet folding
• normally myosin II accumulates on the apical side of cells
• in folded gastrulation (fog) mutants, myosin localisation is patchy, and invagination of the mesoderm fails

1. twist
2. fog
3. RhoGEF2
4. Rho1
5. Rho kinase
6. myosin localisation constriction

Question 12

How does Fog control myosin II localisation?

Answer 12

• secreted Fog is thought to bind to an unknown receptor which activates Rho1 which activates myosin contraction, leading to localisation of myosin filaments

Question 13

What is snail?

Answer 13

• target of twist
• controls loss of epithelial adhesion
• the EMT is too rapid (~15 mins) to be accounted for only by transcriptional mechanisms, so there are likely to be other Twist targets, which regulate the dissolution of existing adherens junction complexes
• snail is turned on by Twist in the ventral presumptive mesoderm
• E-cadherin normally repressed in the mesoderm
• repression depends on snail

Question 14

What is Heartless?

Answer 14

• twist target, aka FGFR
• regulates migration
• heartless is an FGF receptor tyrosine kinase expressed in the mesoderm
• its FGF ligands are expressed in the dorsolateral ectoderm under the control of the transcription factor Dorsal
• mesodermal cells expressing heartless migrate towards the source of the FGFs

Question 15

What are the molecular mechanisms acting downstream of Heartless?

Answer 15

• unknown
• in other situations, activation of receptor tyrosine kinases, like the FGF-receptor, can stimulate F-actin polymerisation via signalling pathways

other pathway

• Cdc42 activates WASP - affects polymerisation of actin
• RTK phosphorylated has PI3-Kinase bind to its tail, phosphorylates PIP2 –> PIP3, can be bound by GEF, GEF activates Cdc42
• theoretical

Question 16

What is a theoretical pathway from growth factor to F-actin?

Answer 16

1. activated FGF-receptor becomes phosphorylated
2. PI3 kinase binds to a phosphorylated FGF-receptor
3. PI3K converts to PIP2 (i.e. PtdIns(4,5)P2) to PIP3
4. GEF is recruited to membrane by binding to PIP3
5. GEF activates Cdc42, which activates WASP, which activates Arp2/3 complex, which stimulates formation of F-actin branches

Question 17

What is pattern formation?

Answer 17

• the process by which a spatial and temporal pattern of gene expression, which regulates cellular activities, is organised within the embryo so that a well ordered structure develops

Question 18

What happens when you get errors in pattern formation?

Answer 18

• can have severe consequences for the development of the organism
• e.g. misregulation of the ultrabithorax gene
• mutation of the HOXD13 gene

Question 19

What did Nusslein-Volhard and Wieschaus?

Answer 19

• in the 1970s
• large number of genes are likely to be involved in embryonic patterning
• they could find these genes by systematically screening mutants that are defective in embryonic patterning
• this ground-breaking study led to the 1995 nobel prize

Question 20

What are model organisms to study development?

Answer 20

• worm
• vinegar fly
• zebra fish
• clawed frog
• chick
• mouse

can’t use humans to study development

developmental genetics of model systems is relevant to human health
this hasn’t changed for 500 million years

Question 21

For what are molecules that control development often also crucial?

Answer 21

• e.g. twist plays similar roles in gastrulation and cancer
• cancer is deregulation of developmental programmes
• yang et al. Twist, a master regulator of morphogenesis, plays an essential role in tumour metastasis

Question 22

Why use Drosophila?

Answer 22

Practical considerations

• cheap and easy to cultivate (small)
• short life cycle (10 days)

Good developmental model

• embryo is easy to visualise
• development is very well characterised

good genetic system

• 4 chromosomes
• small genome (1/25th human size)
• genome sequence and annotated
• sophisticated genetic tools for manipulating gene expression
• easy to do genetic screens

Question 23

What is the life cycle of drosophila?

Answer 23

• embryonic developmnet 1 day
• larval stages, 4 days
• pupations and metamorphosis 4.5 days
• drosophila life cycle is very fast
• at 25ºC adults will hatch in 9.5 days

Question 24

What are early cleavage cell divisions in drosophila?

Answer 24

• the first 13 cell divisions are synchronous and occur in syncytium
• after the 13th cell cycle, the embryo undergoes cellularisation: syncytial blastoderm to cellular blastoderm
• ingrowing cell membrane around nuclei that pinches off
• big yolk in middle

Question 25

What is the product of embryogenesis?

Answer 25

- 1st instar larva | - clearly segmented

Question 26

What is seen in embryonic patterning mutants?

Answer 26

- generate larvae with corresponding cuticular defects - e.g. in fushi-tarazu mutant embryos alternate segments are missing - even-skipped - embryonic patterning defects manifest in the larval cuticle - this allows one to screen larval cuticles to find genes involved in embryonic development

Question 27

What are zygotic mutants?

Answer 27

- mutations in genes that are zygotically expressed | - the phenotype of the embryo is only determined by its own genotype

Question 28

What were the four classes of mutants identified in the initial screen?

Answer 28

1. Gap - several contiguous segments are missing along A-P axis 2. pair rule - alternating segments are missing 3. segment polarity - patterning within each segment is disrupted e.g. posterior half can be replaced by mirror image duplicate of anterior half 4. dorsoventral - loss/gain of denticle belts in the ventral/dorsal regions

Question 29

What are segment polarity mutants?

Answer 29

1. segment polarity - patterning within each segment is disrupted e.g. posterior half can be replaced by mirror-image duplicate of anterior half

Question 30

What was able to be seen in homozygous non-embryonic lethal mutants?

Answer 30

- this gave Nusslein-Volhard and Wieschaus an opportunity to do a maternal screen

Question 31

What are maternal effect mutants?

Answer 31

- maternal effect mutants are mutations in genes that are maternally expressed - the phenotype of the embryo is only affected by the genotype of the female - if she is homozygous for a mutation, the ALL of the progeny is affected

Question 32

What are larval segment specific structures?

Answer 32

- terminal structures (acron and telson) - anterior structures (head and thorax) - posterior structures (abdomen)

Question 33

What groups do maternal effect mutants fall into?

Answer 33

- Dorsal group: ventral side is dorsalised i.e. loss of denticles - ventral group: dorsal side is ventralised, i.e. gain of denticles - anterior group: loss of head/thorax structures - posterior group: loss of abdominal structures - terminal group: loss of terminal structures

Question 34

What are mutant phenotypes affecting anterior/posterior axis?

Answer 34

- anterior structures missing: anterior terminal structures are transformed into posterior terminal structures - posterior structures missing - terminal structures missing

Question 35

What were gap gene mutants arising from the screen?

Answer 35

- kruppel - giant - tailless - hunchback - knirps

Question 36

What were pair-rule gene mutants arising from the screen?

Answer 36

- even-skipped - odd-skipped - fushi-tarazu - runt - hairy

Question 37

What were segment-polarity gene mutants arising from the screen?

Answer 37

- engrailed - gooseberry - hedgehog - wingless

Question 38

What were ventralising gene mutants arising from the screen?

Answer 38

cactus

Question 39

What were dorsalising gene mutants arising from the screen?

Answer 39

- dorsal - pelle - tube - toll - nudel - spatzle - easter - snake - gastrulation defective - pipe

Question 40

What were anterior gene mutants arising from the screen?

Answer 40

- bicoid

Question 41

What were posterior gene mutants arising from the screen?

Answer 41

- oskar - vasa - tudor - nanos - pumilio

Question 42

What were terminal gene mutants arising from the screen?

Answer 42

- torso - torso-like - trunk - L-pole hole