Flashcards in lecture 25 Deck (31)
What are the major players in mechanisms of controlling morphogenetic processes?
- need the FGF-receptor
- Rho GTPase (e.g. Cdc42)
- Rho GTP exchange factor/Rho-GEF
What is the 'aim of the game' re: morphogenetic changes and FGF?
- actin polymerisation
What is the process of FGF signalling?
1. ligand binds to receptor - dimerise RTK
2. kinase domains phosphorylate each other
3. PI3 kinase binds to phosphorylated receptor
4. phosphorylates 3rd position on PIP2 --> PIP3
5. PIP3 is very important signalling molecule
6. Rho-GEF binds to PIP3
7. Exchanges GDP for GTP in Rho-GTPase
8. activated Cdc42 can now bind to WASP and activate it
9. WASP changes shape and binds to Arp2/3
10. Arp2/3 triggers polymerisation of new actin filament
What are examples of zygotic and maternal screens?
- eve-/+ male and eve -/+ female
- eve -/- embryo lethal
- every second segment skipped
- signal comes from within the embryo
- female -/-, male +/+
- embryo lethal -/+
- no abdominal segments
- nanos produced by the mother in the egg - concentrates at one end of the egg
- without this the embryo can't form properly even if it has a copy of the gene
What are the classes of zygotic effect genes?
- gap gene (missing contiguous segments)
- pair-rule (loss of alternate segments)
- segment-polarity (loss of part of segment)
- ventralising (i.e. denticles in dorsal areas)
- dorsalising (i.e. ventral areas bare)
What are classes of maternal effect genes?
- anterior (loss of anterior segments)
- posterior (loss of abdominal segments)
- terminal (loss of acron/telson)
What do we need to know to understand how mutations to these genes create the specific phenotypes?
- where they are expressed
Where are segmentation gene products expressed?
- in the same regions of the embryo that are affected in the mutants
- maternal gene product Bicoid is expressed in anterior half --> mutant bicoid results in complete loss of anterior structures
- Gap gene products Hunchback, Kruppel are expressed in broad regions --> mutant had large segments missing from the embryo, kruppel expressed in middle, mutants had middle segments missing
- pair-rule gene products Even-skipped (Eve) and Fushi tarazu (Ftz) are expressed in alternate segments
- segment polarity gene product: engrailed found in fourteen stripes in the gastrulating embryo
- egg polarity genes, gap genes and pair-rule genes combine to control the expression of homeotic genes
- when these genes are mutated, segment identity is disrupted
What are morphogens?
- puzzle - how to generate this pattern regardless of size
- morphogen: a substance with a varying concentration that controls pattern formation
- the fate of cells depends on the concentration of the morphogen that they are exposed to
- modifications to the morphogen gradient will affect the pattern of differentiated cells
- elevating the gradient --> i.e. increasing the levels of the morphogen movies the pattern to the right
- moving the gradient so that the high point is at the centre will create a mirrored pattern
What happens if the Bicoid dose is changed?
- dose affects position of anterior structures
- the position of the cephalic furrow depends on the levels of bicoid
- increasing bicoid dose moves the furrow posteriorly
- decreasing the dose moves it anteriorly
- when wildtype anterior cytoplasm is injected into the middle of a bicoid mutant embryo, the embryo is repatterned with anterior structures in the middle, and flanked by mirrored thoracic segments
How is bicoid protein expressed?
- as a concentration gradient
- bicoid mRNA is provided maternally and is localised to the anterior end of the embryo
- when bicoid mRNA is translated, Bicoid protein diffuses to form a concentration gradient
What is something does bicoid regulate?
- hunchback transcription
- hunchback: a zygotic mutant with a similar phenotype to bicoid
- analysis of hunchback promoter reveals bicoid binding sites
- hunchback transcription is regulated by bicoid by is only turned on when a certain threshold concentration is present
- increasing the bicoid concentration increases the domain of hunchback expression
What does nanos do?
- controls posterior cell fates
- nanos mRNA is localised to posterior pole
- zygotic translation produces a concentration of nanos protein
- maternal effect gene
- sharply stuck to posterior part of the gene
- nanos regulates hunchback translation
- maternally provided hunchback mRNA is uniformly expressed in the embryo
- nanos acts by repressing hunchback translation in the posterior of the embryo
- maternal contribution of hunchback: mRNA distributed evenly throughout the egg
- if maternal hunchback is removed, nanos is not necessary - evolution doesn't always result in the most efficient solution
In summary, how is hunchback regulated?
- hunchback transcription is activated by Bicoid
- hunchback translation is inhibited by Nanos
How does bicoid also promote anterior fate?
- repressing posterior development
- caudal is important for development of posterior structures
- bicoid represses translation of Caudal protein, producing a gradient of Caudal and preventing posterior structures from developing in the head
What is the symmetrical translational regulation of caudal and hunchback?
- sharp anterior bicoid mRNA
- shap posterior nanos mRNA
- hunchback and caudal mRNA throughout
Early cleavage embryo proteins
- bicoid - anterior to posterior gradient
- nanos posterior to anterior gradient
- hunchback expressed above a certain threshold of bicoid/inhibited at a certain level of nanos
- caudal repressed above a certain level of bicoid
bicoid mRNA --> Bicoid protein --> inhibits translation of caudal mRNA
- nanos mRNA --> nanos protein --> inhibition of hunchback mRNA translation
at this point we have establish the anterior-posterior axis
How can a morphogen gradient generate evenly repeating patterns?
- not a linear concentration gradient of morphogen
- how can the cells at the low concentration end distinguish between very low levels of morphogen?
theory: repeating patterns can be established with reaction-diffusion systems, a beautiful concept - not right
- segmentation is actually achieved by a hierarchical network of gene regulation
What is the hierarchical network of gene regulation?
- gene products at each level can regulate other genes at the same level, AND/OR genes at the next level down
- how a gap genes regulated?
- represses giant
- activates eve
- activates runt
- represses hairy
- represses knirps
- intermediate levels activate kruppel (low or high levels won't)
What do intermediate levels of Hb regulate?
- activate Kr expression
- Kruppel, gap gene normally expressed in the middle of the embryo
- controlled by hunchback
- only turns it on when it is at an intermediate level of concentration
- high levels repress
- in a bicoid mutant (bicoid being the activator of hunchback), we only have the low level of Hb mRNA from the maternal egg so you get kruppel expression all throughout the anterior section
What would happen in a nanos mutant (i.e. where hunchback translation isn't repressed)?
The intermediate levels of Hunchback in the posterior part of the embryo activate Kruppel
How are gap genes regulated?
- they regulate each other
- e.g. Kruppel is repressed in anterior regions by Giant and Hunchback, and in posterior regions by Knirps
What are segments versus parasegments?
- expression of some pair rule genes (e.g. ftz) occurs in regions that are termed parasegments
- these are offset from the segments of the later embryo and adult
- parasegments are comprised of the posterior part of one segment, and the anterior part of the segment behind it
What are pair-rule genes? What is their expression?
- pair-rule genes are expressed in 7 stripes of alternating parasegments
- e.g. alternating stripes of Eve protein and Ftz protein
- 2 classes of pair-rule genes:
-- 1º pair-rule gene expression is directly controlled by gap gene products and by other 1º pair-rule proteins (eve, hairy, and runt)
-- 2º pair-rule gene expression is regulated by 1º pair-rule
- mutations in 1º pair-rule genes affect both 1º pair-rule genes affect both 1º and 2º pair-rule gene expression
- mutation of ftz, a 2º pair-rule gene, does not affect expression of 1º pair-rule genes
What is death of a beautiful concept?
- eve stripe expression is modular
- enhancers for stripes can be separated as seen with lacZ reporter constructs
- every stripe is being individually controlled
What is regulation of eve stripe 2?
- eve stripe 2 enhancer element has binding sites for Bicoid, hunchback, giant and Krüppel
- bicoid and hunchback activate expression in a braod domain
- giant and krüppel repress eve, thereby defining the anterior and posterior limits of expression
What are segment polarity genes? How are they expressed?
- segment polarity genes are expressed in 14 stripes, one in each parasegment, usually 1-2 cells wide
- segment polarity gene expression is initiated by pair-rule proteins, and then maintained by segment polarity proteins
- segment polarity genes reinforce parasegmental periodicity
- segment polarity genes also establish different cell fates within the parasegment
How is segment polarity gene expression initiated?
- two important segment polarity genes are wingless (wg) and engrailed (en)
- en expression is activated by high concentrations of Ftz or Eve and repressed by other pair-rule genes to create en expression in a set of 14 stripes of cells, each stripe initially being precisely one cell wide - only anterior part of parasegment
- wg expression is activated where Ftz and Eve are absent - i.e. only posterior part of parasegment
How are genes regulated in the syncytial blastoderm?
- the bicoid/nanos, gap and pair-rule genes can interact directly since the nuclei that transcribe them are not separated by cell membranes
- this is because the embryo is a syncytial blastoderm
- the pair-rule genes can activate the segment polarity genes directly but, after that, due to cellularisation of the blastoderm, the segment polarity genes interact via signal transduction
How is expression of segment polarity genes maintained?
- the initial expression of engrailed and wingless is maintained by a positive feedback signalling loop
- wg turns on engrailed
- hedgehog transcription initiated by wg
- receptor for hedgehog is Patched
- signalling cascade downstream from patched initiates transcription of wg
How do morphogen gradients pattern the segments?
- gradients of wingless and hedgehog proteins specify cell fate within the segment
- thought these segment polarity genes help determine presence of denticles
- particular concentrations of wg and hedgehog determine cell fate
- mutations to the hedgehog or wingless pathways disrupts the pattern