Ryan Lecture 3 Flashcards
What does cell need to interact with signal
Receptor and machinery to interact with signal
What also effects the way morphogens move
Type of environment
Ex -if thicker = more ecm =harder
Or aqueous = easy
Describe ex of morphogen = activin - gen
Tfg beta fam
Did experiment
Describe ex of morphogen = activin - actual experimental set up
Co culture of animal cap +vegetal part of xenopus blastula
Put activin mRNA into vegetal part - below dotted line
And monitor brachury mRNA expression - Xbra (TF)
Describe ex of morphogen = activin - actual experimental generally facts
Blastula stage
Take pigmented half embryo and separate endoderm from other half = top and bottom
Inject with fluorescent dye
Inject vegetal part with activin mRNA and monitor and look for xbra expression
Describe ex of morphogen = activin results
As activin dose increases distance = further cells turning on genes
Low dose = turn on
Distance of cells turn in on xbra = gets further and further away
THEREFORE = the higher the dose of activin = the further away xbra gene expression is turned on
Describe ex of morphogen = brachyury and goosecoid
Brachyury = further away from activin = expressed, threshold dose = low number of activin/receptors occupied (100)
Goosecoid = turns on with higher levels activin, threshold dose= high number of activin receptors occupied (300)
What is a morphogen
Signalling molecule
Diff responses at diff concentrations
What is a short range inducer
Initiates a sequential chain of secondary signals
Sorting too = each cell has to take it in and determine response and pass it on
Describe a way to test if ligand is a morphogen that DOES NOT WORK
Usually produces a gradient of effects - but morphogens and short range inducers can give same phenotypes
IMPOSSIBLE TO DETERMINE IF A LIGAND IS A MORPHOGEN OR A SHORT RANGE INDUCER BY ADDING IT
Describe an experiment to test if ligand is morphogen
Test by removing intermediate cells
Exp = too cells that cannot respond to sign and put them in, AC sandwich separated by endoderm cells that cannot express brachyury
Describe an experiment to test if ligand is morphogen - the experiment + results = 3
No activin mRNA in lower half = no brachyury expression
Activin mRNA in lower half =brachyury expressed in upper ac
Endoderm cells + cyclohexamide, activin mRNA in lower half = brachyury expression in upper ac (cyclohexamide blocks mRNA translation, so cells do not activate)
Describe an experiment to test if ligand is morphogen = conclusions
Short range inducers cannot affect cells - if remove intermediate cells
Name and describe another way to test if ligand is a morphogen
Test by adding activated receptor
If morphogen = only see in cells its in
If ligand short range inducer = can still see signals
So no longer have a cell producing ligand, just insert an already activated receptors = so for short range inducers (cell with activated receptor will continue to transform signal and pass it on)
Describe d-v patterning in xenopus - gen
Starts at one cell stage xenopus oocyte is radially symmetrical
Animal and vegetal poles phenotypically distinct
Animal pole darkly pigmented
Describe d-v patterning in xenopus - actual
Dorsal organizer formed opposite site of sperm entry - where sperm hits animal cap
Initiates axis,- dorsalizing activity = dorsal side embryo, always forms from cells opposite to where sperm entered
NEXT = stabilize B cat *wnt
Then forms dorsal organizer - which then goes onto form spemann mangold organizer
When does d-v axis formation start in xenopus
With fertilization - 1 hr post fert
Sperm can enter anywhere in animal pole
Dorsal site always forms opposite sperm entry
Fertilization also restores diploid genome
What does sperm entry do - xenopus d-v
Promotes cortical rotation = movement of pigmented cells
Center does not move - inner core yolk mass stays the same
Shift of cortex within 1 hour
Describe fertilization of xenopus oocytes
Animal pole has pigmented surface and contains nucleus
Vegetal Pole not pigmented and is mostly yolk
Egg contains sufficient mRNAs and protein for cleavage phase = enough stored his tones for >10 000 nuclei
What causes cortical rotation and translocation of dorsalizing activity in xenopus
Microtubules = causes cortical rotation
Translocation = caused by cortical rotation
Describe microtubules - xenopus
Sperm centriole acts as microtubule organizing Center - MTOC minus end
Cortex and dorsalizing activity move towards plus end of microtubles
Microtubules that Emirate from Center —> close to periphery of oocyte and acts with cortical rotation to move dorsalizing activity
Microtubules form in shear zone - cortex, outer ring, after fert but before 1st cleavage, see strands of Microtubules - at 0.7 normalized time
Describe precisely what cortical rotation in xenopus does
Translocates dorsalizing activity from vegetal pole to future dorsal side
Cortical rotation includes plasma membrane, cytoskeleton components and ER
Molecules that move along Microtubules and end up at opposite side sperm entered
Does NOT INCLUDE CORE CYTOPLASM
What does not rotate during cortical rotation
Core cytoplasm doe snot rotate
Only vegetal zone, actively transported along microtubule
Describe exp = block microtubule polymerization - xenopus
Result = embryo is ventralized, d-v axis does not develop
Looks like blob
Since dorsal organizer does not form
Block by UV treatment = no dorsal structures forming
What is the dorsalizing activity in xenopus
Key molecule = beta cat, stabilized in dorsal blastomeres, has to get into nucleus
B CAT is stabilized when wnt ligand binds to their receptors
Where does wnt signal come form - xenopus
Maternally deposited wnt 11 mrna is important for dorsalizing activity - b cat stabilization
Describe when wnt vs no wnt
No wnt = b cat degraded by gsk3 beta
Wnt = dsh and gbp are stabilized, gsk3 activity blocked, b cat accumulates and is translocated to the nucleus where it activates gene expression
Describe wnt 11 - xenopus
Initially wnt 11 mrna located at vegetal pole at time of sperm entry
Dispersed from cortex during initial cleavage stage
Wnt 11 is translated and secreted from dorsal vegetal cells
Concentrations of wnt 11 mRNA and protein produced from it = start to be segmented into subset blastomeres = gives higher wnt signal
Describe - all cells -mRNA concept
All cells have diff mRNAs and where they function = controlled by localization in cytoplasm
Like neurons = mRNA already there and can trigger protein translation = easier than propagation along whole neuron
Where is b cat stabilized in xenopus
On dorsal side
But no ventral side
Can see by immunofluroescent standing = anti B cat antibody = see not b cat on ventral side
Describe wnt - pathway/effects
Dishevelled protein = dsh
Brings other components of wnt signalling pathway into region
Dsh moves to plus microtuble end = further away from spear enter
And brings other components wnt in same group of cels
Describe steps of wnt - specifics
Dorsal enrichment of dsh and gbp —> dorsal inhibition of gsk3 —> dorsal enrichment of b cat
Cells in developing blastula that have both wnt stabilized b cat and can now become nuclear with help of other components
Describe whole summary of b cat for xenopus
Nothing ever happens on its own = many things together
1. Dsh and gbp moved along growing end of microtubule by kinesin = fast, stabilizes b cat
2. Wnt 11 mRNA (in vesicles at vegetal pole) moved by cortical rotation to future dorsal side = slow, slower process
3. Wnt 11 secreted by dorsal blastomeres, stabilizes dsh and Gbp = amplifies signal, acts on neighbours = stabilize b cat
4. B cat enters nucleus to activate gene expression = spemann mangold
What does blocking mircotubules do in Xenopus embryos
Ventralizes
Blocking other aspects of these events can also have effect on centralizing embryo
What rescues ventralized xenopus embryos
Transplanting a dorsal vegetal blastomere
Can rescue phenotype
Uv treatment blocks microtubules
Take blastomere from dorsal - untreated and transplant = creates wnt signals nearby
Rescues phenotype
Cut and paste exp
Describe localization of chord in mRNA
In dorsal blastopore lip
Expands through involution = gastrulation
Lines future neural plate
What can also rescue use phenotype
Injection of chordin
Inhibits bmp signaling = dorsalizing activity, so rescues embryo
Describe localization of noggin
Also bmp inhibitor
If give non = embryo ventralized
Need right dose of noggin - optimal
If give too much = get dorsalized
Name all molecules that can rescue uv ventralized phenotype
Wnt pathway = wnts and b cat
Tgf beta = nodal related proteins
BMP antagonists = chordin and notion
How do we test function of these molecules - exp
Manipulate embryos at 4 cell stage then culture them
How do we test function of these molecules - analysis
Look at phenotypes - mRNA expression of markers
Whole mount in situ hybridization
Sizzle expressed ventrally
Six3 expressed in eye, part of cns, to, marks dorsal
Convention = dorsal up, ventral down, ant —> post
Look at late gastrula and early tadpole
How do we test function of these molecules - exp manipulation
Inject each cell of 4 cell xenopus embryo with morphology’s against bmp4 or chordin
Then culture
Recall = bmp4 promotes ventral, chordin promotes dorsal
How do we test function of these molecules - analysis of manipulation
Look at all possibilities =
Reduced bmp, lost marker sizzled and embryo has diff shape = BMP MORPHOLINO, repress ventral and no sizzled
Recused chordin, more sizzled-expansion of ventral and lost dorsal = CHORDIN MORPHOLINOS, repressed dorsal and expands sizzled
How do we test function of these molecules - what is morpholinos
Stable antisense oligonucleotides that block translation of targeted mrna
Usually created over atg so wont start transcription
Can think of 100% effective
BMP morpholinos = blocks bmp
What is anterior posterior patterning in drosophila determined by
Position of egg in ovary
Starts at one cell stage
Describe drosophila dev generally
Early stage eggs - syncytial blastocyst starts as one cytoplasm shared by all nuclei
Nurse cell - makes all mRNAs and stuff deposited into oocyte
Then goes to mature egg with single nucleus
Describe what affects a-p polarity in drosophila
- Maternal effect genes in ovaries
- Anterior posterior protein gradient in embryos
Describe maternal effect genes in ovaries
Cytoplasmic polarity
mRNA products synthesized by nurse cells (maternal) asymmetrically localized in egg prior to fertilization
Anterior = biocid
Posterior = nanos
Happens early = as soon as nurse cells feed oocytes
Depends on genotype of mOM only
Describe bicoid and nanos
Ant = bicoid
Post = nanos
RNA localization important for holding them tight - concentrations of proteins similar to its mRNAs
Tightly held in those regions
Describe mRNA localization - drosophila
Important for generating asymmetric protein distribution
Generates high local protein concentrations - each mRNA translated multiple times
Extremely effective to regulate site of protein activity
Local mRNA pools permit precise temporal control of local protein synthesis in response to a signal
>1500 transcripts had distinct sub cellular localizations
Describe the multi step process of mRNA localization in drosophila
Recognition of cis acting sequences in mrna (localization signals, sequence in mrna recognized by rna binding protein)
RNA protein complexes (rnp) must be packaged into transport particles
Rnp transport particles must be trafficked in the cytoplasm - can move along microtubules and
Rnp particle becomes anchored at destination
Describe asymmetric localization of bicoid
Transported on microtubules to anterior end
Anchored by actin
Describe asymmetric localization of nanos
Nanos transported to posterior end in part by oocyte streaming - as nurse cells pushing all contents in oocyte
Anchored by germ plasm and actin
Describe generally asymmetric localization of bicoid and nanos
Cab see translocation of mrnas along tubules —> subtle differences =
Preferential localiztion of where or how microtubules are
Sufficient to case localized effect in that region
Describe anterior posterior protein gradient in embryos - a-p drosophila
Not as tightly held tho now = can ow move after translation
Asymmetric gradients of protein expression
Describe anterior posterior protein gradient in embryos - a-p drosophila BICOID
RNA binding protein =binds to 3’ Utr and repressed translation of mRNA, like caudal
AND
dna binding protein= regulates transcription,binds rna and dna
= regulates transcription and translation
Describe anterior posterior protein gradient in embryos - a-p drosophila NANOS
RNA binding - binds to 3’ utr
And represses translation of mRNA
Describe anterior and posterior structures in drosophila embryo
My = mouth hook, anterior, see segments and clear anterior structures
Ap = anal plate posterior
Fk = flizkorper: structural specializations of 8th abdominal posterior segment
Describe bicoid and caudal
Note = caudal at post end, in nucleus and cytoplasm
Have complementary a-p expression gradients
What happens if bicoid mutant
No bicoid in anterior = cause to lose ant end
2x fk and 2x ap and no mh
No post end
Recall = genes in drosophila named for phenotype that results when genes mutated
Bicoid = means 2 tailed
What happens when inject bicoid mrna - rescue exp into diff embryos - inject in vitro translated mRNA
Inject in mutant - 2 post ends = inject bicoid to anterior - localized at that end and gives normal
Inject in middle of embryo = 2 tails but ant genes in middle, proteins stay in middle, = head structures
Inject in posterior end = gain of function = 2 headed, 2 ant ends
Describe what causes anterior posterior protein gradients in drosophila - gen
First = was based on asymmetric localization of mRNAs
Now= set up by asymmetric translation mRNAs
Describe what causes anterior posterior protein gradients in drosophila - genes
Maternal effect genes hunchback and caudal mrnas are symmetrically distributed in egg - mrnas not localized
What do Bicoid and nanos do - a-p gradients - 2
Bicoid inhibits translation of caudal mRNA at ant end
Nanos prevents translation of hunchback mRNA at post end
Both are rna binding proteins = prevent translation of mRNA at hey bind to
Diff reason for asymmetry
Describe protein expression in early embryo -drosophila
Asymmetric
Still one cytoplasm - syncytial embryo
Hunchback and caudal not as separated as bicoid and nanos
Since bicoid and nanos have moved away from being held at ends
Also have dorsal ventral gradients at same time - much happening
What does bicoid do to caudal
Repressed translation of caudal
What happens after 13th division drosophila
Cell membranes form to create the cellular blastoderm - a single layer of cells around a yolk core
Cellularization
Get whole other gene expression patterns
Describe gap genes of drosophila
Mutations cause gaps in segmentation pattern
For a-p axis
Regulated by maternal effect genes
Among first genes transcribed in zygote
Turned on in broad stripes
Have v diff expression patterns, see stripes in groups of cellularized cells and compartmentalize diff subsets of rna binding proteins and tfs
Describe pair rule genes and segment polarity genes
Pair rule genes = 7 stripes, a-p
Then segment polarity genes
Regulated by diff concentrations gap genes
Divide embryo into periodic units
Results in stripped pattern fo seven vertical bands, perpendicular to a-p axis
Activate segment polarity genes - 14 stripes , specific stripes of expression
What regulates homeotic selector genes
Gap, pair rule and segment polarity genes regulate Homeotic selector genes to determine developmental fate of each segment
Turns on how genes - diff combos
Describe pair rule genes in drosophila
Fly = v segmented
2.7 hrs after fert
Stripes of eve and ftz
3.5 hrs after fert = gradually stripes get narrower as embryo ages
What are eve striped controlled by
Even skipped = controlled by specific promoter events
Put regions of dna upstream lac z (= makes b galac and cleaves substrate = blue)
Can see where gene enhancer turns on expression
Each turn on diff things - diff enhancers for each stripes, by diff combos tfs
Stripe 1/5 expression in embryo depleted for gap gene giant
Name all 6 steps of a-p axis in drosophila
- Cytoplasmic polarity = asymmetric localization of maternal transcripts
- Asymmetric protein gradients due to translation of asymmetrically localized mRNAs
- Gap genes - restricted zones of expression of zygotic mRNAs
- Stripes of expression of pair rule genes define spatial domains of homeotic genes
- Segment polarity genes turn on Homeotic gene expression, defines segment identity along a-p axis
- Homeotic genes
Describe hox genes in drosophila
Homeotic genes control pattern of body formation during early embryonic dev
Transcription factors that contain a homeobox dna binding domain
Describe hox genes in alls precise
In vertebrates = 4 clusters
Order of genes evolutionary conserved, similar to a-p patterning riles
Comes on in specific pattern
In flies = single hox cluster
Describe hox genes in flies - segmentes
Line of expression. =anterior boundari
Controls phenotypes within segments of flies
On in ant cell and extends posterior
Ant = first to come on and post = later to come on
