Developmental Exam 3 Flashcards
Why can you not work on embryos past gastrulation?
Gastrulation is the point where you get germ layers, cells do things
Functions of frog blastocoel
Allows space for cells to move during gastrulation
Separates layer of cells. Bottom and top separated. Prevents them from interacting
How do cells cleave?
From top to bottom. (From animal side to vegetal side on the bottom)
What does gastrulation achieve?
Establishes three germ layers: endo, meso, ecto
Sets up basic body plan and axes
The morphogenetic movements bring cells into new positions which allows inductive interactions
Steps of gastrulation
- Start with late blastula stage. Blastocoel in animal pole.
- Dorsal blastopore lip (opposite side of where the sperm attached) cells move into blastocoel space along the ectoderm.
The animal pole is moved towards the vegetal pole. Blastocoel is displaced to the ventral side. Space is made from the dorsal blastopore lip called the archenteron and is made from gastrulation movement. Cells are lead by the protein fibronectin. - The ventral blastopore lip moves around until it is on the dorsal side with the dorsal side lip. Have blastopore lip, lateral blastopore lip and ventral blastopore lip on the dorsal side of the organism. Eventually this part of the yolk is plugged. Fibronectin discontinues when the endoderm and not the mesoderm cells are moving. When the endoderm cells are moving a new mechanism takes over called convergent extension.
- Blastopore cavity is filled with internalized endoderm. The yolk starts to plug. Mesenchyme is created between the ectoderm and the endoderm
Tissue movement during (amphibian gastrulation) in order
- Invagination- inward pushing of cells
- Involution- Cell that invaginated are “rolling”/moving (migration) inward to form an underlying layer. Future endoderm/mesoderm rolling in.
- Convergent extension- causes narrowing and lengthening of mesoderm during gastrulation. Directional cell intercalation- rows of cells move between one another
- Epiboly- sheets of epithelial cells spreading by thinning by radical intercalation. Creates tissue that is thinner/longer in different directions. 3D. Makes butt-hole.
Migration of endoderm during gastrulation does what to mesoderm?
Endoderm cells migration “pulls” mesoderm in
The pharyngeal endoderm move out of the way to allow invagination and involution. They move along blastocoel and pull “bottle” cell inward
What group of cells are the first ones to involute?
Chordamesoderm (determines nervous system) and the mesoderm
Where does migration of endoderm and mesoderm (involution) happen first?
At the blastopore
What directs cell migration during gastrulation?
Fibronectin
Does not go around all the way.
How is gastrulation initiated?
It begins at the edge of the grey crescent that is not covered by the thick black cytoplasm.
Begins directionally oposite of where sperm binded.
Grey crescent
sperm attaches and centrosome is in that area and then the other is directionally across it. At the top where the sperm attached there is a thick dark cortical cytoplasm and below that is the grey cytoplasm layer. Microtubules cause cortical rotation and the centrosomes rotate 30 degrees. The thick dark cortical cytoplasm rotates 30 degrees with the rotation. They grey cytoplasm does not.
The grey crescent formation key events
- Cytoplasmic reorganization by microtubules
- Cortical rotation of 30 degrees
Outcomes of grey crescent formation
Gastrulation will happen at/just below the grey crescent/directly across from where sperm initially entered
Hans Spemann’s first experiment
Found when you take a hair and gently cause what looks like cytokinesis to an egg, a nuclei will go to one side and others with be on another side which creates two of the organism.
Outcomes of Hans Spemann’s first experiment
Had two normally developed organisms. Happened when both received the grey crescent cells
or
Had one normally developed organism and one “belly piece”. Happened when one received the grey crescent cells.
Therefore something in the grey crescent is important
Spemann’s transplantation experiments
Used different species of newt with different pigmentations
During gastrulation he transplanted tissue from donor newt to host newt. The transplanted tissue was presumptive ectoderm and he placed the transplants in different locations.
ex. More presumptive ectoderm to region of non-neural ectoderm
Spemann’s transplantation experiment when he used presumptive neural ectoderm from early gastrula and replaced it with presumptive epidermis?
Epidermis formed. Cell developed normal neural plate with normal epidermis.
Call regulation since transported cells were regulated by their environment
Cells adopted correct fate for that region
Spemann’s transplantation experiment when he used presumptive neural ectoderm from late gastrula and replaced it with presumptive epidermis?
Neural plate tissue formed. Had a complete neural plate and neural plate tissue.
Therefore the transplanted cells were already committed and could not be regulated by their environment
Transplanted cells maintained original fate
Hilde Mangold Experiment (1921)
Transferred doral blastopore lip (DSL) cells from one early blastula salamander embryo to another.
The DSL cells made another DBL so the organism grew two! Suggests DBL has inductive powers of axis formation via a morphogen on a morphogen gradient
Spemann-Mangold’s Organizer
Dorsal blastopore lip cells are the organizer
How do we know the DBL cells are the organizer?
- Induced host’s ventral tissues to change fate to form a neural tube and dorsal mesoderm
- Organized host and donor tissue into secondary embryo with clear axes
Possible mechanisms for how organizer cells induce cells to be neural cells
- Organizer promotes nearby ectoderm to become neural ectoderm
- Organizer prevents nearby ectoderm from becoming skin
What is the organizer secreting and how does it induce neural development?
Noggin and Chordin mRNA’s are expressed in DBL which prevents neural ectoderm from becoming epidermis
How to test noggin and chordin mRNA’s in relation to DBL?
Misexpress it with different [morphogen]
1. Subjected vegetal pole to UV radiation to mess up MT’s so no cortical rotation can happen. Get ventralized embryo with no dorsal structures (no head, just belly)
2. Inject different [morphogen] of noggin mRNA. Can you rescue the dorsal structures and allow development?
You can with a certain amount of noggin! Therefore more noggin mRNA=more dorsal (head) structures
BMP-4 role
Expressed everywhere else except neural area. Gradient on dorsal side by mesoderm cells below ectoderm. Promotes ectoderm formation into epidermis
BMP-4 and organizer molecules relationship
Organizer molecules block BMP-4 which blocks BMP-4 from cells near the organizer cells
Creates a gradient which results in cells becoming neural
Absence of BMP-4 causes ectoderm to default to neural fate.
Wnt signaling
When Wnt is missing, GSK3 P’s B-catenin which then is broken down in the proteosome. B-catenin cannot be a txn factor and gene stays off.
When Wnt is there, GSK binds to Axin which binds to P’ed membrane protein. B-catenin is free to go be a txn factor and goes to the gene and turns it on.
Beta-catenin activity turns on txn factors that activate organizer genes (Twin, siamois)
These genes become txn factors which activates expression of organizer genes (noggin).
How do Wnt proteins become localized on the dorsal side of the embryo?
Disheveled recruits GSK3 to membrane and away from beta-catenin, Wnt 11 is the Wnt mRNA. Moves to dorsal side by cortical rotation.
Have the GSK binding protein (GBP) attached to kinesin which is moving to the dorsal side on the microtubule.
These molecules move to the dorsal side through cortical rotation and MT translocation via kinesin (faster)
What region induces organizer region?
Niewkwp center
Is the vegetal endoderm that induces organizer
What does Smad2 do?
It activates organizer genes
It is activated by VegT which is a txn factor which activates to expression of activin/nodel which is ligand for another RTK which activates Smad2
Is also activated by Vg1 which is a ligand for another RTK that activates txn factor Smad2
The organizer
- Initiates movements of gastrulation
- Induces formation of the neural ectoderm; neural tube as well
- Becomes dorsal mesoderm. Will become muscle, bone, cartilage.
Neurulation
Formation of nervous system
Formation of neural tube which becomes the nervous system.
What can ectoderm turn into?
Surface ectoderm (epidermis)
BMP levels are high
Neural crest
BMP levels are moderate
Neural plate/neural tube
BMP levels are low, Sox transcription factors expressed
How can a neural tube be formed?
With a sheet of cells or a ball of cells
Sheet of cells is the most common
Secondary Neurulation
Mesenchymal cells aggregate and coalesce into a tube
Less common version
Primary neurulation
Bending of sheet
Start with a sheet of epithelial cells
Sheet is neural ectoderm/aka. neural plate
Invagination forms tube
Hinge points
Have apical constriction and basal thickening
First step of primary neurulation
- Elongate neural plate and have it start to fold a lil bit
Is elongated in dorsal region of ectoderm
Folding begins at medial hinge points (MHP) where cells are anchored to the notochord and change their shape
Cells become wedged-shaped. Wider at basal end, skinnier at apical end due to asymmetric constriction of actin (apical construction)
Second step of primary neurulation
- Elevation of the neural folds
Neural folds are created by continued bending at MHP of the neural plate and movement of the surface ectoderm toward the middle (toward dorsal mid-line)
Third step of primary neurulation
- Convergence of the neural folds
Need more hinge points to have this happen. Dorsal lateral hinge points (DLHP). Cells there become wedge-shaped
AND surface ectoderm continues movement like before!!
Fourth step of primary neurulation
- Closure/formation of the neural tube
Neural folds adhere to each other to create closed neural tube. Happens via cell adhesion or convergence of surface ectoderm to get on top to join surface ectoderm on other side
Apical constriction
Contraction of actin and myosin motors (actin-myosin) at apical barrier of the cell
Nucleus and other cell components pushed to basal end
Apical constriction at the dorsolateral hinge point (DHLP)
Noggin is expressed in the neural folds
Noggin mutant fails to close neural folds
Noggin expression is sufficient to induce dorsal lateral hinge point (DLHP)
Relationship between Noggin and BMPs
Noggin inhibits BMPs
We get a gradient that is still in place after gastrulation
Noggin gradient comes out at the hinge points. BMP is away from hinge points
How much BMP is needed for hinge?
In control, normal folding because MHP is normal
With dominant negative BMP receptor (non-functional BMP) had more bend at hinge point
Cells with constitutively active BMP receptor (lots of BMP signaling) there is no apical constriction, no bending, no hinge
How is Noggin/BMP hinge point regulated?
With high BMP there is recruitment of proteins that stabilize the junctions. This causes no apical constriction to happen
When there is no BMP actin myosin can constrict because there is no stabilizing proteins. Noggin binds up BMP which inhibits it so then apical constriction can happen. No interaction between BMP and BMP receptor can happen
Seural mechanisms
Zippering of neural folds
Fusion of cell extensions
Continuation of the hinges
Zippering of neural folds
A wave of actomyosin moving from posterior to anterior along boundary of neural cells to epidermal cells
Cranioachischisis
Completely open brain and spinal chord
Defect of neural tube
Anecephaly
Open brain and lack of skull vault
Defect of neural tube
Encephalocele
Herniation of the meninges and brain
Defect of neural tube
Meningocele
Protrusion of the meninges (filled with CSF) through a defect in the spine or skull
Defect of neural tube
How does neural crest migration happen?
EMT (epithelial to mesenchymal transition)
Neural crest derived cells
Peripheral nervous system (PNS) cells
Endocrine and paraendocrine derivatives
Pigment cells
Facial cartilage and bones
Connective tissue
Neural crest regions
Cranial crest, Vagal crest, Cardiac crest, Trunk crest, and sacral crest
Trunk crest
Peripheral nervous system and melanocytes (pigment)
Vagal and Sacral crests
Make up intestinal neurons
Enteric nervous system
Controls intestinal peristalsis
Cranial neural crest
Migrates into frontonasal process and pharyngeal arches to form craniofacial mesenchyme
Facial cartilage and bones
Cranial neurons
Connective tissue
Teeth, inner ear, thymus
What contributes to facial structure?
Cranial neural crest of the pharyngeal arches
What were the possible fate origins for neural crest cells
Since NCC from different regions become many different cell types, their fate orgins could be:
1. Pre-migrating NCC are already specified while in the neural tube
2. NCC are multi-potent stem cells. Are capable of being many things in different environments
Can figure out which one with transplanting, fate maps, etc…
Cre recombinase
Bacterial enzyme that recombines DNA
Recognizes LoxP DNA sequences and cute out the region between the two LoxP sequences and fuses the ends together
What is needed for Cre/Lox lineage tracing?
- Cell specific promoter driving Cre in specific cells
- Target/reporter DNA construct that has LoxP sites flanking it
Ex. If target DNA is stop codon, if Cre is present, gene can be translated beyond the stop codon since Cre recombinase would snip it out
Neural crest fate origin?
They are mostly multipotent
75% of labeled cells using rainbow lineage tracing with Cre/Lox proliferated during/after migration
Clones progeny became many different things. Got several different cell types
What determines neural crest cell fate?
- Its starting position. Along the anterior/posterior axis
- Migratory path. Environment they pass through
How do NCC migrate?
- Change in cell adhesion molecules to leave the neural tubes to separate from one another
Delaminates from the top of neural tube where both E-cadherin and Cadherin6B are expressed. Neural tube cells express N-cadherin and epithelial cells express E-cadherin
Expression of BMP turns on EMT txn. factors. Snail2 txn. repressor inhibits Cad6B expression. Zebra2 txn. repressor inhibits E-cadherin expression - Remodel the cytoskeleton to become migratory
Actin monomers polymerize into actin filaments that push against the cell membrane. Creates protrusions, pushing itself out, attach, drags its butt. Allows cell movement. This is mediated by Rho GTPases.
Ex: RhoA and Rac1
Two Cre cleavage reactions
Cre performs two cleavage reactions (one at each LoxP site) and two ligation reactions.
One ligation results in the cut chromosome being made continuous again, minus the excised sequence between the LoxP sites.
The other ligation brings the ends of the excised sequence together into a little loop. See panel a in the diagram I attached.
RhoGTPases function
Are molecular switches
In on active state: has three phosphates attached to GTP attached to the GTPase
When phosphate is removed it is inactive and is in the off state
What does Rac1 do at front of cells?
Rac1 promotes actin filament protrusions
What does RhoA do at back of cell?
RhoA promotes actin retraction
You hypothesize a population of cells in the tunicate D.occidentalis are a primitive neural crest. What features would you look for in these cells to confirm this?
See if they are multipotent and if they migrate
What are brain vesicles?
Developmental boundaries
What defines A/P brain boundaries?
Different txn. factors define initial A/P boundaries
These txn. factors are determined by organizing regions
A/P boundaries and paracrine factors?
A/P brain boundaries become signaling centers that can correct local patterning of gene expression (via new signaling molecules/ txn. factors)
End up with fined tuned patterns of cell fate which would be certain neuron types since we are in the brain
What is the hindbrain divided into?
Rhombomeres
Rhombomeres
Segments
Is established by signals from the nearby mesoderm
Eph (Receptor)/Ephrin (ligand) expression in alterative rhombomeres generates sharp boundaries
Eph/Ephrin signaling
Ephrin ligand binds Eph receptor on another cell which causes forward and reverse signaling
Hox colinearity in the hindbrain
Rhombomeres are patterned by Hox code
Neurons born in same rhombomeres will take on same/similar fate and extend processes to similar regions
Different regions of spinal chord
Cervical, Thoracic, Lumbar, Sacral
How is the initial expression pattern established for early brain development dorsal-ventral
Neural tube is divided into discrete domains based on txn. factor expression
What do the organizer molecules Chordin, Noggin and Follistatin inhibit?
Epidermal ectoderm spreading to create neural ectoderm
Vegetal from spreading into dorsal endoderm
D/V signals in the neural tube
BMP4 (dorsal side), 7 is in the epidermis and has a gradient towards the notochord
Shh (ventral side) is in the notochord and has a gradient towards the epidermis
This gradient will determine which cells become certain neuron types
What does Shh establish
Ventral fates
What would happen if you removed the notochord?
There would be no floor plate cells
No motor neuron region
No other ventral neuron cell types
Does Shh establish ventral fates?
Yes!
If you transplant a donor notochord with Shh to lateral side of neural tube there will be a duplication of the ventral fates.
Will have two floor plates, secondary set of motor neurons, etc…
Does not have to be the notochord, can be any Shh secreting cells since Shh establishes ventral fates and the notochord does not without secreting Shh.
What happens if you change the concentration of Shh?
Neural tube explants were exposed to different [Shh]
There was a spatial and temporal response (time) to Shh.
Depends on concentration and duration of Shh present
Txn factors Olig2 and Nkx2.2 increase as more Shh is added over time. Txn factor Pax7 decreases since it is present when Shh is not present
Shh signaling
Absence of Shh:
Patched (Shh receptor) receives nothing which inhibits Smooth red receptor
GliR txn. factor stays uncleaved and inhibits Shh target gene expression
Prescence of Shh:
Shh binds to Patched
Allows Smooth red to cleave GliR into Gli-A
This txn. factor goes to the nucleus and allows Shh target gene expression
“Simple model”: Spatial AND temporal response to Shh
Shh induces exp. of ventral patterning factors through a combination of [ ] and duration
Paraxial mesoderm
Makes somites
Temporary block of epithelial cells
vertebrae, ribs, tendons, skeletal muscles, blood vessels
Paraxial mesoderm (somite) development
Cell fate within the somite is determined by position and impacted by exposure to BMP gradient
Organizer molecules are around neural tube area. BMP comes from the surrounding area
