1
Q
Why are gastrulation movements so different among different vertebrates?
A
Eggs are different sizes, Gastrulation is also very different but after the phylotypic stage, The start to look similar
2
Q
What is the phylotypic stage?
A
The stage where embryos of different species begin to look morphologically similar to each other
3
Q
What are characteristic features of the embryo at specific developmental stages? e.g., fertilization, cleavage stage, blastula stage
A
Fertilization:
- Cortical rotation
- Caused by actin polymerization which specifies nieuwkoop center that creates DV axis (uses dorsalizing factor Dvl to make gradient)
- Vitelline membrane lift
Cleavage Stage:
- Synchronous cell division
- No zygotic gene expression, only maternal
Blastula Stage:
- Zygotic genome activation at MBT (mid blastula stage) = cells are specified
- Induction of mesoderm (nodal -> Dorsal mesoderm. BMP -> ventral mesoderm)
- 3 germ layers are present
4
Q
Describe the Saimois Cascade
A
- Wnt ligand activates Dvl which inhibits GSK3
- Dvl stabilizes Beta-Catenin in dorsal cells
- Wnt + Beta-Catenin moves nucleus and regulates transcription of Siamois gene
5
Q
What are characteristic features of the embryo at specific developmental stages?
e.g., gastrula stage, neurula stage and tailbud stage.
A
Gastrula Stage: Involution
- Germ layer rearrangement
- AP axis established
- Neural induction begins
- Forms future gut
Neurula Stage:
- Appearance of neural fold
- Neural tube formation
- Neural crest cells are born
Tailbud Stage:
- Somitogenesis
- Sensory organ precursor formation
- Neural crest cell migration
- 24 hours
6
Q
What happens during cortical rotation? What is the significance of breaking the radial symmetry after fertilization?
A
- Actin polymerization disturbs vegetal region to mix materials toward the equatorial region = breaks radial symmetry
- Specifies the Nieuwkoop center that forms on the opposite side of where sperm entered.
- Allows dorsalizing factors (Dvl: disheveled) which allows B-catenin accumulation in nucleus which will bind to receptors and activate the siamois gene to cause dorsalization
7
Q
What is the difference between Nieuwkoop center and Organizer?
A
8
Q
Comprehend the action of Wnt signaling pathway and how the siamois gene is activated.
A
- Cortical rotation after fertilization causes the dorsalizing factor (Dvl) to move to the dorsal end of the embryo
- If Wnt ligand is present, it activates Dvl and inhibits GSK3. Since it is inhibited, GSK3 cannot degrade B catenin, so we have lots of Bcatenin available
- Bcatenin can now move into nucleus and turn on siamois gene that is expressed in Nieuwkoop center
+ B catenin is NOT a tf
+ If siamois was off: NO HEAD
9
Q
How is fate mapping done in Xenopus embryos? Why is fate mapping important?
A
- Inject high molecular weight dyes into certain cells in the 32 cell blastula stage and observe its final identity after it has fully developed
- Also showed cells were specified already in the blastula stage
- Shows future property of cell behavior
- Shows the cellular potential and potency of a cell
10
Q
What tissues originate from each germ layer?
A
- ABC: ectoderm (animal region) -> epidermis/epidermal derivatives, nervous system
- BC: mesoderm -> notochord, somites (muscle), lateral plate mesoderm (heart, kidney), blood islands (vascular system)
- CD: endoderm (vegetal region) -> gut (epithelial gut lining of trachea, lungs, salivary glands, liver, pancreas)
11
Q
What evidence supports the notion that mesoderm is actually induced by endoderm.
A
- A piece of the ectoderm becomes the epidermis
- A piece of mesenchyme becomes ventral mesoderm
- A piece of notochord becomes dorsal mesoderm
- A piece of vegetal tissue becomes endoderm
12
Q
Comprehend both BMP and TGF-signaling pathways.
A
- Mesoderm induction from the endoderm requires a signal from the vegetal (yolky)region. Evidence: when animal and vegetal explants are separated by a filter, induction of mesoderm still occurs, showing that the mesoderm inducing signal is a small diffusible molecule (Nodal and BMP)
- There are 2 different mesoderm that are independently regulated inducing signals:
13
Q
What evidence supports that mesoderm inducing factor is a member of the TGFb signaling family?
A
- If you injected a mutant TGFb/Nodal type 1 receptor mrna (truncated receptor missing kinase bottom domain) into a 2 cell stage embryo, you don’t get proper mesoderm formation showing that nodal signaling is required
- You don’t even get a signal if you have 1 defective receptor attached to 1 normal one bc this is a dominant negative mutation.
14
Q
What is a dominant negative mutant? How is it different from regular loss-of-function
mutation?
A
- A mutation that prevents the normal wt gene product even though it interacts with other elements and it can still dimerize. So, set up can still occur but you don’t get the original wt gene product so you don’t get the signal.
- It is different from a regular LOF
15
Q
Comprehend the molecular steps leading to gastrulation.
A
- Midblastula stage: bcatenin AND VegT-> Mesoderm induction: -> high nodal -> Gastrulastage: dorsal mesoderm -> organizer
- Midblastula stage: Vegt only -> Mesoderm induction: -> low nodal -> Gastrula Stage:ventral mesoderm
16
Q
Distinguish between Archenteron and blastocoel
A
- Archenteron: future gut cavity in animal pole (top) seen in the late gastrula stage
- Blastocoel: cavity in vegetal region (bottom) that appears in late gastrula stage and disappears later
17
Q
What are three major morphogenic movements that control gastrulation movements?
A
- Involution: sheets of cells corresponding to future mesoderm and endoderm roll into gastrula through the blastopore
- Conversion: once inside blastopore, mesodermal and endodermal cells converge
- Extension: they then extend under the dorsal ectoderm
18
Q
How do prechodal plate mesoderm and chordamesoderm migrate during gastrulation?
A
- Mesoderm under the ectoderm has some power to specify tissue of ectoderm
- Prechordal plate mesoderm = anterior mesoderm: induces 2nd head, produces Noggin and Chordin BLOCK BMP to make neuroectoderm
- Chordamesoderm = posterior mesoderm: induces a 2nd trunk, produces WNT and retinoic acid to make trunk
19
Q
What are the functions of prechordal plate mesoderm and chordamesoderm?
A
- Prechordial plate mesoderm will give rise to head structures in the embryo. Plays a role in facial structure. (mesenchymal cells)
- Chordamesoderm gives rise to the central nervous system (notochord cells). Also helps with formation of neural tube which becomes brain and spine
20
Q
Visualize the 3D movments of amphibian gatrulation.
A
21
Q
What is the significance of the organizer transplantation?
A
- Organizer induces the body’s axis by recruiting host tissues. (AP axis formation)
22
Q
Which tissues are induced by the organizer? How was that shown?
A
+ Notochord is all donor derived
+ Somites are both donor and host derived
+ Nervous system is all host derived
- Was shown by taking a piece of the organizer tissue from a donor and transplanting it into a host cell during gastrulation stage which resulted in forming a secondary axis
23
Q
How is neural tissue induced in the early xenopus embryo?
A
- The organizer and prechordal plate mesoderm secretes the neural inducers called noggin and chordin. When expressed, they block BMP so ectoderm -> neuroectoderm
- If there is no noggin and chordin, BMP is not blocked and turns the ectoderm -> epidermis
24
Q
Compare between Xenopus and drosophila neural induction and identify the roles of Noggin and Chordin.
A
- Frog: noggin and chordin are secreted by organizer and prechordal plate mesoderm which block BMP activity so that the ectoderm -> neuroectoderm
25
How would you experimentally demonstrate that different mesodermal regions (prechoral + Chordamesoderm) of a gastrulating embryo have different neural inducing activities?
- You can take a tissue sample of the prechordal plate mesoderm and transplant it into a gastrulating embryo and see what develops. Since the prechordal plate secretes noggin and chordin, you should see a neural ectoderm develop from the ectoderm since BMP was inhibited
- You should do the same with a tissue sample from chordamesoderm and should see the induced anterior neural ectoderm transform into a more posterior character and make a trunk. This is because it secretes Wnt and retinoic acid
26
Describe the relationship between neural ectoderm neural plate, neural fold, notochord, neural tube, and neural crest.
- Neural ectoderm: outside layer of ectoderm that will undergo neurulation
- Neural plate: layer of neural ectoderm that will get folded -> neural fold
- Neural fold: 1st sign of neurulation that encloses neural plate
- Notochord: patterns neural tube, later surrounds neural tube, later will be part of intervertebral discs, formed along dorsal midline
- Neural tube: forms above notochord (dorsal)
- Neural crest:
27
Why is avian development after fertilization is so different from that of Xenopus?
- At time of egg laying, blastoderm is already formed and is a disc of cells that sits at the top of the yolk
28
How does chick break a radial symmetry?
- Egg rotates through hen’s uterus every 6 minutes as the embryo develops which causes the blastoderm to tip in the direction of rotation to create A-P axis and allowing primitive streak to form from posterior and migrate to anterior
29
Identify the functions of epiblast, hypoblast, posterior marginal zone, Koller’s sickle, and Hensen’s node.
- Epiblast: develops into future embryo
- Hypoblast: develops into extraembryonic structures like yolk sac
- Posterior marginal zone: forms the junction of the area pellucida and area opaca and defines dorsal side and posterior end of embryo. Also where gastrulation is initiated.
- Koller’s sickle: defines position of primitive streak, secrete Vg1 and Wnt ligands
- Hensen’s node: equivalent to organizer in frogs (recall: organizer creates AP secondaryaxis)
30
When is notochord formed in chick?
During neurulation and somites form on one side of the notochord, one pair every 90 minutes
31
What are amnion and allantois, and what are the functions of these tissues?
- These are extraembryonic tissues that are absent in frogs
1. Amnion: provides mechanical protection
2. Allantois: receives excretory products, site of O2/CO2 exchange
3. Yolk sac: surrounds embryo and gives it nutrients via vittelin vien
32
The posterior marginal zone is said to be similar to Nieuwkoop center. Why is that?
- The PMZ defines the dorsal side and posterior end of the embryo similar to the NKC that established the DV axis in a frog
33
Compare and contrast the similarities between chick and frog gastrulation
34
Describe the origin of cranial neural crest and trunk neural crest cells. What do these neural crest give rise to?
- Origins: neural fold
- Cranial neural crest cells: give rise to bone and connective tissue of face
- Trunk neural crest cells: give rise to melanocytes, sensory and autonomic neurons, glia,neuroendocrine cells, etc
35
What are rhombomeres? How does each rhombomere acquire a specific identify?
- The vertebrate hind region is organized into 8 segment boundaries called rhombomeres. Each one behaves like a compartment and they share some adhesive properties to define boundaries resulting in lineage restriction in each one.
- They acquire a specific identity by expressing Hox genes in a well defined pattern
- Retinoic acid turns on hox genes in posterior rhombomeres
36
How is the neural crest cell migration orchestrated in the hindbrain?
- NCCs acquire positional identity before they migrate out of the hindbrain from Hox genes and they end up in different branchial arches
- They have a meeting (NCCs at origin) get orders from capt hox and go where they need to go
37
Describe the types of tissues the somite can give rise to.
- Vertebrae and rib
- Bone and cartilage of the trunk
- Skeletal muscle
- Dermis of the skin
38
What is presomitic mesoderm? When and where do you find them?
- Presomitic mesoderm: an unsegmented mesoderm between the regressing node and last formed somite. They have positional identity before somite formation and are controlled by Hox genes
39
What experiment support the notion that anterior posterior patterning of the skeleton along
the body axis is determined.
- Somite formation shows that the molecular information and timing/fate is laid down early by an AP axis signal. The experiment shows that the chick mesoderm forms A -> P as the Hensen’s node regresses.
- Experiment: take a portion of the presomitic mesoderm, flip it 180 degrees and implant it into another embryo. It shows that the pattern still follows. Mesoderm does not change temporal sequence of somite formation because the molecular timing and fate is laid down by an earlier signal
40
How is positional values along anteroposterior axis of somite established?
- A presomitic mesoderm has a positional identity before somites are even formed due to a signal. This is seen in the experiment when a presomitic mesoderm from a thoracic vertebrae forming region is transplanted into a cervical region. Ribs begin to grow at the cervical region
41
Describe how the somite gives rise to different mesodermal derivatives such as cartilage, muscle or dermis along the medio-lateral axis.
- Dorsal and lateral regions of somites form: dermomyotome
- Myotome forms: muscle cells
- Dermomyotome forms: dermis
- Sclerotome forms: cartilage of vertebrae and ribs
42
What is the role of notochord and floor plate during somite differentiation?
- Signals from the lateral plate mesoderm and ectoderm induce the dermomyotome (->dermis)- The notochord promotes sclerotome formation (-> cartilage)- Floor plate also promotes sclerotome formation ( -> cartilage)
43
Compare the term blastula, blastoderm, and blastocyte. What organism do they refer? What features do they have? How do they differ?
- Blastomere: single cell in blastula
- Blastoderm: disc like structure that sits on yolk in avians
- Blastocyst: 3.5 days after fertilization, cleavage made lots of cells in the morula ->blastocyst, 2 big parts: ICM and Trophectoderm
Mouse
Features and differences:
44
When does zygotic genome activation occurs in mouse embryos?
Before cleavage (cell stage 1-2)
45
What are inner cell mass and trophectoderm cells?
- Blastocyst becomes:
1. Inner cell mass: becomes embryo (ESC - Pluripotent: can make any cell but not extraembryonic ones like placenta)
2. Trophectoderm: becomes embryonic tissues (placenta)
46
Describe an experiment that shows the cleavage stage mouse embryo is totipotent.
- Isolated inner mass cells from the blastocyst are cultured in a cocktail which stops them from differentiating
- Won a nobel
47
What is the difference between totipotent and pluripotent cells?
Totipotent: before Morula (8 cell stage) = can become any cell type including extraembryonic structures like placenta
Pluripotent: ESC (true stem cells): can become any cell but not placenta/extramembrane structures
48
What is the function of primitive streak?
- It elongates P->A then regresses back P and gives rise to the node
49
Follow the lineage of primitive endoderm, mural and polar trophectoderm.
- Polar trophectoderm -> extraembryonic ectoderm -> placenta
- IMC: Primitive endoderm -> parietal and visceral endoderm (touches embryo) ->head/anterior org region
- Mural trophectoderm -> trophoblast giant -> placenta
50
Understand how AVE is formed and what it does.
- Visceral endoderm moves to anterior and signals epiblast to specify anterior ectoderm. It secretes Wnt and Nodal antagonists that make ectoderm -> head (recall: ectoderm is inside!)
- DVE: -> gut
51
How are ES cells derived?
- At the blastocyst stage, it becomes ICM and trophectoderm. ICM is made of ES cells that are pluripotent and can become anything but extraembryonic tissue (placenta).
- Can isolate them in culture
52
How do you knockout a gene using a traditional homologous recombination approach?
53
How is CRISPR/Cas9 approach used to mutate genes?
1. Make a guide rna that can be complementary to the gene of interest and therefore targetit. Bind that rna to enzyme Cas9
2. Cas9 guides to the target DNA and makes a double strand break in DNA
3. Can rejoin by
a. Non homologous end joining (induce random mutation
b. Homology directed repair (adding new DNA)
54
What is the genetic cause of situ inversus?
- Mouse: IV gene mutation causes reversal of “handedness” of internal organs by reversing the flow of fluid affects dynein protein function
1. Normally: motile cilia push fluid to the Right to Left which activates sensorycilia on L side of node. This causes L side increase of Ca2+ -> L side increase ifNodal -> activates pitx2 tf expression
2. Mutation: motile cilia moves fluid L to R, affecting dynein protein function ->randomized organ asymmetry
- Humans: 25% of people with situs inversus have primary ciliary dyskinesia
55
How is left-right asymmetry achieved in chick and mouse? Compare and contrast the similarities and differences.
56
Compare human and mouse developmental time and understand the difference
57
How are yolk sac and amnion formed?
58
How does monozygotic twins develop?
- Everything splits during cleavage/blastula stage
- ICM splits within the blastocyst at the epiblast stage
- Can have these types:
a. Split at 2 Cell Stage: 2 separate everything
b. Split at early blastocyst stage: separate amnions only
c. Later split: share everything
59
How is IVF carried out?
- Stimulate ovary hormones, Take out an egg from mom, add sperm on culture to get embryo, place back into uterus
60
What might be the pros and cons of PGC?
Developmental Biology
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