Final exam - Xenopus Development Flashcards
(129 cards)
1
Q
Phylotytpic stage
A
- when embryos are similar in appearance and structures
- stage where the embryo possesses a distinct head, neural tube and somites
2
Q
Vertebral column
A
- the segmented backbone surrounding the spinal cord and brain, which is enclosed in a skull
- composed of a succession of vertebrae
3
Q
Bilateral symmetry
A
- mirror image at the dorsal midline
- type of symmetry possessed by animals with a main axis of symmetry running from head to tail and the two sides of the body being mirror images of each other
4
Q
Head
A
-the structure located at the anterior end of a bilaterally symmetrical animal, such as an arthropod or a vertebrate, that typically houses the brain, various organs and the mouth
5
Q
Notochord
A
- rod-shaped tissue running along the dorsal midline
- a transient stiff, rod-like cellular structure in vertebrate embryos that runs from head to tail and lies centrally beneath the neural tube
- derived from mesoderm and its cells eventually become incorporated into the vertebral column
6
Q
Neural tube
A
- precursor of central nervous system
- neural tube sinks below the epidermis during neurulation
- The anterior neural tube becomes the brain
- Middle and posterior becomes the spinal cord
- derived from the ectoderm
- tubular structure that forms along the dorsal midline of a vertebrate embryo and gives rise to the nervous system
- guides the central nervous system, including the brain
7
Q
Somites
A
- segmented blocks of mesoderm tissue on either side of the notochord
- blocks of mesoderm that segment from the mesoderm on either side of the notochord
- give rise to trunk and limb muscles, the vertebral column and ribs, and the dermis
8
Q
Cleavage
A
- a series of rapid cell divisions without cell growth that occurs after fertilization and divides the embryo up into a number of small cells called blastomeres
- First cleavage: along the animal-vegital axis, divides egg into the future left and right
- Second cleavage: 90 degree to the first
- Third cleavage: equitoral and asymmetric, four smaller animal cells and four large vegital cells
- The 8-cell stages has 8 blastomeres
9
Q
Gastrulation
A
- the morphogenic process occurring in three dimensions where endoderm and mesoderm are internalized by entering through the blastopore and migrate via involution toward the future anterior of the embryo
- The first cells to enter the blastopore are most anterior and later cells are posterior in patterning
- development will cease if these tissues are not brought into the cell (most important part of development)
- the process in animal embryos in which prospective endodermal and mesodermal cells move from the outer surface of the embryo to the inside, where they give rise to internal organs
- during gastrulation, the anterior-posterior axis is laid down perpendicular to the dorsal-ventral axis
- The first cells to enter the blastopore are dorsal mesoderm
- Mesoderm and endoderm move towards the future anterior aspect of the embryo
10
Q
Notochord formation
A
-during post-gastulation, the dorsal mesoderm starts to develop the notochord
11
Q
Neurulation
A
- the process in vertebrates in which the future brain and spinal cord are formed from the ectodermal neural plate
- the neural plate develops a central groove (neural groove) which folds rising up on either side (neural folds)
- folds eventually meet and fuse along the midline to form a tubular structure (neural tube) that develops into the brain and spinal cord
- in birds and mammals, the neural plate gives rise to the brain and the spinal cord is formed from the stem zone
12
Q
Somitogenesis
A
-formation of somites
13
Q
Organogenesis
A
- the development of specific organs such as limbs, eyes and heart
- eyes and ears start to develop, then three branchial arches which form our jaws and facial features, then endoderm forms the lining of the intestine (liver, pancreas, and lungs)
14
Q
Xenopus laevis life cycle
A
1) egg (animal-vegetal axis)
2) cleavage
3) blastulation (blastomeres)
4) blastula (germ layers)
5) gastrulation
6) gastrula
7) neurula (neurulation, notochord, neural tube, somites)
8) organogenesis
6) tailbud embryo stage
7) free-swimming tadpole
8) metamorphosis
9) adult
15
Q
Xenopus laevis egg
A
- egg has a distinct polarity from animal to vegital axis
- Dark pigmented animal region
- Heavy and yolky vegital region
- At fertilization, the sperm enters the animal region
- Breaking of radial symmetry of the egg
16
Q
Animal region
A
- dark and pigmented region where the sperm enters during fertilization
- in eggs of amphibians, the hemispherical end of the egg where the nucleus resides, away from the yolk
- the most terminal part of this region is the animal pole, which is directly opposite the vegetal pole at the other end of the egg
- in Xenopus, the pigmented animal half is called the animal cap
- contains the ectoderm
17
Q
Vegetal region
A
- heavy and yolky region
- the yolky lower hemisphere of amphibian eggs and blastulas, and the region from which the endoderm will develop
- contains endoderm
18
Q
Animal-vegetal axis
A
-axis that runs from the animal pole to the vegetal pole in an egg or early embryo
19
Q
Radial symmetry
A
-the symmetry around the central axis in cylindrical structures such as plant stem and roots
20
Q
Blastulation
A
-formation of the blastula stage
21
Q
Blastomeres
A
-any of the cells formed by the cleavage of the fertilized egg
22
Q
Blastula
A
- early stage in the development of some embryos (amphibians, sea urchins), which is the outcome of cleavage
- it is a hollow ball of cells composed of an epithelial layer of cells enclosing a fluid-filled cavity, the blastocoel
- reached after 12 divisions (4096 cells)
- 3 germ layers are apparent: ectoderm (largely animal region), mesoderm (marginal zone) and endoderm (part of the marginal zone and vegital region, gives rise to digestive tract)
- Mesoderm is induced from the ectoderm through signals provided by the endoderm
23
Q
Blastocoel
A
-fluid-filled cavity that develops in the interior if a blastula
24
Q
Marginal zone
A
- equatorial ring around the embryo separating the animal and vegital regions
- contains mesoderm and endoderm
- the belt-like region of presumptive mesoderm at the equator of the late blastula of an amphibian embryo
25
Blastopore
- A small slit-like opening close to the marginal zone on the future dorsal side of the embryo
- slit-like or circular invagination on the surface of amphibian and sea urchin embryos, at which the mesoderm and endoderm move inside the embryo at gastrulation
26
Embryonic organizer / Spemann-Mangold organizer
- Located on the dorsal lip of the blastopore
- a signalling center on the dorsal side of the amphibian's early embryo and similar organizing regions in other vertebrates (node in the chick) that can direct the development of a complete embryo
- signals from this center can organize new antero-posterior and dorso-ventral axes
- mesoderm is patterned along the anterior-posterior axis via the action of this organizer during late gastrulation stage
- The organizer is patterning the embryo during gastrulation
- The properties of the organizer change over time (if taken later in development)
- not a uniform group of cells, they are changing over time
- receives the highest levels of Nodal signalling (concentration and time period)
27
Involution
-a type of cell movement that occurs at the beginning of the amphibian gastrulation, when a coherent sheet of cells (mesoderm and endoderm) enters the interior of the embryo at the blastopore by rolling in under itself
28
Epiboly
-the process during gastrulation in which the ectoderm extends to cover the whole of the embryo
29
Archenteron
- the second cavity formed inside the embryo when the endoderm and mesoderm invaginate during gastrulation
- it forms the gut or digestive tract
30
Organizer / organizing region / organizing center
- a signalling center that directs the development of the whole embryo or of part of the embryo, such as a limb
- in amphibians, the organizer usually refers to the Spemann-Mangold organizer
- the organizing center in pants refers to the cells underlying the central zone of the meristem, which maintains the stem cells of the central zone
- not a uniform group of cells, they are changing over time
31
Neurula
- the stage of neurulation in vertebrate embryonic development at the end of gastrulation when the neural tube is forming
- neural plate forms neural folds which rise towards the midline and fuse to form the neural tube
- The neural tube sinks below the epidermis
- The anterior neural tube becomes the brain
- Middle and posterior becomes the spinal cord
- Neural tube closure is second most important stage in development!
32
Lateral plate mesoderm
- Mesoderm located on the lateral parts of the embryo
- mesoderm in vertebrate embryos that lies lateral and ventral to the somites and gives rise to the tissues of the heart, kidney, gonads, blood vessels and cells, and the limb connective tissues
33
Neural plate
- is the ectoderm located above the notochord
- This plate forms neural folds which rise towards the midline and fuse to form the neural tube
- an area of thickened dorsal ectodermal epithelium at the anterior of a vertebrate embryo that gives rise to the nervous system through the process of neurulation
34
Neural folds
-the two folds that rise up at each edge of the neural plate at the beginning of neurulation and will eventually fuse to form the neural tube, which gives rise to the nervous system
35
Neural tube closure
- second most important stage in embryo development
- the coming together and fusion of the dorsal tips of the neural folds to form the neural tube that occurs during neurulation
36
Tail bud stage
- tailbud=the structure at the posterior end of vertebrate embryos containing stem-like cells that give rise to the post-anal tail
- after neurulation, the early tail bud stage occurs
- brain is divided into forebrain, midbrain and hindbrain
37
Forebrain
-the anterior part of the vertebrate embryonic brain that will give rise to the cerebral hemispheres, the thalamus, and the hypothalamus
38
Midbrain
-the middle section of the embryonic vertebrate brain that gives rise to the tectum (in amphibians and birds) and similar structures in mammals, which are the sites of integration and relay centers for signals coming to and from the hindbrain, and also for inputs from the sensory organs
39
Hindbrain
-the most posterior part of the embryonic brain, which gives rise to the cerebellum, the pons, and the medulla oblongata
40
Branchial arches
- structures that develop on each side of the embryonic head and give rise to the gill arches in fishes, and to the jaws and other facial structures in other vertebrates
- three branchial arches form our jaws and facial features
41
Chordates
- Animals with backbone in development
| - Pass through a phylotypic stage
42
Chordate synapomorphies?
- head
- notochord
- neural tube (guides the central nervous system, including brain)
- Somites (blocks of mesoderm on either side of the notochord)
- Vertebral column (segmented backbone surrounding the spinal cord and brain, which is enclosed in a skull)
- Anterior-posterior axis (head to tail axis)
- Dorsal-ventral axis (back to belly)
- Bilateral symmetry (mirror image at the dorsal midline)
43
Why use Xenopus laevis in studies?
- a model amphibian embryo
- embryos are large (0.5mm)
- large embryos allow for dissection of early embryo
- dissected tissues were easy to culture
44
Anterior-posterior axis vs Dorsal-ventral axis
Anterior-posterior axis=head to tail, established during gastrulation
Dorsal-ventral axis=back to belly, established during fertilization and elaborated on during gastrulation
45
All vertebrate embryos undergo what similar pattern of development?
1) gametogenesis
2) fertilization
3) cleavage
4) gastrulation
5) notochord formation
6) neurulation
7) somitogenesis
8) organogenesis
46
How is radial symmetry broken in the Xenopus laevis egg?
at fertilization, the sperm enters the animal region
47
How many blastomeres does the 8-cell stage of the embryo contain?
8 blastomeres
48
Xenopus laevis undergoes which steps in order ****
1) fertilization of egg
2) cleavage into blastomeres
3) blastulation into blastula
4) gastrulation into gastrula
5) neurulation into neurula
6) early tail bud stage
7) organogenesis
49
Which is the most important step in embryo development?
gastrulation since development will cease if endoderm and mesoderm are not internalized
50
Dorsal lip
- a section of the blastopore
- First visual sign of dorsal/ventral polarity and the beginning of the gastrula stage
- not a uniform group of cells, they are changing over time
- different cells occupy the lip at different times
51
Lateral mesoderm
mesoderm spreads to the left and right of the embryo to cover the inside of the archenteron
52
Late gastrulation (end of gastrulation)
- The blastopore closes (the future anus)
- The mesoderm contacts both the ectoderm and endoderm along the anterior-posterior axis
- Mesoderm is patterned along the Anterior-Posterior axis via the action of the Spemann-Mangold organizer
- Ectoderm now covers the embryo (gives rise to skin and nervous system)
- Yolk remains a food source for the embryo
53
Post-gastrulation
- the dorsal mesoderm starts to develop the notochord
- Somites start to form
- Lateral plate mesoderm forms
54
Which is the second most important step in embryo development?
neural tube closure
55
Sperm entry
- breaks radial symmetry of egg (fertilization)
- anywhere in animal hemisphere (ventral region)
- Future dorsal side is opposite of sperm entry
- Cortical rotation is enacted
- The cortex loosens from the membrane and rotates 30 degrees in direction away from the sperm
- dorsal-ventral axis is the first to be defined
- Nuclear localization of B-catenin is the major signalling output of cortical rotation
- Polarity of the egg changes so that there is a directed movement of mRNA and proteins from vegetal region to future dorsal side opposite of sperm entry
56
Establishment of the dorsal side
- is opposite from sperm entry
- cortical rotation occurs
- directed movement of mRNA and proteins from the vegetal region to the future dorsal side, opposite of sperm entry
57
Maternal factors
- protein or RNA that is deposited in the egg by the mother during oogenisis
- production of these maternal proteins and RNAs is under the control of maternal genes
- specify endoderm and ectoderm
- Vg-1, XWnt-11, VegT
58
Mid-blastula transition
- in amphibian embryos, the stage in development when the embryo's own genes begin to be transcribed, cleavages become asynchronous, and the cells of the blastula become motile
- marks the trasnition between maternal and zygotic expression
- Vg-1 mRNAs in Xenopus are localized in the vegetal cortex
- Vg-1 mRNAs are translated upon fertilization
59
Vg-1 (vegetalizing factor-1)
- a maternal factor
- Maternal mRNA and proteins that are deposited in egg prior to fertilization
- Genes are not yet under control of the embryonic genome (zygotic)
- Transforming growth factors (TGFB) family
- Localized to the vegetal cortex
- Assists in mesoderm induction (mesoderm inducer)
- a TGFB signalling molecule
60
Vegetal cortex
- where Vg-1 mRNAs are localized in Xenopus
| - a region of actin-rich cytoskeleton lying beneath the plasma membrane
61
XWnt-11
- a maternal factor
- wingless or Wnt lignand
- a ligand for the Wnt pathway
- mRNAs localized to the vegetal cytoplasm
- aids in specification of dorsal-ventral axis
- with Dishevelled, they specify the dorsal side of the embryo = dorsalizing factor
62
Vegetal cytoplasm
-where XWnt-11 (wingless or Wnt ligand) is localized
63
VegT
- a maternal factor
- T-box family of transcription factors
- Upregulates the transcription of Nodal proteins (Xnr)s
- mRNA is localized to the vegetal hemisphere
- a factor required for endoderm
- is sufficient and necessary for endoderm differentiation
- If mRNA translation is blocked, endoderm is lost = necessary
- Sufficient in that Veg T mRNA injected into the cells of the animal cap result in expression of endoderm
- with nuclear B-catenin, they stimulate Xnr transcription
- The highest Xnr protein levels are produced by the Nieuwkoop centre where Veg-T and B-catenin overlap
64
transcription factors
- a protein required to initiate or regulate the transcription of a gene into RNA
- act within the nucleus of a cell by binding to specific sites in the regulatory regions of the gene
65
Vegetal hemisphere
-where VegT is localized
66
Cortical rotation
- cortex loosens from the membrane and rotates 30 degrees in direction away from the sperm
- Polarity of the egg changes so that there is a directed movement of mRNA and proteins from vegital region to future dorsal side opposite of sperm entry
- a movement of the cortex of the fertilized amohibian egg that occurs immediately after fertilization
- the cortex rotates with respect to underlying cytoplasm away from the point of sperm entry
- Nuclear localization of B-catenin is the major signalling output of cortical rotation
67
canonical Wnt/B-catenin signalling pathway
- an intracellular signalling pathway stimulated by members of the Wnt family of signalling proteins that leads to the stabilization of B-catenin and its entry into nuclei, where it acts as a transcriptional co-activator
- Nuclear B-catenin has its highest concentration on the future dorsal side of the embryo
- Wnt signalling increases in the dorsal region and decreases in the ventral
- Wnt signalling decreases in the anterior region and increases in the posterior
- Signalling is separated by time and mode of action
- In blastula, increases Wnt signalling establishes dorsal region, with high [B-catenin] in nuclei
- During gastrulation, the time that cells are resident in the organizing region is patterning the anterior-posterior axis
68
B-catenin
- protein that functions both as a transcriptional co-activator and as a protein that links adhesive cell junctions to the cytoskeleton
- as a transcriptional co-activator, B-catenin is activated in early development in many embryos as the end result of a Wnt signalling pathway
- nuclear B-catenin has its highest concentration on the future dorsal side of the embryo
- B-catenin is a transcription co-factor for TCF/LEF 1 (transcription factors)
- with VegT they stimulate Xnr transcription
- The highest Xnr protein levels are are highest in the future Nieuwkoop centre where Veg-T and B-catenin overlap
69
Dutch embryologist Pieter Nieuwkoop
-discovered the Nieuwkoop Center
70
Siamois
- a transcription factor
| - is up-regulated and sets up the dorsal-ventral polarity of the blastula
71
Dorsalizing factors
- in the early vertebrate embryo, a protein or RNA that promotes the formation of dorsal structures, such as maternal components of the Wnt signalling pathway in Xenopus
- X Wnt-11 and Dishevelled specify the dorsal side of the embryo
- “Necessary” for the Spemann-Mangold organizer and Nieuwkoop Centre
- Localized stabilization of B-catenin
72
Dishevelled
-with XWnt-11, they specify the dorsal side of the embryo = dorsalizing factor
73
Ventralizing factors
-UV exposure of ventral side of embryo results in: Failure of cortical rotation, Failure resulted in a ventralized embryo, Deficient dorsal structures at the expense of ventral tissues (like blood forming tissue)
74
Lithium chloride
- inhibits activity of GSK-3B
| - Treatment with LiCl results in dorsalization of the embryo at the expense of the ventral structures
75
What are the three main stages of vertebrate development?
1) setting up the main body axes (anterior-posterior and dorsal-ventral)
2) specification of the three germ layers (ectoderm, mesoderm and endoderm)
3) germ layer patterning (mesoderm and early nervous system)
76
Explain the canonical Wnt/B-catenin signalling pathway when Wnt (ligand) is absent.
***
1) nothing binds to the receptor Frizzled
2) Dishevelled does not act as a co-receptor (no role)
3) APC/Axin/GSK-3B complex does B-catenin degradation
4) nothing enters the nucleus and there is no transcription
- Axin is a scaffolding protein
77
Explain the canonical Wnt/B-catenin signalling pathway when Wnt (ligand) is present.
***
1) Wnt (diffusible ligand) binds to Frizzled receptor
2) Dishevelled arrives (an adapter/co-receptor) and phosphorylates
3) APC/Axin/GSK-3B complex (B-catenin destruction complex) also binds to Dishvelled
4) B-catenin degradation is inhibited
5) free B-catenin (transcription co-factor) accumulates
6) stabilization of the transcription co-factor B-catenin (involved in polarity) and enters the nucleus and binds to TCF/LEF1 (a transcription factor), which then becomes and enhancer for transcription
7) transcription begins
- Axin is a scaffolding protein
- TCF-LEF1 is repressor growcho
* ***B-catenin transitions the TCF/LEF1 complex into transcription activators
78
An experiment was done where they took the Spemann-Mangold Organizer from one embryo and transplanted it into another (like the ventral region). What were the results of this experiment? What happens it was taken at different stages of development?
What happens if you were to transplant the Nieuwkoop Centre?
- resulted in the formation of a second dorsal axis (twinned embryo that are connected)
- Early gastrulation can create head structures, as well as mid-body and tail
- A later blastopore (like dorsal lip) becomes limited in its capacity to create anterior structures and only a tail bud results with transplantation
- If taken mid gastrulation, then only a trunk and tail will be created
-gives the same result of a second dorsal axis (twinned embryo that are connected), like the Spemann-Mangold organizer
79
Morphogen
any substance active in pattern formation whose spatial concentration varies and to which cells respond differently at different threshold concentrations
80
Morphogen gradients can be interpreted in which two ways?
1) increase in B-catenin concentration
2) time that cells experience high Wnt signalling
- this is particularly relevant for migrating populations of cells that can move away from a point source of a signal
81
Nieuwkoop Center
- Blastula organizer, the precursor to the Spemann-Mangold organizer
- Pietre Nieuwkoop found this
- It is the combined action of B-catenin and maternal factors Veg T on the dorsal side of the vegetal region that is the first signalling centre
- A transcription factor siamois is up-regulated and set up the dorsal-ventral polarity of the blastula
- Spemann-Mangold organizer is located just above the Nieuwkoop Centre
- This forms later in the blastula stage
- Molecular evidence suggests Nieuwkoop Centre “maybe” a precursor to the organizer
- The highest Xnr protein levels are produced by the Nieuwkoop centre where Veg-T and B-catenin overlap
82
Necessary
take it away then can’t develop, needed for development
83
What would happen if you were to transplant the dorsal cytoplasm to the ventral side of the embryo?
a second dorsal axis due to dorsalizing factors (twinned embryo)
84
What would happen if you were to inject B-catenin mRNA into the ventral side of the embryo?
- B-catenin=dorsalizing factor
- A Nieuwkoop Centre
- Spemann-Mangold organizing Centre
- Dorsal axis
- results in a twinned embryo with a duplicated dorsal axis (second dorsal axis)
85
Xfz-7
- Frizzled receptor in the canonical Wnt/B-catenin signalling pathway
- Loss of function of this frizzled receptor
- Loss of dorsal structure
86
Animal pole
-the most terminal part of the animal region, which is directly opposite the vegetal pole
87
Ectoderm
-the embryonic germ layer that gives rise to the epidermis and the nervous system
88
Vegetal pole
-is the central point on the surface of the vegetal region, directly opposite the animal pole
89
Endoderm
-the embryonic germ layer that gives rise to the gut and associated organs, such as the lungs and liver in vertebrates
90
Mesoderm
-germ layer that gives rise to the skeleto-muscular system, connective tissues, the blood, and internal organs such as the kidney and heart
91
Animal cap assay
-Method where the animal region of the blastula was dissected away and cultured
92
Ectodermin
- a factor required for ectoderm
- Ectodermin counteracts the mesoderm inducing signal and limits the spatial range of mesoderm induction
- restricts the ventral signal to the marginal zone
93
Foxl1e
- a factor required for ectoderm
- Foxl 1e is expressed in late blastula, is a transcription factor that maintains region identity of the ectoderm
- If Foxl 1e is removed, ectodermal cells start to mix with the other germ layers and start to differentiate according to the new position
94
Three signal model of mesoderm induction and patterning
1) induction of mesoderm and organizer; VegT, B-catenin and Xenopus nodal-related proteins (Xnr); brachyury and goosecoid
2) BMP-4 and XWnt-8 ventralize mesoderm
3) organizer secretes signals that inhibit BMP-4 or XWnt-8 activity
95
Xenopus Nodal-related proteins (Xnr)
- mediate and are required for mesoderm induction in all vertebrates (mesoderm inducers)
- part of the TGFB family of signalling molecules
- are expressed by endoderm
- their transcription is stimulated by VegT and nuclear B-catenin
- The highest Xnr protein levels are produced by the Nieuwkoop centre where Veg-T and B-catenin overlap
96
Derriere
-a mesoderm inducer that can be added to the animal cap and induce mesoderm formation
97
Bone Morphogenic Proteins (BMPs)
-a mesoderm inducer that can be added to the animal cap and induce mesoderm formation
98
Activin
- Activin could be applied to the isolated animal cap and this was sufficient for mesoderm induction
- This confirmed the role of the Nodal-related pathway in mesoderm induction
- a mesoderm inducer that can be added to the animal cap and induce mesoderm formation
99
Brachyury
- T-box family transcription factor
- required for mesoderm development
- Highly conserved in mesoderm induction
- Earliest marker of mesoderm induction
- Xbra in Xenopus
- First expressed in all presumptive mesoderm
- Later confined to notochord and tail bud
- Necessary for posterior mesoderm development
100
Goosecoid
- transcription factor required for mesoderm development
- This is a transcription factor that is expressed in the organizer
- Is sufficient to make an organizer
- Injection of goosecoid mRNA into the ventral region of the blastula results in a second organizer and a second embryonic axis
101
Threshold responses (concentration gradients) to Nodal-related protein Signalling
- pattern the mesoderm
- A graded growth factor signalling is patterning the dorsi-ventral axis
- This is providing positional information to the mesoderm
- In the animal cap assay, beads soaked in activin (part of the Nodal family) can activate Nodal receptors
- Increasing concentration of activin activates different mesodermal fates...
- No or very low activin=epidermal genes (keratin)
- Intermediate concentration=Brachyury and muscle specific gene (somites)
- Highest concentration=goosecoid expression and tissues like the notochord and formation of the organizer
* Direct relationship between the amount of activin in the beads and the pattern of mesoderm
102
Activin soaked beads
- In the animal cap assay, beads soaked in activin (part of the Nodal family) can activate Nodal receptors
- Direct relationship between the amount of activin in the beads and the pattern of mesoderm
- Increasing concentration of activin activates different mesodermal fates...
- No or very low activin=epidermal genes (keratin)
- Intermediate concentration=Brachyury and muscle specific gene (somites)
- Highest concentration=goosecoid expression and tissues like the notochord and formation of the organizer
103
BMP-4
- along with XWnt-8, ventralize mesoderm
- its antagonists are Noggin, Chordin, Follistatin
- ventral signal is largely formed by BMP-4
- if activity blocked, results in dorsal mesoderm at the expense of ventral mesoderm
- overexpression of BMP-4 ventralizes embryo
104
XWnt-8
- along with BMP-4, ventralize mesoderm
- its antagonsist is Frizzled-related protein (Frzb)
- is required for ventral fate
105
Ventralize mesoderm
-BMP-4 and XWnt-8
106
Noggin
- an antagonistic signal of BMP-4 secreted by the organizer
- prevents BMP-4 from binding its receptor
- was first discovered as a factor that could rescue ventralized embryos caused by ventral UV exposure
107
Chordin
-an antagonistic signal of BMP-4 secreted by the organizer
108
Follistatin
-an antagonistic signal of BMP-4 secreted by the organizer
109
Frizzled-related protein (Frzb)
-an antagonistic signal of XWnt-8 secreted by the organizer
110
Mesoderm induction
- Is mediated by a family of signalling molecules (TGFB family) represented by Nodal-related proteins= Xnr (Xenopus Nodal-related)
- Nodal-related proteins are required for mesoderm induction in all vertebrates
- the blastula embryo (prior to gastrulation) can be manipulated to determine tissues or signals that are directing germ layer specification
- In the animal cap assay, the animal cells were cultured with the vegetal cells
- It was noted that ectoderm could now take on a mesoderm fate
- The ectoderm is receiving a signal to form mesoderm = mesoderm induction
- Scientists repeated the experiment with ectoderm and endoderm separated by a porous membrane to allow for diffusion of small molecules (such as proteins and hormones)
- Mesoderm was still induced
- Activin could be applied to the isolated animal cap and this was sufficient for mesoderm induction
- This confirmed the role of the Nodal-related pathway in mesoderm induction
- Animal cap cells combine with different regions of the endoderm give rise to different types of mesoderm
- Dorsal endoderm induces dorsal mesoderm (notochord)
- Ventral endoderm induces ventral mesoderm (blood cells, vessels)
111
If you were to use the animal cap assay to culture and isolate the animal pole cells, what would be the result? What about vegetal cells and marginal zone cells?
- Animal pole cells will give rise to ectoderm when cultured in isolation, this suggests that animal cells are specified towards ectoderm
- Vegetal cells in isolation give rise to endoderm (specified)
- The marginal zone could give rise to ectoderm or mesoderm
112
Which factors are required for endoderm specification?
- Veg T mRNA is sufficient and necessary for endoderm differentiation
- If mRNA translation is blocked, endoderm is lost = necessary
- Sufficient in that Veg T mRNA injected into the cells of the animal cap result in expression of endoderm
113
Which factors are required for ectoderm specification?
- Ectodermin counteracts the mesoderm inducing signal and limits the spatial range of mesoderm induction
- Foxl1e is expressed in late blastula, is a transcription factor that maintains region identity of the ectoderm
- If Foxl1e is removed, ectodermal cells start to mix with the other germ layers and start to differentiate according to the new position
114
Draw a cross-section through an embryo to observe how mesoderm is divided into regions along the dorsal-ventral axis.
Dorsal of embryo:
- Neural tube (at the very top, gives rise to spinal cord) = ectoderm
- Notochord (directly underneath neural tube) = most dorsal mesoderm fate
- Somites (on either side of notochord, gives rise to muscles) = next most dorsal mesoderm fate
- Lateral plate mesoderm (coming off of each somite, gives rise to blood) = lateral to ventral fate
- Gut (directly underneath notochord) = endoderm
115
Which three signals pattern the mesoderm along the dorsal-ventral axis?
1) vegetal region produces a signal that induces the mesoderm and establishes the Spemann-Mangold organizer (most dorsal mesoderm) on the dorsal side
2) a distinct set of signals specifies the ventral mesoderm
3) the organizer releases a second signal that inhibits ventral fate, limits ventral fate to ventral side so that the mesoderm adjacent to the organizer can give rise to notochord and somites
116
Mesoderm inducers
- Mesoderm inducers were defined as factors that could be added to the animal cap and induce mesoderm formation
- TGFB growth factors
- Xenopus Nodal-related proteins (Xnr)
- Derrière
- BMPs (bone-morphogenic proteins)
- Vg-1
- Activin
117
TGFB family
- a family of growth factors and signalling molecules
- are mesoderm inducers that could be added to the animal cap and induce mesoderm formation
- includes Xenopus Nodal-related proteins (Xnr) as part of the family
118
Nodal TGFB signalling pathway
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- members of the TGFB family like Nodal and the Nodal-related proteins, bone morphogenetic proteins (BMPs) and activin are dimeric ligands that act at cell-surface receptors (heterodimers)
- binding of these ligands phosphorylates the receptor
- this in turn phosphorylates intracellular signalling proteins called Smads
- they then form a transcriptional regulatory complex that enters the nucleus to either activate or repress target genes
- if BMP ligand binds to receptor, the result is ventralization of the mesoderm
- if Nodal, TGFB or Activin ligands bind, then dorsal mesoderm induction occurs
119
How are Nodal-related proteins activated?
- VegT activates the expression of Nodal-related proteins and Derriere
- if VegT is depleted, no mesoderm develops and ectoderm forms throughout the embryo
- injection of nodal-related and derriere mRNAs can rescue mesoderm formation
- if B-catenin (from the Wnt signalling pathway) and VegT are both present, then Nodal-related protein concentrations are high and the organizer forms
- if only VegT is present, , Nodal-related protein concentrations are low and the ventral mesoderm forms
120
Why is the Wnt/B-catenin signalling pathway important in terms of B-catenin concentrations?
- B-catenin needs to be kept in check otherwise could lead to cancers (proliferation of cells)
- kept in check by GSK-3B/APC/Axin: the B-catenin destruction complex
121
What two signalling pathways are required to form the Nieuwkoop center?
Wnt and Nodal signalling pathways
122
Which are the most ventral to most dorsal structures of the developing embryo?
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Most ventral:
-blood, vessels
-kidneys, heart
-somites, muscles
-notochord
-organizer
Most dorsal:
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123
Inhibitory mechanisms
- Inhibits mesoderm production because otherwise ectoderm cells would completely become mesoderm
- Control the Nodal signals that transform cells into mesoderm
124
Nodal and Wnt signalling pathways working together
- The first signal induces mesoderm and specifies the organizer
- Xnrs, Derriere, BMPs, Vg-1, activin
- Veg-T is necessary and sufficient for endoderm and mesoderm specification
- One output is expression of Xenopus Nodal-related proteins (Xnr)
- This family is expressed in the endoderm and induces mesoderm
125
Which structure of the developing embryo receives the highest levels of Nodal signalling (concentration and time period)?
the Spemann-Mangold organizer
126
Which transcription factors are required for mesoderm development?
Brachyury and goosecoid, Xnot, Xlim1
127
Ventral signal
- Largely formed by BMP-4
- Initially uniform
- Then restricted to marginal zone by ectodermin
- If blocked BMP-4 activity, results in dorsal mesoderm at the expense of ventral mesoderm
- Overexpression of BMP-4 ventralizes embryo
- This signal is counter-acted by the organizer
- Organizer secretes Noggin that inhibits BMP-4
- Noggin was first discovered as a factor that could rescue ventralized embryos caused by ventral UV exposure
- Noggin prevents BMP-4 from binding its receptor
- The actual mechanism is complex
128
Name the conserved signalling molecules and transcription factors in vertebrates.
Genes encoding transcription factors:
Xenopus gastrula=brachyury and goosecoid
Mouse gastrua=brachyury and goosecoid
Genes encoding secreted proteins:
Xenopus gastrula=Chordin, noggin and Cerberus
Mouse gastrula= chordin, noggin and Cerberus-related
129
Mesoderm induction and patterning - Three signal model
- mesoderm induction and patterning are occurring simultaneously
1) VegT transcription factors are activating the Nodal-related proteins (ligands for TGFB signalling). Wnt isactivated in the very dorsal part of the embryo and stabilizes B-catenin. This caused Nodal to be highest in the dorsal endoderm and mesoderm
2) BMP-4 = ventral, signal for mesoderm, formation of blood tissues and some muscle. Without Nodal signalling embryo becomes ventralized (so NO notochord or organizer forming)
3) Upon formation of the organizer, it secretes molecules that inhibit BMP-4. These include Noggin, Chordin and Follistatin
