Organogenesis (Prior) Flashcards
`What is organogenesis?
Organogenesis is the process by which organs arise from one of three germ layers during the later stages of embryonic development
This requires input from genetic programs, cell-cell interactions, and mechanical forces
What are the timings of blastocyst and gastrula formation in mouse and humans?
Mouse:
Blastocyst - E3.5
Gastrula - E6.5-7.0
Human:
Blastocyst - Day 5
Gastrula - Week 2-3
What do blastocysts and gastrula contain?
Blastocysts contain the ICM which gives rise to the 3 germ layers and the embryo proper:
- Endoderm
- Mesoderm
- Ectroderm
They also contain trophectoderm, which are the outer cells that contribute to extra embryonic tissue
Gastrula contains the embryonic disc which encompasses the 3 germ layers
What organs do the three germ layers give rise to?
Endoderm:
lungs / intestines / thyroid / pancreas / bladder
Mesoderm:
heart / blood / bones / muscles / kidneys
Ectroderm:
epidermis / nervous system / eyes / inner ears
What is the route the ICM takes to produces its derivatives?
What is the process of heart development?
The vascular system-the heart, blood vessels, and blood cells- is the first organ system to develop in vertebrate embryos.
Early development - oxygen and nutrients need to be delivered to the rapidly developing tissues and subsequently to developing organs.
The heart begins development from the first mesodermal cells which migrate from the site of gastrulation toward the anteriolateral border of the trilaminar embryonic disc.
While migrating, these mesodermal cells will be rendered competent for differentiation toward the cardiac lineage
Day 21/22 the tubular heart starts to beat
At the beginning of the fourth week of development (CS10) the straight heart tube undergoes looping
When does the eye begin to develop?
Embryo at the end of 4 weeks of development showing the otic and optic vesicles
The vertebrate eye is essentially an extension of the forebrain
It extends from the ectoderm
What is the master gene for eye development?
Pax6
E.g.
The ectopic expression of mouse Pax6 in a Drosophila antennal disc results in compound eye structures developing on the antenna
Mouse timings and process of endodermal organogensis
E7: Endoderm specification:
- Endodermal tube is formed
E8: Hepatic specification
- Hepatoblasts
- Liver progenitors in posterior foregut
E9: Liver bud growth
Depending on where along the anterior posterior axis the progenitors sit, different organs will arise
Anterior foregut
- Lungs
Posterior foregut
- Liver
- Pancreas
Midgut
- Small intestine
Hindgut
- Colon
The liver and pancreas originate from adjacent regions of the definitive endoderm that gives rise to three diverticula (buds)
Formation of the organ buds and their outgrowth from the foregut endoderm are controlled by mesodermal signals.
These specify endoderm cells to hepatic and pancreatic fates and promote proliferation of the budding cells.
Pancreas development
The pancreas arises from ventral and dorsal endodermal buds located caudally to the liver
Cell proliferation rapidly generates a population of multipotent progenitors that differentiate to the mature epithelial cell types: endocrine, acinar, and ductal cells.
Do organs develop in isolation
No, organs do not develop in isolation
The gallbladder, hepatic, cystic, and common bile ducts, develop from the same endodermal region as the ventral pancreas.
As the ventral pancreas starts rotating around the gut to merge with the dorsal pancreas, the extrahepatic biliary tract develops separately from the pancreas while maintaining a connection with the common pancreatic duct.
What are the two main functional cells in the liver?
Hepatoblasts give rise to:
- Hepatocytes
- Ductal cells
Hepatocytes are responsible for detoxification and bile production
Ductal cells are bile ducts that collect the bile ducts produced by hepatocytes and take it away for digestion
How was the liver progenitor identified?
Using a process called lineage tracing
Lineage tracing is where in certain cells, you can turn on a florescent reporter
This was done in the liver at E9.5 in mice, at a gene called Lgr5+ using the reporter “TdTomato” (red)
The embryo is then left until E11.5
At E11.5 red cells were found in the liver
This tells us there is a progenitor in the liver at E9.5 that is positive for the gene Lgr5+
The embryo was then left to be born, and after a month red cells were located in the liver including hepatocytes and ductal cells
What happens if progenitors are destroyed in the liver and the pancreas?
Using a specific transgenic mouse, embryonic liver and pancreas progenitors can be targeted for destruction
After such treatment, the embryonic liver grows back to a normal size, indicating that it does not arise from a fixed number of cells
In contrast, if some of the cells of the pancreas in a mouse embryo are destroyed (by the technique described above) after the pancreatic ‘bud’ has formed, a smaller than normal pancreas develops.
What are planarians?
Planarians are tiny flatworms with the ability to regrow the entire organisms
Planarians contains lots of multipotent progenitors that reside within the adult called neoblasts
These neoblasts are able to migrate to the site of injury to differentiate into the required materials
What are hydra?
Hydra have similar regeneration to planarians but use a different technique
Although Hydra is devoid of pluripotent stem cells, it has three stem cell types (ectodermal and endodermal epithelial stem cells and interstitial stem cells) throughout the body
What is regeneration?
Regeneration is the reactivation of developmental mechanisms in postembryonic life to restore missing or damaged tissues
Do mammals exhibit regeneration?
Do mammals exhibit regeneration?
Mammals have a very limited regenerative capacity
Severe damage to tissues or organs (e.g., hearts, limbs, or spinal cords) does not induce regenerative responses but rather induces healing concomitant with fibrotic scarring
However, they can regenerate certain structures
How does liver regeneration occur?
In the mammalian liver, no regenerating blastema is formed
The liver regenerates the same volume as it lost. Acute damage -each cell appears to generate its own cell type
A reserve population of multipotent progenitor cells divides when these tissues cannot regenerate the missing portion
How do somatic stem cells know when its time to regenerate?
When cells are damaged, they release soluble chemoattractants, such as chemokines, which recruit somatic stem cells to the site of injury
These cells may then differentiate into the target tissue cell type
Somatic stem cells can induce local changes, such as the generation of new blood vessels that promote wound healing
The regenerative capacity of endogenous somatic stem cells may not always be sufficient to repair diseased or damaged tissue
What things are required to grow tissues in a lab?
- A 3D cellular cluster that is derived from stem/progenitor cells that are able to self-renew and self-organise
- A similar cellular architecture to primary tissue that is genetically stable
- Developmental signals to direct the developmental pathW
What is an organoid?
Organoid is an artificial, self-organising tissue that resembles an organ
Being like an organ, it must satisfy criteria that also define an organ
2 main types:
- Pluripotent stem cell derived
- Adult derived
They generally contain multiple cell types but can contain just 1
How can an organoid be grown?
Organoids can be generated from adult stem cells.
They can also be generated from PSCs by directed differentiation
In both cases, stem cells or progenitors are grown in an environment that mimics their in vivo counterpart, allowing for self-organisation into a tissue with a remarkably similar structure to that in vivo
What are the easiest organoids to make?
Ectrodermal tissues are the easiest to make
If you place the 3D cell aggregate and don’t do anything with it, cerebral organoids will be generated
The use of growth factors and small molecules such as dual-SMAD will drive brain-region specific orgnaoid generation
The use of Wnt inhibition + dissolved MG will result in a retinal organoid
The use of Wnt inhibition + dissolved MG + low FGF and BMP inhibiton results in inner ear organoids
Kidney organoid generation
Early activation of Wnt and FGF9 drives kidney organoid generation
What are some adult tissue derived mesodermal organoids that can be generated?
Bone
- -serum
- +TGFß1
Fallopian tube
- Wnt
- RSP01
- EGF
- FGF10
- noggin
Endometrial
- RSPO1
- EGF
- FGF10
- noggin
- TGFß inhibitor
What are some PCS derived endodermal tissue organoids?
Gut
- EGF
- noggin
- RSPO
Thyroid
- EGF
- FGF
- LGF
- insulin
- TSH
Stomach
- EGF
- noggin
- RA
- (Wnt)
Liver bud
- HGF
- dexamethasone
- oncostatin
Lung
- noggin
- FGF
- Wnt
- TGFB1
What are some adult-tissue derived endodermal tissue organoids?
Gut
- RSPO
- EGF
- noggin
- TGFB1
Stomach
- RSPO
- EGF
- noggin
- FGF
- TGFB1
Pancreas
- RSPO
- EGF
- noggin
- FGF
- TGFB1
Liver
- RSPO
- EGF
- HGF
- FGF
- TGFB1
- Nic
- FSK
Lung
- RSPO
- noggin
- FGF
- TGFB1
- Nic
- p38i
- RhoKi
How are intestinal organoids formed?
By isolating Lgr5+ stem cells from the crypts of the intestinal epithelium and growing in matrigel with the correct growth factors, an organoid with all of the required cell types for the intestines will be generated
How are pancreas organoids formed?
Duct within pancreas is isolated and grown within matrigel with correct growth factors
Organoid generated is a single layered structure, the same as would be in an in vivo pancreatic duct
What are some reasons to form pancreatic organoids?
Investigate biology of healthy tissue
Model diseases
Investigate cell therapy to cure pancreatic diseases
Liver hepatocyte organoid generation
Liver ducts can be isolated and grown in 3D environment to form a ductal liver organoid
This can then be exposed to growth factors to differentiate into hepatocytes
Liver organoid generation overview
What is a Body Plan?
A body plan is the general structure, arrangement, and organization of the different parts of an organism.
Includes aspects such as symmetry (bilateral, radial, etc.), the presence and type of body cavity (coelom, pseudocoelom, acoelomate), segmentation, and limb disposition.
Determines how an organism’s body is organized and functions, influencing its development, evolution, and adaptation to the environment.
Where does a chick embryo develop?
In an egg, consisting of an eggshell, yolk, and an embryonic disk on top of the yolk.
What is the area of cytoplasm on top of the yolk called, and what are its two main regions?
It is the area of the small disk of cytoplasm, with the area pellucida (gives rise to the embryo) and the area opaca (does not give rise to the embryo).
What is the significance of the posterior marginal zone?
It is where the Koller’s sickle forms, initiating the primitive streak, which determines the anterior-posterior axis.
What is the primitive streak, and why is it important?
The primitive streak is a structure that extends from the posterior marginal zone towards the midline of the area pellucida, forming Hanson’s node, which acts as the organizer and establishes the anterior-posterior axis.
Describe Koller’s sickle and its role in development.
Koller’s sickle is a structure at the posterior margin of the chick embryo that initiates the formation of the primitive streak, crucial for axis development.
What happens as the primitive streak regresses in the chick embryo?
The neural tube is laid down, and the mesoderm on either side begins to segment into somites, forming the segmented body plan.
What is Hanson’s node, and why is it important?
Hanson’s node is the condensation of cells at the anterior end of the primitive streak, serving as the primary organizer in the embryo.
What did graft experiments using quail and chick embryos demonstrate?
Early quail organizer transplanted into chick embryos induces the formation of a body axis, showing the organizer’s potency to induce embryonic cells.
What do the results of graft experiments reveal about early vs. late organizers?
Early organizers can induce extensive body structures (head, trunk, tail), while later organizers primarily induce tail structures due to decreased potency.
How does gravity influence the formation of the posterior marginal zone in chick embryos?
The rotating egg during development positions the lighter components (embryonic disk) at the top, specifying the posterior marginal zone.
What were the results of the space shuttle experiment on chick embryo development?
In low gravity conditions, most embryos did not develop normally due to the lack of proper posterior marginal zone formation; only one out of eight embryos developed normally in space.
Describe the process of left-right asymmetry establishment in chick embryos.
The left side is specified by Sonic Hedgehog, which activates Caronte, BMP, and then Pitx2, while the right side is inhibited by activin, preventing Sonic Hedgehog signaling.
What does in situ hybridization show about left-right asymmetry in chick embryos?
In situ hybridization shows that Caronte is found on the left side when viewed ventrally, indicating the left side specification by Sonic Hedgehog pathway.
How is left-right asymmetry established in other vertebrates like fish and amphibians?
It’s influenced by the flow of embryonic fluids driven by cilia, creating a vortex that determines the left side.
What role do human embryonic stem cells play in similar experiments?
When cultured to form an organizer-like structure and transplanted into chick embryos, they can induce axis formation, demonstrating cross-species induction potential.
Explain the significance of gravity in chick embryo development.
Gravity ensures the proper formation of the posterior marginal zone by positioning the lighter embryonic disk at the top during the egg’s rotation through the oviduct.
What happens when chick embryos develop in low gravity or no gravity environments?
The absence of gravity leads to improper formation of the posterior marginal zone, resulting in abnormal development of most embryos.
How does the primitive streak establish the body plan in chicks?
It sets up the anterior-posterior axis, and as it regresses, it helps lay down the neural tube and segment the mesoderm into somites.
What is the significance of the organizer’s potency?
Early organizers can induce extensive body structures (head, trunk, tail), while later organizers primarily induce tail structures due to decreased potency.
Explain the concept of the organizer in human and chick embryo experiments.
The organizer from human embryonic stem cells, when transplanted into chick embryos, can induce axis formation, showcasing evolutionary conservation of developmental mechanisms.
Describe the role of Koller’s sickle in primitive streak formation.
Koller’s sickle marks the posterior end of the chick embryo and initiates the formation of the primitive streak, which is essential for setting up the body axes.
How do cilia contribute to left-right asymmetry in vertebrates?
Cilia in vertebrate embryos create a fluid flow that establishes the left-right axis by moving embryonic fluids and determining the left side.
How is the anterior-posterior axis formed in chick embryos?
The axis is established by the primitive streak, which originates from the posterior marginal zone, with Hanson’s node forming at the anterior end and regressing to lay down the neural tube and somites.
What experimental evidence supports the role of the organizer in axis formation?
Graft experiments, such as transplanting quail organizer cells into chick embryos, demonstrate the ability of the organizer to induce axis formation and embryonic development.
What are the implications of the space shuttle experiment for understanding embryo development?
The experiment shows that gravity is crucial for proper posterior marginal zone formation and subsequent embryo development, highlighting the role of physical forces in developmental biology.
How does Sonic Hedgehog influence left-right asymmetry in chick embryos?
Sonic Hedgehog activates Caronte on the left side, which then activates BMP and Pitx2, leading to left-side specification. On the right side, activin inhibits Sonic Hedgehog, preventing this pathway.
What was observed in embryos with left-right asymmetry using in situ hybridization?
In situ hybridization showed that Caronte expression is localized to the left side, confirming the role of Sonic Hedgehog in left-right asymmetry.
What is the outcome of transplanting early and late organizers in quail and chick graft experiments?
Early organizers induce the formation of head, trunk, and tail structures, while late organizers primarily induce tail structures. This demonstrates the higher potency of early organizers.
What is the importance of the left-right asymmetry in vertebrates?
Left-right asymmetry ensures the correct positioning of organs such as the heart and liver, which are essential for proper physiological functioning.
How did the space shuttle experiment test the effects of low gravity on embryo development?
A: Embryos were fertilized and developed in low gravity conditions aboard the Endeavour Space Shuttle, and the results showed that most embryos did not develop normally without proper gravity.
